WO2007023461A2 - Method for detection of micro-organisms and antibiotic resistance markers and nucleic acid oligonucleotides therefor - Google Patents

Method for detection of micro-organisms and antibiotic resistance markers and nucleic acid oligonucleotides therefor Download PDF

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Publication number
WO2007023461A2
WO2007023461A2 PCT/IB2006/052919 IB2006052919W WO2007023461A2 WO 2007023461 A2 WO2007023461 A2 WO 2007023461A2 IB 2006052919 W IB2006052919 W IB 2006052919W WO 2007023461 A2 WO2007023461 A2 WO 2007023461A2
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WIPO (PCT)
Prior art keywords
seq
nos
sequence
pair
antibiotic resistance
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PCT/IB2006/052919
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French (fr)
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WO2007023461A3 (en
Inventor
Gerd Luedke
Johannes Bacher
Jens-Peter Seher
Holger Engel
Jürgen LAUBER
André Bahr
Helge Lubenow
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Philips Intellectual Property & Standards Gmbh
Koninklijke Philips Electronics N. V.
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Priority to JP2008527574A priority Critical patent/JP2009505651A/en
Publication of WO2007023461A2 publication Critical patent/WO2007023461A2/en
Publication of WO2007023461A3 publication Critical patent/WO2007023461A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • NA nucleic acids
  • NA sequences are not yet performed on a routine basis, as is the case, for instance, in the measurement of blood glucose concentration of diabetics.
  • well-equipped laboratories and well-trained staff are necessary and careful protocols have to be used in order to give reliable results.
  • the present methods of analysis are not only laborious, but also time consuming.
  • a current procedure for DNA or RNA analysis takes several days due to, amongst other thing, the requirement of various systems for the taking of samples, the culturing of samples, the isolation of DNA or RNA from the sample, the subsequent assay for the analysis of the presence, absence, or amount of the target sequences in the sample, the processing of any results obtained and the corresponding presentation of the results.
  • One embodiment of the present invention is a method of detecting one or more micro-organisms and/or one or more antibiotic resistance markers in a sample, comprising identifying the presence of distinct nucleic acid regions.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region of a micro-organism is in the 23S RNA gene.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region is identified using nucleic acid amplification.
  • Another embodiment of the present invention is a method as described above, wherein multiplex PCR is used to detect two or more distinct nucleic acid regions.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region is identified using hybridisation.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterobacter cloacae, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 3 and 4 or SEQ ID NOs: 5 and 6.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterobacter cloacae, comprising the use of a hybridisation probe corresponding to a sequence represented by any of SEQ ID NOs: 3 to 6.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 1 or 2, and a microorganism is Enterobacter cloacae.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterococcus faecalis, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, or SEQ ID NOs: 15 and 11.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterococcus faecalis, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 9 to 15.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 7 or 8, and a microorganism is Enterococcus faecalis.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterococcus faecium, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 18 and 19, SEQ ID NOs: 19 and 20, or SEQ ID NOs: 20 and 21.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterococcus faecium, comprising the use of a probe corresponding to the sequences represented by SEQ ID NOs: 19 to 21.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 16 or 17, and a microorganism is Enterococcus faecium.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Escherichia coli, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 24 and 25, SEQ ID NOs: 24 and 26, SEQ ID NOs: 27 and 29, or SEQ ID NOs: 28 and 29.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Escherichia coli, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 24 to 29.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 22 or 23, and a microorganism is Escherichia coli.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Klebsiella pneumoniae, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36 or SEQ ID NOs: 37 and 33.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Klebsiella pneumoniae, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 32 to 37.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 30 or 31, and a microorganism is Klebsiella pneumoniae.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Pseudomonas aeruginosa, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 40 and 41 or SEQ ID NOs: 40 and 42.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Pseudomonas aeruginosa, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 40 to 42.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 38 or 39, and a micro-organism is Pseudomonas aeruginosa.
  • Another embodiment of the present invention is a method as described above, wherein a micro-organism is Staphylococcus aureus, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, SEQ ID NOs: 50 and 51.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Staphylococcus aureus, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 45 to 51.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 43 or 44, and a micro-organism is Staphylococcus aureus.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Staphylococcus epidermidis, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, or SEQ ID NOs: 63 and 61.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Staphylococcus epidermidis, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 54 to 63.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 52 or 53, and a microorganism is Staphylococcus epidermidis.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Candida albicans, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, or SEQ ID NOs: 70 and 71.
  • Another embodiment of the present invention is a method as described above, wherein said micro-organism is Candida albicans, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 66 to 71.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 64 or 65, and a micro-organism is Candida albicans.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is bla ges -2, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 74 and 75 or SEQ ID NOs: 76 and 77.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is bla ges .2, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 74 to 77.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 72 or 73, and an antibiotic resistance marker is bla ges -2.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is bla s h v , comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 80 and 81 or SEQ ID NOs: 82 and 83.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is bla s h v , comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 80 to 83.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 78 or 79, and an antibiotic resistance marker is bla s/ , v .
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is mecA, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 86 and 87 or SEQ ID NOs: 88 and 89.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is mecA, comprising the use of a probe corresponding to the sequences represented by SEQ ID NOs: 86 or 89.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 84 or 85, and an antibiotic resistance marker is mecA.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is spA, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 92 and 93 or SEQ ID NOs: 94 and 95.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is spA, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 92 to 95.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 90 or 91, and an antibiotic resistance marker is Spa.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanA, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 98 and 99 or SEQ ID NOs: 100 and 101.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanA, comprising the use of a probe corresponding to the sequences represented by SEQ ID NOs: 98 to 101.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 96 or 97, and an antibiotic resistance marker is VanA.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanB, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 104 and 105 or SEQ ID NOs: 106 and 107.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanB, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 104 to 107.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 102 or 103, and an antibiotic resistance marker is VanB.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanC, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 110 and 111 or SEQ ID NOs: 112 and 113.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanC, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 110 to 113.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 108 or 109, and an antibiotic resistance marker is VanC.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is MDR-I, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 116 and 117 or SEQ ID NOs: 118 and 119.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is MDR-I, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 116 to 119.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 114 or 115, and an antibiotic resistance marker is MDR-I.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is CDR- 1, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 122 and 123 or SEQ ID NOs: 124 and 125.
  • Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is CDR- 1, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 122 to 125.
  • Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 120 or 121, and an antibiotic resistance marker is CDR-I.
  • Another embodiment of the present invention is a container preloaded with one or more pairs of amplification primers, selected from the sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6,SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, SEQ ID NOs: 15 and 11,SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, SEQ ID NOs: 28 and 29, SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 33, SEQ ID NOs: 40 and 41, SEQ ID NOs: 40 and 42, SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and
  • Another embodiment of the present invention is a container preloaded with one or more probes, selected from the sequences represented by any of SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
  • SEQ ID NOs: 3 to 6 SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID
  • kits comprising one or more pairs of amplification primers, selected from the sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6,SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, SEQ ID NOs: 15 and 11,SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, SEQ ID NOs: 28 and 29, SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 33, SEQ ID NOs: 40 and 41, SEQ ID NOs: 40 and 42, SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49,
  • kits comprising one or more probes selected from the sequences represented by SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
  • Another embodiment of the present invention is a kit comprising one or more containers as described above.
  • Another embodiment of the present invention is a device comprising one or more pairs of amplification primers selected from the sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6,SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, SEQ ID NOs: 15 and 11,SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, SEQ ID NOs: 28 and 29, SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 33, SEQ ID NOs: 40 and 41, SEQ ID NOs: 40 and 42, SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49,
  • Another embodiment of the present invention is a device comprising one or more probes, selected from the sequences represented by SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
  • Another embodiment of the present invention is a use of a container, kit or device as described above for detecting one or more micro-organisms and/or one or more antibiotic resistance markers in a sample.
  • compositions comprising a probe selected from the sequences represented by: SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
  • a probe selected from the sequences represented by: SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs
  • Another embodiment of the present invention is a composition comprising two or more probes selected from the sequences represented by: SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125..
  • compositions comprising a pair of amplification primers selected from the sequences represented by: SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, SEQ ID NOs: 51 and 52, SEQ ID NOs: 55 and 56, and SEQ ID NOs: 59 and 60.
  • a pair of amplification primers selected from the sequences represented by: SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs:
  • compositions comprising two or more pairs of amplification primers selected from the sequences represented by: SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, SEQ ID NOs: 51 and 52, SEQ ID NOs: 55 and 56, and SEQ ID NOs: 59 and 60.
  • Another embodiment of the present invention is a sequence of 23S RNA gene selected from the sequences represented by SEQ ID NOs: 131 to 157.
  • Another embodiment of the present invention is a sequence of antibiotic resistance marker selected from the sequences represented by SEQ ID NOs: 158 to 261.
  • Another embodiment of the present invention is a method as described above, wherein said sequence(s) represented by said SEQ ID NO(s) is (are) the complement(s) of said SEQ ID NO(s).
  • Another embodiment of the present invention is a method as described above, wherein said sequence(s) represented by said SEQ ID NO(s) is (are) an homologous sequence(s) of said SEQ ID NO (s).
  • Another embodiment of the present invention is a container, kit, device or use as described above wherein said sequence(s) represented by said SEQ ID NO(s) is (are) the complement(s) of said SEQ ID NO(s).
  • Another embodiment of the present invention is a container, kit, device or use as described above wherein said sequence(s) represented by said SEQ ID NO(s) is (are) an homologous sequence(s) of said SEQ ID NO(s).
  • Another embodiment of the present invention is a composition as described above wherein said sequence(s) represented by said SEQ ID NO(s) is (are) the complement(s) of said SEQ ID NO(s).
  • Another embodiment of the present invention is a composition as described above, wherein a sequence represented by a SEQ ID NO is an homologous sequence of said SEQ ID NO.
  • Another embodiment of the present invention is a sequence of 23S RNA gene as described above, wherein said sequence represented by said SEQ ID NO is the complement(s) of said SEQ ID NO.
  • Another embodiment of the present invention is a sequence of 23S RNA gene as described above, wherein said sequence represented by said SEQ ID NO is an homologous sequence of said SEQ ID NO.
  • Another embodiment of the present invention is a sequence of an antibiotic resistance marker as described above, wherein said sequence(s) represented by said SEQ ID NO(s) is(are) the complement(s) of said SEQ ID NO(s).
  • Another embodiment of the present invention is a sequence of an antibiotic resistance marker as described above, wherein said sequence(s) represented by said SEQ ID NO(s) is(are) an homologous sequence(s) of said SEQ ID NO(s).
  • Figure 1 Sequences and alignments of 23S RNA sequences of microorganisms.
  • Figure 2 Sequences and alignments of antibiotic resistance genes.
  • the present invention relates to sequences of 23S RNA genes of micro- organisms, and to antibiotic resistance genes and their use as templates for hybridisation and/or nucleic acid amplification reactions, and/or other identification methods in order to detect the presence of one or more micro-organisms and/or antibiotic resistance genes in a sample.
  • the invention further relates to nucleic acid amplification primers and hybridisation probes suitable for amplification of and hybridisation to said sequences.
  • the sample may be any sample of interest. It may be derived from animals
  • sample e.g. human, agricultural livestock, domestic livestock, scientific livestock, zoological livestock
  • non-animal e.g. solid and liquid consumables, water systems, sewerage systems, soil, heating / cooling systems
  • sample may be for example, blood, saliva, urine, faeces, any bodily fluid or tissue.
  • the sample is any that warrants investigation, and is capable of providing template nucleic acid.
  • species of microorganisms useful according to the invention are one or more of Staphylococcus aureus, Staphylococcus epidermidis, Enterobacter cloacae, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Enterococcus faecium, Klebsiella pneumoniae, and Candida albicans.
  • antibiotic resistance markers useful according to the invention are one or more of mecA (methicillin resistance gene, confers resistance to B-lactams), vanA (vancomycin resistance gene A), vanB (vancomycin resistance gene B), vanC (vancomycin resistance gene C), bla s h v (beta- lactam resistance gene), bla ges - 2 (beta-lactam resistance gene), spA (Staphylococcus-aureus protein A), MDR-I (multi-drug resistance gene-1 in Fungi), and CDR-I (multi-drug resistance gene-2 in Fungi).
  • mecA methicillin resistance gene, confers resistance to B-lactams
  • vanA vancomycin resistance gene A
  • vanB vancomycin resistance gene B
  • vanC vancomycin resistance gene C
  • bla s h v beta- lactam resistance gene
  • bla ges - 2 beta-lactam resistance gene
  • spA Staphylococc
  • nucleic acid sequences of 23S RNA or antibiotic resistance markers serve as templates for sequence- specific nucleic acid detection methods.
  • a detection method involves detecting the presence of one or more distinct regions i.e. a region of 23S RNA gene (including 23S RNA per se), or a region of an antibiotic marker sufficiently distinct to enable identification of a species or an antibiotic resistance marker by detecting the presence of the region. Detection may be by amplification of at least a portion the distinct region. Alternatively, or in addition, detection may be by the use of an anti-nucleic acid antibody or sequencing. Alternatively, or in addition, detection may be by hybridisation using a probe. Detection may occur when total nucleic acid from other species or taxonomical groups is present in the sample.
  • the distinct regions may, for example,
  • sequence portions which are unconserved between species - comprise sequence portions which are unconserved between antibiotic resistance markers
  • a distinct region includes homologous sequences in which one or more bases have been deleted, substituted and/or inserted.
  • the number of substitutions, deletions and/or insertions permitted in an homologous sequence may be less than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 residues.
  • the number is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of residues.
  • An homologous sequence still permits identification of the distinct region based on some or all of the unchanged residues.
  • a distinct region also includes the complement sequence of the distinct region.
  • primers pairs are of such sequence and length to provide amplification products (amplicons) only when one or more distinct regions of the species or resistance genes are present.
  • the primers may provide a product of particular length or pattern for the species of interest, distinguishable from amplification products arising from the amplification of other sequences.
  • the amplification primers may provide a relative quantity of product, enabling identification of the species (e.g. one or more strong bands on a electrophoretic gel). Any method of matching the result of an amplification to the presence of the nucleic acid of interest is within the scope of the invention.
  • nucleic acid amplification methods such as the PCR process are well known in the art (see U.S. Pat. Nos. 4,683,195 and 4,683,202 which are incorporated herein by reference) and although a variety of commercial vendors, such as Roche, Invitrogen, Qiagen, Promega sell PCR reagents and publish PCR protocols, some general PCR information is provided below for purposes of clarity.
  • the target nucleic acid in the sample is denatured
  • Denaturation is typically achieved by heating the samples up to about 95 0 C.
  • the next step in PCR involves hybridising the separated strands with primers that flank the target region or subsequence by lowering the temperature of the sample below the melting temperature T M .
  • the primers are then extended to form complementary copies of the target strands by increasing the sample temperature up to the temperature for optimum extension (e.g. 70 to 75 deg C), and the cycle of denaturation, hybridisation, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.
  • Template-dependent extension of primers in PCR is catalysed by a polymerising agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of the appropriate salts, metal cations, and pH buffering system.
  • Suitable polymerising agents are enzymes known to catalyse template-dependent DNA synthesis. For example, if the template is RNA, a suitable polymerising agent to convert the RNA into a complementary DNA
  • cDNA sequence is reverse transcriptase (RT), such as arian myeloblastosis virus RT.
  • suitable polymerases include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, and Taq polymerase, a heat stable DNA polymerase isolated from Thermus aquaticus. The latter enzyme, Taq DNA polymerase, is widely used in the amplification and sequencing of nucleic acids.
  • the reaction conditions for using DNA polymerases are known in the art, and are described in, for example, the treatise Methods in Enzymology, and in Maniatis et al., Molecular Cloning: A Laboratory Manual.
  • the temperature is carefully controlled so that strand separation and primer annealing and extension occur in equilibrium. The control of temperature is typically achieved using dry heat generated from a thermocycler.
  • the reaction is catalysed by a thermostable DNA polymerase enzyme, such as Taq DNA polymerase, and carried out at an elevated temperature.
  • a thermostable DNA polymerase enzyme such as Taq DNA polymerase
  • the preferred temperature is one at which the enzyme is thermostable, and at which the nucleic acids are in an equilibrium of single and double strands, so that sufficient primer will anneal to template strands to allow a reasonable rate of polymerisation Strand separation is achieved by heating the reaction to a sufficiently high temperature for sufficient time to cause the denaturation of the duplex, but not to cause an irreversible denaturation of the polymerase.
  • the PCR method can be performed in a step-wise fashion, where after each step new reagents are added, or in a fashion where all of the reagents are added after a given number of steps. For example, if strand separation is induced by heat, and the polymerase is heat- sensitive, then the polymerase will have to be added after every round of strand separation. However, if, for example, a helicase is used for denaturation, or if a thermostable polymerase is used for extension, then all of the reagents may be added initially, or, alternatively, if molar ratios of reagents are of consequence to the reaction, the reagents may be replenished periodically as they are depleted by the synthetic reaction.
  • Methods for detecting the presence, size and/or quantity of the PCR product include the use of electrophoresis, chromatography, capillary-zone electrophoresis, analytical centrifugation etc.
  • Such method may be in combination with the use of labels (e.g. fluorescent, chemiluminescence, radioisotope, enzyme-labels (such as horse radish peroxidases or alkaline phosphatase), dye, antibody, etc).
  • labels e.g. fluorescent, chemiluminescence, radioisotope, enzyme-labels (such as horse radish peroxidases or alkaline phosphatase), dye, antibody, etc).
  • Detection may also be achieved by the use of hybridisation probes (mentioned below) either in solution or immobilised on a solid support or antibodies directed against the DNA to be detected.
  • a container may be single sample or multiple-sample.
  • Single sample containers include, tubes, vials, Eppendorf tubes etc., as known to the skilled person.
  • Multi- sample containers include, but are not limited to multi-well plates, solid phase slides, solid phase membrane (e.g. nylon or nitrocellulose), microspheres, glass slides, microarrays, chips etc.
  • Single sample containers may use the aforementioned substrates in a single sample mode.
  • the multiple- sample containers permit, for example, several or a large number of PCRs to proceed simultaneously using a single thermal cycling block.
  • the containers may be compatible with high-throughput screening or microarray devices.
  • a container may be provided which already comprises one or more pairs of primers.
  • Such preloaded containers may comprise combinations of primers for detection of specified micro-organisms and/or resistance genes.
  • a preloaded container may comprise a combination of primers suited for detection of a limited combination of distinct regions which are of interest to the operator (e.g. for the detection of just E. coli and vanA or vanB or vanC). Several variations for the detection of particular combinations may be made available.
  • Such preloaded containers may be available as part of a kit, or available separately.
  • two or more pairs of amplification primers are used to detect simultaneously the presence of two or more different species, or two or more different antibiotic resistance markers, or at least one species and at least one antibiotic resistance marker.
  • the amplification products obtained thereby are sufficiently different in property to enable identification of the presence of said species and/or antibiotic resistance marker.
  • the simultaneous amplifications may be performed under the same temperature cycling conditions, but in different wells or spaces (e.g. on a microarray having separate wells for different primer pairs).
  • the buffers may be the same for the different pairs of amplification primers.
  • the different primer pairs are designed of certain sequences and length, to function under identical conditions of temperature and optionally buffer.
  • the amplification primers occupy the same well or space, i.e. all the primers are present in the same reaction (multiplexed).
  • the multiplex mode involves the simultaneous amplification of different target regions using more than one set of amplification primer pairs. Therefore, conditions such as temperature and optionally buffers are identical for pairs of multiplex PCR primers.
  • the primers are thus further designed to preclude cross-reaction between primer pairs, to have similar thermal melting points, and operate in identical buffer conditions.
  • all pairs for multiplexed PCR have Tms (hybdridisation melting temperatures) within 8 deg C of each other and that the average Tm is between 45 deg C and about 70 deg C. with preference for an average Tm of between 60 deg C and 66 deg C.
  • the amplification primers may be completely complementary to the target i.e. there are no mismatches. It is also within the scope of the invention that a primer does not fully complement the target, but still allows amplification thereof.
  • the primers may bind where there are one or more mismatches, deletions and/or insertions in the target template. The number of mismatches, deletions and/or insertions permitted in the target template may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues within the native complementary region, and which still allow amplification of the target region.
  • the number may be less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of template residues within the native complementary region.
  • Such values depend upon the length and composition of the primers as known by the skilled person.
  • the mismatches, deletions and/or insertions are restricted to sequences from the middle of the complementary region towards the 3' end of a template.
  • the primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted.
  • the number of substitutions, deletions and/or insertions permitted in a primer may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues within the native complementary region.
  • the number is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of primer residues within the ends of native complementary region.
  • Such values depend upon the length and composition of the probes as known by the skilled person.
  • the mismatches, deletions and/or insertions are restricted to sequences from the middle of the complementary region towards the 5' end of a primer.
  • an amplification primer may be chemically modified, for example, with modified bases or backbones ⁇ e.g. phosphorothiates, alkylphosphorothiates, peptide nucleic acids, or may contain intercalating agents). Variations or modifications introduced may necessitate adaptations with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However, the eventual results of the amplification reaction will be essentially the same.
  • an amplification primer may be extended in the 5' direction with one or more additional bases, modified bases, or chemical groups (e.g. tags). Such modifications are known to the skilled person and do not normally affect the amplification.
  • identification comprises double amplification i.e. amplification of a region which encloses the distinct region, followed by the amplification of the distinct region e.g. by nested PCR.
  • the product from the first reaction may be applied as a template in the second reaction.
  • the first reaction may be allowed to proceed for a limited number of cycles, before primers pertinent to the second reaction are added to the same reaction container.
  • primers pertinent to the second reaction are added to the same reaction container.
  • the probe may be of such sequence and length that hybridisation is indicated only when nucleic acid of a distinct region is present in the reaction. Methods and protocols to achieve selective binding of hybridisation probes are known to the skilled person. Alternatively, or in addition, the probe may provide a particular relative strength of signal to enable identification of the species against background or other hybridisation. Any method of matching the result of a hybridisation reaction to the presence of the nucleic acid of interest is within the scope of the invention. The methods and conditions for performing a hybridisation reaction are known in the art, and can be found, for example, in Molecular Cloning: A Laboratory Manual (Third Edition) (Joseph Sambrook, Peter MacCallum, David Russell, Cold Spring Habor Laboratory Press).
  • the stability of the [probe: target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished, for example, by avoiding long AT-rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and %GC result in a Tm about 2 to 10 deg C higher than the temperature at which the final assay is be performed.
  • the base composition of the probe is significant because G-C base pairs exhibit greater thermal stability compared with A-T base pairs due to additional hydrogen bonding. Thus, hybridisation involving complementary nucleic acids of higher C-C content will be more stable at higher temperatures.
  • Conditions such as ionic strength and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that the degree of hybridisation will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridisation. Destabilisation of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridisation for probes of about 10 to 50 bases in length occurs approximately 5 deg C below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridise and can therefore result in reduced specificity.
  • the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid.
  • the degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid.
  • Hybridisation is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand: It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridisation may be greatly increased. Computer programs are available to search for this type of interaction. However, in certain instances, it may not be possible to avoid this type of interaction.
  • Methods for detecting the presence of sequences may include, but are not limited to Southern blot, Northern blot, affinity chromatography and solid-phase assays. Methods may include the use of fluorescent markers, radioisotope markers, enzyme linked markers, dyes, antibodies, enzymes linked to the probe as understood by the person skilled in the art.
  • a container may be single sample or multiple- sample.
  • Single sample containers include, tubes, vials, Eppendorf tubes etc., as known to the skilled person.
  • Multi- sample containers include, but are not limited to multi-well plates, solid-phase support, solid phase slides, solid phase membrane (e.g. nylon or nitrocellulose), porous structures, microspheres, glass slides, microarrays, chips etc.
  • Single sample containers may use the aforementioned substrates in a single sample mode. Sample may be applied, for example, using an multiple applicator, soft lithography or microcontact printing, inkjet technology, etc.
  • One or more probes or samples may be immobilised onto a container (e.g. onto a solid phase support).
  • the multiple- sample solid supports permit, for example, several or a large number of hybridisations to proceed simultaneously using a single hybridisation oven or platform and/or single set of reagents.
  • the solid support may be compatible with high-throughput screening or microarray devices.
  • a container may be provided which already comprises one or more probes.
  • Such preloaded containers may comprise combinations of probes for detection of specified micro-organisms and/or resistance genes.
  • a preloaded container may comprise a combination of probes suited for detection of a limited combination of distinct regions which are of interest to the operator (e.g. for the detection of just E. coli and vanA or vanB or vanC). Several variations for the detection of particular combinations may be made available.
  • Such preloaded containers may be available as part of a kit, or separately.
  • two or more hybridisation probes are used to detect simultaneously the presence of two or more different species, or two or more different antibiotic resistance markers, or at least one species and at least one antibiotic resistance marker.
  • the hybridisation products obtained thereby are sufficiently different in property to enable identification of the presence of said species and/or antibiotic resistance marker.
  • the simultaneous hybridisation may be performed under the same temperature conditions, but in different wells or spaces (e.g. on a microarray having separate wells for different primer pairs).
  • the buffers may be the same for the different probes.
  • the different probes are thus designed of certain sequences and length, to function under identical conditions of temperature and optionally buffer.
  • the hybridisation probes occupy the same well or space, i.e. all the probes are present in the same reaction. Therefore, conditions such as temperature and optionally buffers are identical for the probes.
  • the probes are thus further designed to preclude cross-reaction, and to provide a result allowing the presence of two or more species, DNA resistance gene or both to be reliably identified.
  • Such simultaneous hybridisation enables detection of a range of bacterial species and/or antibiotic resistance markers on a single microarray, or in a single reaction, with the likelihood of false results from cross-binding minimized.
  • the hybridisation probes may be completely complementary to the target i.e. there are no mismatches. It is also within the scope of the invention that the probes do not fully complement the target, but still allow identification and discrimination thereof.
  • the probe may bind when there are one or mismatches, deletions and/or insertions in the target template.
  • the number of mismatches, deletions and/or insertions permitted in the target template may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues within the ends of the native complementary region.
  • the number is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of template residues within the ends of the native complementary region. Such values depend upon the length and composition of the probes as known by the skilled person.
  • a sequence of a probe includes homologous sequences in which one or more bases have been deleted, substituted and/or inserted.
  • the number of substitutions, deletions and/or insertions permitted in a probe may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues within the ends of the native complementary region.
  • the number is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of primer residues within the ends of native complementary region.
  • Such values depend upon the length and composition of the probes as known by the skilled person.
  • a probe may be chemically modified, for example, with modified bases or backbones ⁇ e.g. phosphorothiates, alkylphosphorothiates, peptide nucleic acids, or may contain intercalating agents). Variations or modifications introduced may necessitate adaptations with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However, the eventual results of the hybridisation will be essentially the same. The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridisation kinetics, reversibility of hybridisation, biological stability of oligonucleotide molecules etc.
  • a hybridisation probe according to the present invention is capable of annealing to a sequence of any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases of the distinct region, or the complement thereof.
  • the template of hybridisation is an amplification product. That is to say nucleic acid enclosing a distinct region is first amplified and the hybridisation proceeds using the product of the amplification.
  • a distinct region of Enterobacter cloacae 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 1 or 2) indicated in Tables 1 and 2.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Table 1 Sequence of distinct region of 23S RNA gene of Enterobacter cloacae.
  • An example of a substitution according to the invention is T1502C (underlined).
  • W is a nucleotide with an adenine or thymine base.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO:1.
  • a pair of amplification primers is capable of amplifying any region between residues 1251 and 2050 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1279 and 1998 (Table 2, SEQ ID NO: 2).
  • a pair of amplification primers is a pair capable of amplifying the region between residues 1279 and 1998 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1279 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1998 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting Enterobacter cloacae comprise the sequences in Table 3.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NO: 3 (F) and SEQ ID NO: 4 (R); SEQ ID NO: 5 (F) and SEQ ID NO: 6 (R) as indicated in Table 3, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Amplification primer examples for amplifying distinct region of 23S RNA gene of Enterobacter cloacae, length and melting temperature TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 1, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 1279 and 1998 inclusive (SEQ ID NO: 2), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1279 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1998 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting Enterobacter cloacae comprise the sequences represented by SEQ ID NOs: 3 to 6, and the complements thereof.
  • Another aspect of the invention is a method for identifying Enterobacter cloacae by amplification of nucleic acid using primer pairs of Table 3, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 3 to 6.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 3.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • a distinct region of Enterococcus faecalis 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 7 or 8) indicated in Tables 4 and 5.
  • a distinct region is an homologous sequence of said SEQ ID NOs.
  • Table 4 Sequence of distinct region of 23S RNA gene of Enterococcus faecalis. Examples of substitutions according to the invention are Tl 320Y and/or Y1337C (underlined), where Y is a nucleotide with a pyrimidine base.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 7.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1259 and 1978 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1268 and 1940 (Table 5, SEQ ID NO: 8).
  • Table 5 Sequence of distinct region of 23S RNA gene of Enterococcus faecalis. Examples of substitutions according to the invention are Tl 320Y and/or Y1337C (underlined).
  • a pair of amplification primers is a pair capable of amplifying the region between residues 1268 and 1940 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1268 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1940 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting Enterococcus faecalis comprise the sequences in Table 6.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 9 (F) and 11 (R); SEQ ID NOs: 9 (F) and 12 (R); SEQ ID NOs: 13 (F) and 14 (R); SEQ ID NOs: 15 (F) and 12 (R); SEQ ID NOs: 15 (F) and 11 (R) as indicated in Table 6, though other primer pair combinations are possible given the similarity in melting temperatures. Such combination may be present in a composition.
  • Table 6 Amplification primer examples for amplifying distinct region of 23S RNA gene of Enterococcus faecalis, length and melting temperature.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • hybridisation probe is capable of annealing to SEQ ID NO:7, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 1279 and 1998 inclusive (SEQ ID NO: 8), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1279 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1998 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting Enterococcus faecalis comprise the sequences represented by SEQ ID NOs: 9 to 15, and the complements thereof.
  • Another aspect of the invention is a method for identifying Enterococcus faecalis by amplification of nucleic acid using primers pairs of Table 6, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 9 to 15.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 6.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • a distinct region of Enterococcus faecium 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 16 or 17) indicated in Tables 7 and 8.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 16.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1381 and 1560 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1392 and 1547 (Table 8, SEQ ID NO: 17).
  • Table 8 Sequence of distinct region of 23S RNA gene of Enterococcus faecium
  • a pair of amplification primers is a pair capable of amplifying the region between residues 1392 and 1547 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1392 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1547 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting Enterococcus faecium comprise the sequences in Table 9.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 18 and 19; SEQ ID NOs: 19 and 20; SEQ ID NOs: 20 and 21 as indicated in Table 9, though other primer pair combinations are possible given the similarity in melting temperatures.
  • RNA gene of Enterococcus faecium length and melting temperature.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • hybridisation probe is capable of annealing to SEQ ID NO: 16, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 1392 and 1547 inclusive (SEQ ID NO: 17), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1392 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1547 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting Enterococcus faecium comprise the sequences represented by any of SEQ ID NOs: 18 to 21, and the complements thereof.
  • Another aspect of the invention is a method for identifying Enterococcus faecium by amplification of nucleic acid using primers pairs of Table 9, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 18 to 21.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 9.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • a distinct region of Escherichia coli 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 22 or 23) indicated in Tables 10 and 11.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Table 10 Sequence of distinct region of 23S RNA gene of Escherichia coli.
  • deletion of G1294 (underlined).
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 22.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1201 and 1740 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1265 and 1667 (Table 11, SEQ ID NO: 23).
  • a pair of amplification primers according to another embodiment of the invention is a pair capable of amplifying the region between residues 1265 and 1667 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1265 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1667 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting Escherichia coli comprise the sequences in Table 12.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 24 (F) and 25 (R); SEQ ID NOs: 24 (F) and 26 (R); SEQ ID NOs: 27 (F) and 29 (R); SEQ ID NOs: 28 (F) and 29 (R) as indicated in Table 12, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 12 Amplification primer examples for amplifying distinct region of
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:22, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 1265 and 1667 inclusive (SEQ ID NO: 23), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1265 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1667 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting Escherichia coli comprise the sequences represented by SEQ ID NOs: 24 to 29, and the complements thereof.
  • Another aspect of the invention is a method for identifying Escherichia coli by amplification of nucleic acid using primers pairs of Table 12, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 24 to 29.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 12.
  • Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • a distinct region of Klebsiella pneumoniae 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 30 or 31) indicated in Tables 13 and 14.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Table 13 Sequence of distinct region of 23S RNA gene of Klebsiella pneumoniae, wherein R (underlined) is G or A and Y (underlined) is C or T.
  • R (underlined) is G or A
  • Y (underlined) is C or T.
  • Example of a substitution according to the invention is C1354T (underlined).
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 30.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1251 and 1600 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1281and 1560 (Table 14, SEQ ID NO: 31).
  • Table 14 Sequence of distinct region of 23S RNA gene of Klebsiella pneumoniae, wherein R (underlined) is any purine (G or A) and Y (underlined) is any pyrimidine (C or T).
  • R (underlined) is any purine (G or A)
  • Y (underlined) is any pyrimidine (C or T).
  • Example of a substitution according to the invention is C1354T (underlined).
  • a pair of amplification primers is a pair capable of amplifying the region between residues 1281 and 1560 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1281 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1560 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting Klebsiella pneumoniae comprise the sequences in Table 15.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 32 (F) and 34 (R); SEQ ID NOs: 32 (F) and 33 (R); SEQ ID NOs: 35 (F) and 36 (R); SEQ ID NOs: 37 (F) and 33 (R) as indicated in Table 12, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 15 Amplification primer examples for amplifying distinct region of 23S RNA gene of Klebsiella pneumoniae, length and temperature.
  • Note R is any purine (G or A)
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:30, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 1281 and 1560 inclusive (SEQ ID NO: 31), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1281 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1560 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting Klebsiella pneumoniae comprise the sequences represented by any of SEQ ID NOs: 32 to 37, and the complements thereof.
  • Another aspect of the invention is a method for identifying Klebsiella pneumoniae by amplification of nucleic acid using primers pairs of Table 15, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 32 to 37.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 15.
  • Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • a distinct region of Pseudomonas aeruginosa 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 38 or 39) indicated in Tables 16 and 17.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Table 16 Sequence of distinct region of 23S RNA gene of Pseudomonas aeruginosa.
  • Example of a substitution according to the invention is T374C (underlined).
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 38.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 51 and 750 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 104 and 704 (Table 17, SEQ ID NO: 39).
  • Table 17 Sequence of distinct region of 23S RNA gene of Pseudomonas aeruginosa.
  • Example of a substitution according to the invention is T374C (underlined).
  • a pair of amplification primers is a pair capable of amplifying the region between residues 104 and 704 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 104 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 704 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting Pseudomonas aeruginosa comprise the sequences in Table 18.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 40 (F) and 41 (R); SEQ ID NOs: 40 (F) and 42 (R) as indicated in Table 18, though other combinations are possible given the similarity in melting temperatures. Such combination may be present in a composition.
  • Table 18 Amplification primer examples for amplifying distinct region of 23S RNA gene of Pseudomonas aeruginosa, length and melting temperature.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • hybridisation probe is capable of annealing to SEQ ID NO:38, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 104 and 704 inclusive (SEQ ID NO: 39), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 104 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 704 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting Pseudomonas aeruginosa comprise the sequences represented by any of SEQ ID NOs: 40 to 42, and the complements thereof.
  • Another aspect of the invention is a method for identifying Pseudomonas aeruginosa by amplification of nucleic acid using primers pairs of Table 18, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a sequence corresponding to any of SEQ ID NOs: 40 to 42.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 18.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 7.
  • a distinct region of Staphylococcus aureus 23S RNA gene comprises a nucleotide sequence (SEQ ID NO: 43 or 44) indicated in Tables 19 and 20.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Table 19 Sequence of distinct region of 23S RNA gene of Staphylococcus aureus
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 43.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1021 and 1320 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1037 and 1263 (Table 20, SEQ ID NO: 44).
  • Table 20 Sequence of distinct region of 23S RNA gene of Staphylococcus aureus
  • a pair of amplification primers is a pair capable of amplifying the region between residues 1037 and 1263 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1037 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1263 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting Staphylococcus aureus comprise the sequences in Table 21.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 45 (F) and 46 (R); SEQ ID NOs: 48 (F) and 47 (R); SEQ ID NOs: 48 (F) and 49 (R); SEQ ID NOs: 48 (F) and 51 (R); SEQ ID NOs: 50 (F) and 51 (R) as indicated in Table 21, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 21 Amplification primer examples for amplifying distinct region of
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:43, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 1037 and 1263 inclusive (SEQ ID NO: 44), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1037 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1263 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting Staphylococcus aureus comprise the sequences represented by any of SEQ ID NOs: 45 to 51, and the complements thereof.
  • Another aspect of the invention is a method for identifying Staphylococcus aureus by amplification of nucleic acid using primers pairs of Table 21, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 45 to 51.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 21.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • a distinct region of Staphylococcus epidermidis 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 52 or 53) indicated in Tables 22 and 23.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Table 22 Sequence of distinct region of 23S RNA gene of Staphylococcus epidermidis.
  • Example of a substitution according to the invention is T859C (underlined).
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 52
  • a pair of amplification primers is a pair capable of amplifying any region between residues 501 and 1050 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1037 and 1263 (Table 23, SEQ ID NO: 53).
  • Table 23 Sequence of distinct region of 23S RNA gene of Staphylococcus epidermidis.
  • Example of a substitution according to the invention is T859C (underlined).
  • a pair of amplification primers is a pair capable of amplifying the region between residues 1037 and 1263 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1037 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1263 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting Staphylococcus epidermidis comprise the sequences in Table 24.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 54 (F) and 55 (R); SEQ ID NOs: 54 (F) and 56 (R); SEQ ID NOs: 54 (F) and 57 (R); SEQ ID NOs: 58 (F) and 57 (R); SEQ ID NOs: 58 (F) and 59 (R); SEQ ID NOs: 58 (F) and 60 (R); SEQ ID NOs: 58 (F) and 61 (R); SEQ ID NOs: 58 (F) and 62 (R); SEQ ID NOs: 63 (F) and 59 (R); SEQ ID NOs: 63 (F) and 60 (R); SEQ ID NOs: 63 (F) and 61 (R) as indicated in Table 24, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 24 Amplification primer examples for amplifying distinct region of 23S RNA gene of Staphylococcus epidermidis, length and melting temperature.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:52, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 1037 and 1263 inclusive (SEQ ID NO: 53), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1037 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1263 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting Staphylococcus epidermidis comprise the sequences represented by any of SEQ ID NOs: 54 to 63, and the complements thereof.
  • Another aspect of the invention is a method for identifying Staphylococcus epidermidis by amplification of nucleic acid using primers pairs of Table 24, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 54 to 63.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 24.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • a distinct region of Candida albicans 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 64 or 65) indicated in Tables 25 and 26.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Table 25 Sequence of distinct region of 23S RNA gene of Candida albicans.
  • Y is a nucleotide with a pyrimidine base.
  • the nucleotides “XX” may both be absent, may be "TT” or may be a single nucleotide "A”.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 64.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 181 and 778 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 214 and 739 (Table 26, SEQ ID NO: 65).
  • SEQ ID NO: 65 SEQ ID NO: 65
  • Table 26 Sequence of distinct region of 23S RNA gene of Candida albicans Y is a nucleotide with a pyrimidine base.
  • the nucleotides “XX” may both be absent, may be "TT” or may be a single nucleotide "A”.
  • a pair of amplification primers is a pair capable of amplifying the region between residues 214 and 739 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 214 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 739 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting Candida albicans comprise the sequences in Table 27.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 66 and 67; SEQ ID NOs: 68 and 69; SEQ ID NOs: 70 and 71 as indicated in Table 27, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • hybridisation probe is capable of annealing to SEQ ID NO: 64, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 214 and 739 inclusive (SEQ ID NO: 65), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 214 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 739 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting Candida albicans comprise the sequences represented by any of SEQ ID NOs: 66 to 71, and the complements thereof.
  • Another aspect of the invention is a method for identifying Candida albicans by amplification of nucleic acid using primers pairs of Table 27, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 66 to 71.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 27.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • a distinct region of a bla ges -2 gene comprises a nucleotide sequence (SEQ ID NOs: 72 or 73) indicated in Tables 28 and 29.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Table 28 Sequence of distinct region of bla ges . 2 gene.
  • R (underlined) is any purine (G or A). Examples of deletions according to the invention include deleting of any of Rl 12 and/or R313.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 72.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 653 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 140 and 482 (Table 26, SEQ ID NO: 73).
  • a pair of amplification primers is a pair capable of amplifying the region between residues 140 and 482 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 140 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 482 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting the bla ges . 2 gene comprise the sequences in Table 30.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 74 (F) and 75 (R); SEQ ID NOs: 76 (F) and 77 (R) as indicated in Table 30, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 30 Amplification primer examples for amplifying a distinct region of the bla.ges-2 gene, length and melting temperature.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:72, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 140 and 482 inclusive (SEQ ID NO: 73), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between 140 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 482 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting the bla ges . 2 gene comprise the sequences represented by any of SEQ ID NOs: 74 to 77, and the complements thereof.
  • Another aspect of the invention is a method for identifying the bla ges -2 gene by amplification of nucleic acid using primers pairs of Table 30, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 74 to 77.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 30.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • bla shv (beta-lactam resistance gene)
  • a distinct region of a bla shv gene comprises a nucleotide sequence (SEQ ID NO: 78 or 79) indicated in Tables 31 and 32.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 78.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 693 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 149 and 350 (Table 32, SEQ ID NO: 79).
  • Table 32 Sequence of distinct region of the bla s h v gene.
  • Y is any pyrimidine (C or T).
  • Y is any pyrimidine (C or T)
  • R is any purine (A or G)
  • S is a (C or G). Examples of deletions according to the invention includes the deletion of one or more of Y240, R583, R588 and S669.
  • a pair of amplification primers is a pair capable of amplifying the region between residues 149 and 350 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 149 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 350 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting the bla s/ , v gene comprise the sequences in Table 33.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 80 and 81; SEQ ID NOs: 82 and 83 as indicated in Table 33, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 33 Amplification primer examples for amplifying a distinct region of the bla s hv gene, length and melting temperature.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 78, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 149 and 350 inclusive (SEQ ID NO: 79), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 149 ( ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 350 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting the bla s/ , v gene comprise the sequences represented by any of SEQ ID NOs: 80 to 83, and the complements thereof.
  • Another aspect of the invention is a method for identifying the bla s h v gene by amplification of nucleic acid using primers pairs of Table 33, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 80 to 83.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 33.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 12. mecA (methicillin resistance gene)
  • a distinct region of a mecA gene comprises a nucleotide sequence (SEQ ID NOs: 84 or 85) indicated in Tables 34 and 35.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 84.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 611 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 184 and 484 (Table 35, SEQ ID NO: 85).
  • Table 35 Sequence of distinct region of the mecA gene.
  • a pair of amplification primers is a pair capable of amplifying the region between residues 184 and 484 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 184 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 484 (+10, 9, 8, 7, 6, 5, 4, 3,
  • amplification primers suitable for detecting the mecA gene comprise the sequences in Table 36.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 86 (F) and 87 (R); SEQ ID NOs: 88 (F) and 89 (R) as indicated in Table 36, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Amplification primer examples for amplifying a distinct region of the mecA gene, length and melting temperature are either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 84, or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 149 and 349 inclusive (SEQ ID NO: 85), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 184 ( ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 484 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting the mecA gene comprise the sequences represented by any of SEQ ID NOs: 86 to 89, and the complements thereof.
  • Another aspect of the invention is a method for identifying the mecA gene by amplification of nucleic acid using primers pairs of Table 36, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 86 to 89.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 36.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • spA Staphylococcus-aureus protein A
  • a distinct region of a spA gene comprises a nucleotide sequence (SEQ ID NOs: 90 or 91) indicated in Tables 37 and 38.
  • a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 90
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 501 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 292 and 409 (Table 38, SEQ ID NO: 91).
  • Table 38 Sequence of distinct region of the spA gene. Note Y is pyrimidine (C or T), H is (A, C or T). Example of deletions according to the invention include the deletion of H349, Y356, Y359, Y362 and/or Y387.
  • a pair of amplification primers is a pair capable of amplifying the region between residues 292 and 409 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 292 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 409 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting the spA gene comprise the sequences in Table 39.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 92 (F) and 93 (R); SEQ ID NOs: 94 (F) and 95 (R) as indicated in Table 39, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 39 Amplification primer examples for amplifying a distinct region of the spA gene, length and melting temperature. .
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 90 or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 292 and 409 inclusive (SEQ ID NO: 46), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 292 ( ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 409 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting the spA gene comprise the sequences represented by any of SEQ ID NOs: 92 to 95, and the complements thereof.
  • Another aspect of the invention is a method for identifying the spA gene by amplification of nucleic acid using primers pairs of Table 39, in the combinations indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 92 to 95.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 39.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 14.
  • VanA vancomycin resistance gene A
  • a distinct region of a VanA gene comprises a nucleotide sequence (SEQ ID NOs: 96 or 97) indicated in Tables 40 and 41.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • AAAGTTGCAATACTGTTTGGGGGTTGCTCAGAGGAGCATGACGTATCGGT AAAATCTGCAATAGAGATAGCCGCTAACATTAATAAAGAAAAATACGAGC CGTTATACATTGGAATTACGAAATCTGGTGTATGGAAAATGTGCGAAAAA CCTTGCGCGGAATGGGAAAACGACAATTGCTATTCAGCTGTACTCTCGCC GGATAAAAAAATGCACGGATTACTTGTTAAAAAGAACCATGAATATGAAA TCAACCATGTTGATGTAGCATTTTCAGCTTTGCATGGCAAGTCAGGTGAA GATGGATCCATACAAGGTCTGTTTGAATTGTCCGGTATCCCTTTTGTAGG CTGCGATATTCAAAGCTCAGCAATTTGTATGGACAAATCGTTGACATACA TCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCTTTTGGGTTATTAAT AAAGATGATAGGCCGGTGGCAGCTACGTTTACCTATCCTGTTTTTGTTAA GCCGGCGCGTTGGCG
  • Table 40 Sequence of distinct region of VanA gene. Note B is (T, C or G).
  • Example of a deletion according to the invention includes the deletion of B528.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 96
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 944 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 138 and 641 (Table 41, SEQ ID NO: 97).
  • Table 41 Sequence of distinct region of the VanA gene. Note B is (T, C or G).
  • Example of deletion according to the invention includes the deletion of B528.
  • a pair of amplification primers is a pair capable of amplifying the region between residues 138 and 641 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 138 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 641 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting the VanA gene comprise the sequences in Table 42.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 98 (F) and 99 (R); SEQ ID NOs: 100 (F) and 101 (R) as indicated in Table 42, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 42 Amplification primer examples for amplifying a distinct region of the VanA gene, length and melting temperature.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • hybridisation probe is capable of annealing to SEQ ID NO: 96 or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 138 and 641 inclusive (SEQ ID NO: 97), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 138 ( ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 641 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting the VanA gene comprise the sequences represented by any of SEQ ID NOs: 98 to 101, and the complements thereof.
  • Another aspect of the invention is a method for identifying the VanA gene by amplification of nucleic acid using primers pairs of Table 42, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 98 to 101.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 42.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • VanB vancomycin resistance gene B
  • a distinct region of a VanB gene comprises a nucleotide sequence (SEQ ID NOs: 102 or 103) indicated in Tables 43 and 44.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Table 43 Sequence of distinct region of VanB gene. Note B is (T, C or G), and R is purine (G or A). Examples of deletions according to the invention include one of more of B418, R540 and R696.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 102.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 741 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 126 and 574 (Table 44, SEQ ID NO: 103).
  • Table 44 Sequence of distinct region of the VanB gene. Note B is (T, C or G), and R is purine (G or A). Examples of deletions according to the invention include one of more of B418 and R540.
  • a pair of amplification primers is a pair capable of amplifying the region between residues 126 and 574 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 126 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 574 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • amplification primers suitable for detecting the VanB gene comprise the sequences in Table 45.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 104 (F) and 105 (R); SEQ ID NOs: 106 (F) and 107 (R) as indicated in Table 45, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 45 Amplification primer examples for amplifying a distinct region of the VanB gene, length and melting temperature.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 102 or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 126 and 574 inclusive (SEQ ID NO: 103), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 126 ( ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 574 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
  • probes suitable for detecting the VanB gene comprise the sequences represented by SEQ ID NOs: 102 and 103, and the complements thereof.
  • Another aspect of the invention is a method for identifying the VanB gene by amplification of nucleic acid using primers pairs of Table 45, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 104 to 107.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 45.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 16.
  • VanC vancomycin resistance gene C
  • a distinct region of a VanC gene comprises a nucleotide sequence (SEQ ID NOs: 108 or 109) indicated in Tables 46 and 47.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 108
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 438 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 27 and 407 (Table 47, SEQ ID NO: 109).
  • a pair of amplification primers is a pair capable of amplifying the region between residues 27 and 407 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 27 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 407 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive.
  • amplification primers suitable for detecting the VanC gene comprise the sequences in Table 48.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 110 (F) and 111 (R); SEQ ID NOs: 112 (F) and 113 (R) as indicated in Table 48, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Table 48 Amplification primer examples for amplifying a distinct region of the VanC gene, length and melting temperature.
  • TYPE is either forward (F) or reverse (R) primer
  • PAIR is a paired primer SEQ ID NO. for amplification
  • LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 108 or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 27 and 407 inclusive (SEQ ID NO: 109), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 27 ( ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 407 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive.
  • probes suitable for detecting the VanC gene comprise the sequences represented by any of SEQ ID NOs: 110 to 113, and the complements thereof.
  • Another aspect of the invention is a method for identifying the VanC gene by amplification of nucleic acid using primers pairs of Table 48, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NO: 110 to 113.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 48.
  • Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • a distinct region of a MDR-I gene comprises a nucleotide sequence (SEQ ID NOs: 114 or 115) indicated in Tables 49 and 50.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Y is any nucleotide with a pyrimidine base
  • R is any nucleotide with a purine base
  • W is any nucleotide with an adenine or thymine base.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 108.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 201 and 850 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 275 and 834 (Table 50, SEQ ID NO: 115).
  • Table 50 Sequence of distinct region of the MDR-I gene.
  • a pair of amplification primers is a pair capable of amplifying the region between residues 275 and 834 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 275 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 834 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive.
  • amplification primers suitable for detecting the MDR-I gene comprise the sequences in Table 51.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 116 (F) and 117 (R); SEQ ID NOs: 118 (F) and 119 (R) as indicated in Table 51, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Amplification primer examples for amplifying a distinct region of the MDR-I gene, length and melting temperature are either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 114 or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 275 and 834 inclusive (SEQ ID NO: 115), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 275 ( ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 834 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive.
  • probes suitable for detecting the MDR-I gene comprise the sequences represented by any of SEQ ID NOs: 116 to 119, and the complements thereof.
  • Another aspect of the invention is a method for identifying the MDR-I gene by amplification of nucleic acid using primers pairs of Table 51, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NO: 116 to 119.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 51.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 18.
  • CDR l CDR l
  • a distinct region of a CDR l gene comprises a nucleotide sequence (SEQ ID NOs: 120 or 121) indicated in Tables 52 and 53.
  • a distinct region is a complement of said SEQ ID NOs.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • Y is any nucleotide with a pyrimidine base
  • R is any nucleotide with a purine base
  • K is any nucleotide with a thymine or guanine base
  • W is any nucleotide with an adenine or thymine base
  • M is any nucleotide with a cytosine or adenine base
  • S is any nucleotide with a cytosine or guanine base.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 120.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 387 inclusive.
  • a pair of amplification primers is a pair capable of amplifying any region between residues 111 and 178 (Table 53, SEQ ID NO: 121).
  • Table 53 Sequence of distinct region of the CDR-I gene. Sequence of distinct region of CDR-I gene. Y is any nucleotide with a pyrimidine base.
  • a pair of amplification primers is a pair capable of amplifying the region between residues 111 and 178 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 111 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 178 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive.
  • amplification primers suitable for detecting the CDR l gene comprise the sequences in Table 54.
  • Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 122 (F) and 123 (R); SEQ ID NOs: 124 (F) and 125 (R) as indicated in Table 54, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
  • Amplification primer examples for amplifying a distinct region of the CDR-I gene, length and melting temperature are either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
  • a hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 120 or the complement thereof.
  • hybridisation probe is capable of hybridising to the region between residues 111 and 178 inclusive (SEQ ID NO: 121), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 111 ( ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 178 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive.
  • probes suitable for detecting the MDR-I gene comprise the sequences represented by any of SEQ ID NOs: 122 to 125, and the complements thereof.
  • Another aspect of the invention is a method for identifying the CDR-I gene by amplification of nucleic acid using primers pairs of Table 54, in the combination indicated or other suitable combination of forward and reverse primers.
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NO: 122 to 125.
  • Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 54.
  • homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • Figure 1
  • the Figure shows an alignment of sequences 23S RNA genes of each of Staphylococcus aureus, Staphylococcus epidermidis, Enterobacter cloacae, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Enterococcus faecium, Klebsiella pneumoniae, and Candida albicans, and an indication of identity (marked with *).
  • a distinct region of a 23S RNA gene of a micro-organism comprises a nucleotide sequence (SEQ ID NOs: 131 to 157) indicated in Figure 1.
  • a distinct region is a complement of said SEQ ID NOs.
  • One aspect of the present invention is nucleotide acid corresponding to a sequence represented by any of SEQ ID NOs: 131 to 157 indicated in Figure 1, or complement thereof.
  • a distinct region of a 23S RNA gene of a micro-organism comprises a nucleotide sequence indicated in Figure 1, corresponding to a distinct region represented by any of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), 64 and 65 (Candida albicans).
  • a distinct region of a 23S RNA gene of a micro-organism comprises a nucleotide sequence indicated in Figure 1, obtainable using a pair of amplification primers specific to said micro-organism.
  • Said amplification primer are mentioned above, and are, depending on the micro-organism: Enterobacter cloacae: SEQ ID NOs: 3 and 4, or SEQ ID NOs: 5 and 6,
  • Enterococcus faecalis SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12 or SEQ ID NOs: 15 and 11,
  • Enterococcus faecium SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, or SEQ ID NOs: 20 and 21, Escherichia coli: SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs:
  • Klebsiella pneumoniae SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, or SEQ ID NOs: 37 and 33,
  • Pseudomonas aeruginosa SEQ ID NOs: 40 and 41 or SEQ ID NOs: 40 and 42
  • Staphylococcus aureus SEQ ID NOs: 45 and 46
  • SEQ ID NOs: 48 and 47 SEQ ID NOs: 48 and 47
  • SEQ ID NOs: 48 and 49 SEQ ID NOs: 48 and 51, or SEQ ID NOs: 50 and 51,
  • Staphylococcus epidermidis SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, or SEQ ID NOs: 63 and 61,
  • Candida albicans SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, or SEQ ID NOs: 70 and 71.
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of a SEQ ID NO in Figure 1, a complement thereof, or an homologous sequence of said region or complement.
  • a pair of amplification primers is a pair capable of amplifying a region of a sequence listed in Figure 1 corresponding to a distinct region represented by any of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), 64 and 65 (Candida albicans).
  • the primers are capable of amplifying the aforementioned corresponding region in Figure 1, +10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues from either or both ends.
  • a hybridisation probe according to the present invention is capable of hybridising to a region of a sequence listed in Figure 1, corresponding to a distinct region, or complement thereof, represented by any of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), 64 and 65 (Candida albicans). It is within the scope of the invention that the probes are capable of binding to the aforementioned corresponding sequences of Figure 1, +10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues from either or both ends.
  • Another aspect of the invention is a method for identifying a distinct region by amplification of nucleic acid using a pair of primers directed towards a region of a sequence in Figure 1 corresponding to a distinct region, or complement thereof, represented by any of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), 64 and 65 (Candida albicans).
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification.
  • homologous sequences of the above mentioned regions corresponding to distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • the Figure shows an alignment of sequenced genes of each of mecA, vanA, vanB, vanC, bla shv , bla ges - 2 , spA, MDR-I, and CDR-I, together with a consensus sequence.
  • a distinct region of an antibiotic resistance gene of a comprises a nucleotide sequence (SEQ ID NOs: 158 to 261) indicated in Figure 2.
  • a distinct region is a complement of said SEQ ID NOs.
  • One aspect of the present invention is nucleotide acid corresponding to a sequence represented by any of SEQ ID NOs: 158 to 261 indicated in Figure 2, or complement thereof.
  • a distinct region of an antibiotic resistance gene comprises a nucleotide sequence indicated in Figure 2, corresponding to a distinct region represented by any of SEQ ID NOs: 72 or 73 (bla ges - 2 ), 78 or 79 (bla s hv), 84 or 85 (mecA), 90 or 91 (spA), 96 or 97 (vanA), 102 or 103 (vanB), 108 or 109 (vanC), 114 or 115 (MDR-I), 120 or 121 (CDR-I).
  • a distinct region of an antibiotic resistance gene comprises a nucleotide sequence indicated in Figure 2, obtainable using a pair of amplification primers specific to said micro-organism.
  • Said amplification primer are mentioned above, and are, depending on the marker: bla ges - 2 marker: SEQ ID NOs: 74 and 75, or SEQ ID NOs: 76 and 77, bla s hv marker: SEQ ID NOs: 80 and 81, or SEQ ID NOs: 82 and 83, mecA marker: SEQ ID NOs: 86 and 87, or SEQ ID NOs: 88 and 89, spA marker: SEQ ID NOs: 92 and 93, or SEQ ID NOs: 94 and 95, VanA marker: SEQ ID NOs: 98 and 99, or SEQ ID NOs: 100 and 101,
  • a distinct region is an homologous sequence of the distinct region or complement thereof.
  • a pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of a SEQ ID NO in Figure 2, a complement thereof, or an homologous sequence of said region or complement.
  • a pair of amplification primers according to another embodiment of the invention is a pair capable of amplifying a region of a sequence listed in Figure 2 corresponding to a distinct region represented by any of SEQ ID NOs: 72 or 73 (bla ges - 2 ), 78 or 79 (blashv), 84 or 85 (mecA), 90 or 91 (spA), 96 or 97 (vanA), 102 or 103 (v ⁇ nB), 108 or 109 (yanC), 114 or 115 (MDR-I), 120 or 121 (CDR-I).
  • the primers are capable of amplifying the aforemention corresponding region in Figure 2, +10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues from either or both ends.
  • a hybridisation probe according to the present invention is capable of hybridising to a region of a sequence listed in Figure 2, corresponding to a distinct region, or complement thereof, represented by any of SEQ ID NOs: 72 or 73 (bla ges - 2 ), 78 or 79 (bla s h v ), 84 or 85 (mecA), 90 or 91 (spA), 96 or 97 (vanA), 102 or 103 (v ⁇ nB), 108 or 109 (vanC), 114 or 115 (MDR-I), 120 or 121 (CDR-I).
  • the probes are capable of binding to the aforementioned corresponding sequences of Figure 2, +10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues from either or both ends.
  • Another aspect of the invention is a method for identifying a distinct region by amplification of nucleic acid using a pair of primers directed towards a region of a sequence in Figure 2 corresponding to a distinct region, or complement thereof, represented by any of SEQ ID NOs: 72 or 73 (bla ges - 2 ), 78 or 79 (bla shv ), 84 or 85 (mecA), 90 or 91 (spA), 96 or 97 (vanA), 102 or 103 (v ⁇ nB), 108 or 109 (vanC), 114 or 115 (MDR-I), 120 or 121 (CDR-I).
  • a further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification.
  • homologous sequences of the above mentioned regions corresponding to distinct regions, amplification primers and hybridisation probes are within the scope of the invention.
  • the distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
  • the present invention also relates to the simultaneous detection of two or more (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) distinct regions of nucleic acid.
  • One embodiment of the invention is a method for detection of two or more (e.g.
  • Staphylococcus aureus Staphylococcus epidermidis
  • Enterobacter cloacae Escherichia coli
  • Enterococcus faecalis Enterococcus faecalis
  • Pseudomonas aeruginosa Enterococcus faecium
  • Klebsiella pneumoniae Klebsiella pneumoniae and Candida albicans by detecting nucleic acid corresponding to distinct regions of 23S RNA therein.
  • Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting nucleic acid corresponding to two or more ⁇ e.g. at least 3, 4, 5, 6, 7, 8, or 9) of:
  • SEQ ID NOs: 22 or 23 (Escherichia coli), SEQ ID NOs: 30 or 31 (Klebsiella pneumoniae),
  • SEQ ID NOs: 38 or 39 (Pseudomonas aeruginosa)
  • SEQ ID NOs: 64 and 65 Candida albicans.
  • Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) of nucleic acid sequences listed in Figure 1, each sequence corresponding to the micro-organism for detection.
  • Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting at least two (e.g. at least 3, 4, 5, 6, 7, 8, or 9) regions of a nucleic acid listed in Figure 1, each region corresponding a distinct region of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), or 64 and 65 (Candida albicans).
  • SEQ ID NOs: 1 or 2 Enterobacter cloacae
  • 7 or 8 Enterococcus faecalis
  • 16 or 17 Enterococcus
  • Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by using two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) primer pairs, wherein the two or more organisms for detection and the primer pairs are selected from the following, preferably, though not necessarily, one primer pair selected for one micro-organism:
  • Enterobacter cloacae SEQ ID NOs: 3 and 4, or SEQ ID NOs: 5 and 6,
  • Enterococcus faecalis SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12 or SEQ ID NOs: 15 and 11, Enterococcus faecium: SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, or SEQ ID NOs: 20 and 21,
  • Pseudomonas aeruginosa SEQ ID NOs: 40 and 41 or SEQ ID NOs: 40 and 42,
  • Staphylococcus aureus SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, or SEQ ID NOs: 50 and 51, Staphylococcus epidermidis: SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and
  • SEQ ID NOs: 54 and 57 SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, or SEQ ID NOs: 63 and 61,
  • Candida albicans SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, or SEQ ID NOs: 70 and 71.
  • composition comprising two or more ⁇ e.g. at least 3, 4, 5, 6, 7, 8, or 9) of the following primer pairs:
  • Enterobacter cloacae SEQ ID NOs: 3 and 4,
  • Enterococcus faecalis SEQ ID NOs: 9 and 12
  • Enterococcus faecium SEQ ID NOs: 18 and 19
  • Escherichia coli SEQ ID NOs: 24 and 25,
  • Klebsiella pneumoniae SEQ ID NOs: 32 and 33,
  • Pseudomonas aeruginosa SEQ ID NOs: 40 and 42,
  • Staphylococcus aureus SEQ ID NOs: 48 and 49
  • Staphylococcus epidermidis SEQ ID NOs: 54 and 55
  • Candida albicans SEQ ID NOs: 66 and 67.
  • Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by using two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) single probes corresponding to said two or more organisms for detection below, preferably, though not necessarily, one probe selected for one micro-organism:
  • Enterobacter cloacae any of SEQ ID NOs: 3 to 6,
  • Enterococcus faecalis any of SEQ ID NOs: 9 to 15,
  • Enterococcus faecium any of SEQ ID NOs: 18 to 21,
  • Escherichia coli any of SEQ ID NOs: 24 to 29, Klebsiella pneumoniae: any of SEQ ID NOs: 32 to37,
  • Pseudomonas aeruginosa any of SEQ ID NOs: 40 to 42,
  • Staphylococcus aureus any of SEQ ID NOs: 45 to 51,
  • Staphylococcus epidermidis any of SEQ ID NOs: 54 to 63
  • Candida albicans SEQ ID NOs: any of 66 to 71.
  • compositions comprising two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) of the following probes, preferably, though not necessarily, one probe selected for one micro-organism:
  • Enterobacter cloacae SEQ ID NOs: 3 or 4
  • Enterococcus faecalis SEQ ID NOs: 9 or 12
  • Enterococcus faecium SEQ ID NOs: 18 or 19,
  • Klebsiella pneumoniae SEQ ID NOs: 32 or 33,
  • Pseudomonas aeruginosa SEQ ID NOs: 40 or 42
  • Staphylococcus aureus SEQ ID NOs: 48 or 49
  • Staphylococcus epidermidis SEQ ID NOs: 54 or 55,
  • Candida albicans SEQ ID NOs: 66 or 67.
  • Another embodiment of the invention is a method for detecting two or more (e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10) of the antibiotic resistance markers mecA, SpA, vanA, vanB, vanC, bla s h v , bla ges - 2 , MDR-I, CDR-I by detecting nucleic acid corresponding to distinct regions therein.
  • Another embodiment of the invention is a method for identifying at least two antibiotic resistance markers in a sample by detecting nucleic acid corresponding to two or more (e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10) of SEQ ID NOs: 72 or 73 (bla ges - 2 marker),
  • SEQ ID NOs: 120 or 121 Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting two or more ((e.g. at least 3, 4, 5, 6, 7, 8, or 9) of nucleic acid sequences listed in Figure 2, each sequence corresponding to the micro-organism for detection. Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting at least two (e.g.
  • each region corresponding a distinct region of SEQ ID NOs: 72 or 73 (bla ges - 2 marker), SEQ ID NOs: 78 or 79 (bla shv marker), SEQ ID NOs: 84 or 85 (mecA marker), SEQ ID NOs: 90 or 91 (spA marker), SEQ ID NOs: 96 or 97 (VanA marker), SEQ ID NOs: 102 or 103 (VanB marker), SEQ ID NOs: 108 or 109 (VanC marker), SEQ ID NOs: 114 or 115 (MDR-I marker), or SEQ ID NOs: 120 or 121 (CDR-I marker).
  • Another embodiment of the invention is a method for identifying at least two antibiotic resistance markers in a sample by using two or more (e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10) primer pairs wherein the antibiotic resistance markers for detection and the primer pairs are selected from the following preferably, though not necessarily, one primer pair selected for one marker: bla ges - 2 marker: SEQ ID NOs: 74 and 75, or SEQ ID NOs: 76 and 77, bla s hv marker: SEQ ID NOs: 80 and 81, or SEQ ID NOs: 82 and 83, mecA marker: SEQ ID NOs: 86 and 87, or SEQ ID NOs: 88 and 89, spA marker: SEQ ID NOs: 92 and 93, or SEQ ID NOs: 94 and 95,
  • VanA marker SEQ ID NOs: 98 and 99, or SEQ ID NOs: 100 and 101,
  • VanB marker SEQ ID NOs: 104 and 105, or SEQ ID NOs: 106 and 107,
  • VanC marker SEQ ID NOs: 110 and 111, or SEQ ID NOs: 112 and 113, MDR-I marker: SEQ ID NOs: 116 and 117, or SEQ ID NOs: 118 and 119, and
  • CDR l marker SEQ ID NOs: 122 and 123, or SEQ ID NOs: 124 and 125.
  • compositions comprising two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) of the following primer pairs: bla ges - 2 marker: SEQ ID NOs: 76 and 77, bla shv marker: SEQ ID NOs: 80 and 81 , mecA marker: SEQ ID NOs: 88 and 89, spA marker: SEQ ID NOs: 92 and 93,
  • VanA marker SEQ ID NOs: 100 and 101,
  • VanB marker SEQ ID NOs: 106 and 107
  • VanC marker SEQ ID NOs: 112 and 113
  • MDR-I marker SEQ ID NOs: 116 and 117
  • CDR l marker SEQ ID NOs: 124 and 125.
  • Another embodiment of the invention is a method for identifying at least two antibiotic resistance markers in a sample by using two or more (e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10) probes wherein the antibiotic resistance markers for detection and the probes are selected from the following, preferably, though not necessarily, one probe selected for one micro-organism: bla ges - 2 marker: any of SEQ ID NOs: 74 to 77, bla s hv marker: any of SEQ ID NOs: 80 to 83, mecA marker: any of SEQ ID NOs: 86 to 89, spA marker: any of SEQ ID NOs: 92 to 95,
  • VanA marker any of SEQ ID NOs: 98 to 101,
  • VanB marker any of SEQ ID NOs: 104 to 107
  • VanC marker any of SEQ ID NOs: 110 to 113
  • MDR-I marker any of SEQ ID NOs: 116 to 119,
  • CDR l marker any of SEQ ID NOs: 122 to 125.
  • composition comprising two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) of the following probes, preferably, though not necessarily, one probe selected for one marker: bla ges - 2 marker: SEQ ID NOs: 76 or 77, bla s hv marker: SEQ ID NOs: 80 or 81, mecA marker: SEQ ID NOs: 88 or 89, spA marker: SEQ ID NOs: 92 or 93, VanA marker: SEQ ID NOs: 100 or 101,
  • VanB marker SEQ ID NOs: 106 or 107
  • VanC marker SEQ ID NOs: 112 or 113
  • MDR-I marker SEQ ID NOs: 116 or 117
  • CDR l marker SEQ ID NOs: 124 or 125.
  • Another embodiment of the invention is a method for detecting at least one
  • Another embodiment of the invention is a method for identifying at least one micro-organism and at least one antibiotic resistance marker in a sample, by detecting the distinct regions corresponding to the SEQ ID NOs mentioned above.
  • Another embodiment of the invention is a method for identifying at least one micro-organism and at least one antibiotic resistance marker in a sample, by using two or more of the primer pairs or single probes corresponding to the SEQ ID NOs mentioned above.
  • Another embodiment of the invention is a method for identifying one microorganism and at least one antibiotic resistance marker in a sample, by using one of the primer pairs or single probes corresponding to the SEQ ID NOs mentioned above relating to 23S RNA and two or more ⁇ e.g. 3, 4, 5, 6, 7, 8, 9 or 10 or more) primer pairs or single probes corresponding to the SEQ ID NOs mentioned above relating to antibiotic resistance genes.
  • Another embodiment of the present invention is a composition comprising one or more ⁇ e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) of the following primer pairs:
  • Enterobacter cloacae SEQ ID NOs: 3 and 4,
  • Enterococcus faecalis SEQ ID NOs: 9 and 12,
  • Enterococcus faecium SEQ ID NOs: 18 and 19, Escherichia colt SEQ ID NOs: 24 and 25,
  • Klebsiella pneumoniae SEQ ID NOs: 32 and 33,
  • Pseudomonas aeruginosa SEQ ID NOs: 40 and 42,
  • Staphylococcus aureus SEQ ID NOs: 48 and 49,
  • Staphylococcus epidermidis SEQ ID NOs: 54 and 55
  • Candida albicans SEQ ID NOs: 66 and 67
  • bla s hv marker SEQ ID NOs: 80 and 81
  • mecA marker SEQ ID NOs: 88 and 89
  • spA marker SEQ ID NOs: 92 and 93
  • VanA marker SEQ ID NOs: 100 and 101,
  • VanB marker SEQ ID NOs: 106 and 107
  • VanC marker SEQ ID NOs: 112 and 113
  • MDR-I marker SEQ ID NOs: 116 and 117
  • CDR l marker SEQ ID NOs: 124 and 125.
  • compositions comprising one or more (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) of the following probes, preferably, though not necessarily, one probe selected for one micro-organism:
  • Enterobacter cloacae SEQ ID NOs: 3 or 4,
  • Enterococcus faecalis SEQ ID NOs: 9 or 12,
  • Enterococcus faecium SEQ ID NOs: 18 or 19,
  • Escherichia col ⁇ SEQ ID NOs: 24 or 25, Klebsiella pneumoniae: SEQ ID NOs: 32 or 33,
  • Pseudomonas aeruginosa SEQ ID NOs: 40 or 42,
  • Staphylococcus aureus SEQ ID NOs: 48 or 49,
  • Staphylococcus epidermidis SEQ ID NOs: 54 or 55,
  • Candida albicans SEQ ID NOs: 66 or 67. and one of more ⁇ e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) of the following probes, preferably though not necessarily, one probe selected for one marker: bla ges - 2 marker: SEQ ID NOs: 76 or 77, bla s hv marker: SEQ ID NOs: 80 or 81, mecA marker: SEQ ID NOs: 88 or 89, spA marker: SEQ ID NOs: 92 or 93,
  • VanA marker SEQ ID NOs: 100 or 101,
  • VanB marker SEQ ID NOs: 106 or 107
  • VanC marker SEQ ID NOs: 112 or 113
  • MDR-I marker SEQ ID NOs: 116 or 117
  • CDR l marker SEQ ID NOs: 124 or 125.
  • a composition according to the present invention may be a solution, a mixture, an admixture, or may constitute the components in or on a container or device of the invention.
  • Another embodiment of the present invention is a container comprising two or more (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) primer pairs as defined in the above method and composition embodiments.
  • Another embodiment of the present invention is a container comprising two or more probes (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) as defined in the above method and composition embodiments.
  • Another embodiment of the present invention is a kit comprising two or more (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) primer pairs as defined in the above method and composition embodiments.
  • kits comprising two or more probes (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) as defined in the above method and composition embodiments.
  • a method detects the presence of one or more antibiotic resistance genes a micro-organism. According to another aspect of the invention, a method detects the presence of one or more antibiotic resistance genes a micro- organism and also the micro-organism.
  • the primers advantageously permit simultaneous or multiplexed PCR of template sequences in a single reaction, without the formation of primer dimer or cross reactions.
  • the length of a product is particular to a species or antibiotic resistance marker. This allows the product of an amplification reaction to be separated, for example, by electrophoresis, and identification of several genes by evaluating the length attributed to one or more bands.
  • Multiplex detection of target sequences not only has the benefit of increasing throughput, but also allows differential diagnosis and monitoring of therapy in clinical applications.
  • the disease of bacteraemia and septicaemia is caused by one or more different pathogens and the therapy strongly depends on the detection of the involved bacteria and involved antibiotic resistant bacterial strains, which will guide the application of the right antibiotics. Therefore, for a proper and effective therapy it is essential to very specific and sensitive detection of the presence, absence or amount of specific NA sequences of one or more pathogens and their antibiotic resistant species, - the so called panel of pathogens and antibiotic resistant species. It is well known to the person skilled in the art, that the treatment of bacteraemia and septicaemia will only be successful in a very narrow timeframe after the outbreak of the disease.
  • the present invention includes products comprising one or more primers or probes, suitable for use in a device enabling identification of the distinct regions mentioned herein.
  • Such products include, for example, - one or more containers (e.g. microarray or multi-sample container) preloaded with one or more pairs of amplification primers.
  • a multi-sample container allows simultaneous detection of distinct regions either in the same reaction or as separate reactions,
  • kits comprising one or more pairs of primers and optionally buffers, reagents and containers for performing amplification reactions, - a kit comprising one or more containers and optionally buffers, reagents for performing amplification reactions,
  • one or more containers preloaded with one or more preloaded with one or more probes.
  • a multi-sample container allows simultaneous detection of distinct regions either in the same reaction or as separate reactions, ,
  • kits comprising one or more probes and optionally buffers, reagents and containers for performing hybridisation
  • a kit comprising one or more containers and optionally buffers, reagents and containers for performing hybridisation
  • - a device comprising one or more probes for performing hybridisation.
  • compositions comprising one or more primer pairs as mentioned herein. a composition comprising one or more probes as mentioned herein.
  • Example 1 Sample preparation 200 ⁇ l of patient' s blood was pipetted into a vessel containing glass beads, acid-washed (Sigma- Aldrich) with a diameter 106 ⁇ m and finer. On a commerical IKA MS2 Minishaker the suspension was vortexed for 1 to 3 min at ambient temperature.
  • Example 2 Nucleic acid extraction and purification The extraction and purification of nucleic acid was performed on a modified commercial biorobot EZl (Qiagen). The vortexed blood sample was incubated with lysostaphin at 37 0 C for 10 min. In a following reaction step the nucleic acid was immobilized on magnetic beads.
  • the immobilized nucleic acid By actuating the magnetic beads via external magnetic fields in different washing and rinsing solutions, the immobilized nucleic acid will be freed from cell debris and other cell proteins.
  • the nucleic acid will be separated from the magnetic particles and then mixed in a separate vessel with a multiplex PCR Mastermix (Qiagen). After mixing, the solution will be distributed to strip vessels with multiplex primer pairs and human control primer pairs.
  • Example 3 Multiplex PCR
  • Multiplex PCR was performed on a commercial thermal cycler, e.g. Perkin Elmer 9700, with a Qiagen Multiplex PCR kit. Following the universal multiplex cycling protocol, a first initial activation step was performed at 95 0 C for 15 min. It followed a 3 step cycling with denaturing at 94 0 C for 30 sec, annealing at 61 0 C for 90 sec, and extension at 72 0 C for 90 sec. The cycle number was between 30 and 45, dependend on the sensitivity requirements. A final extension at 72 0 C for 10 min finished the amplification.
  • a commercial thermal cycler e.g. Perkin Elmer 9700
  • the detection of the amplified nucleic acid was performed on a DNA 1000 kit.
  • the chip, which is part of the kit, was read out with a 2100 bioanalyzer. Kits and Analyzer were purchased from Agilent Technologies.
  • the chip was prepared and loaded with the references and amplified nucleic acids and then inserted in the 2100 bioanalyzer. After an analysis run time of 30 min, proprietary software running on a PC read out and analyzed the data from the bioanalyzer.

Abstract

The present invention relates to methods of detecting one or more micro¬ organisms and/or one or more antibiotic resistance markers in a sample, comprising identifying the presence of distinct nucleic acid regions. Primers and probes suitable for use in such methods are provided.

Description

METHOD FOR DETECTION OF MICRO-ORGANISMS AND ANTIBIOTIC RESISTANCE MARKERS AND NUCLEIC ACID OLIGONUCLEOTIDES THEREFOR
Since the discovery of nucleic acids (NA), the technology relating to the detection of the presence, absence, or amount of specific DNA or RNA sequences in a sample has gained tremendous interest in academia, as well as in industry. The invention of amplification techniques, especially the Polymerase Chain Reaction (PCR) and hyridisation have contributed enormously to the development of assays of all types for the detection of the presence, absence, or amount of NA sequences. At present, it is possible to collect NA containing samples from an organism and determine the presence, absence, or amount therein of certain specific NA sequences (target sequences). Technology is available to perform such analysis for multiple target sequences at the same time, so-called multiplex detection of target sequences to thereby increase throughput as well as to improve the diagnostic significance.
At present, detection based on NA sequences is not yet performed on a routine basis, as is the case, for instance, in the measurement of blood glucose concentration of diabetics. Generally, well-equipped laboratories and well-trained staff are necessary and careful protocols have to be used in order to give reliable results. Furthermore, the present methods of analysis are not only laborious, but also time consuming. Typically, a current procedure for DNA or RNA analysis takes several days due to, amongst other thing, the requirement of various systems for the taking of samples, the culturing of samples, the isolation of DNA or RNA from the sample, the subsequent assay for the analysis of the presence, absence, or amount of the target sequences in the sample, the processing of any results obtained and the corresponding presentation of the results.
This time consuming analysis can be dramatically improved by applying highly specific hybridisation and amplification methods to the whole or parts of the target sequences so enabling the presence, absence, or amount of certain specific NA sequences to be determined. A highly sensitive and specific PCR and/or hybridisation can be applied, which in turn requires highly specific primers to certain specific NA target sequences. NA sequences of some bacterial strains are known in the art, for example, Staphylococcus aureus NA sequences are disclosed in J Clin Microbiol. (2000), 38(2), 781-8, Journal of Microbiological Methods (2004), 58, 403- 411, J Clin Microbiol. (2004), 42(3), 1048-57, US 5,582,975, WO 90/14444, WO 03/095677, and US 5,958,679; Staphylococcus epidermidis NA sequences are disclosed in Journal of Microbiological Methods (2004), 58, 403- 411 and WO 03/095677; Pseudomonas aeruginosa NA sequences are disclosed in Journal of Microbiological Methods (2004), 58, 403- 411, J Clin Microbiol. (2004), 42(3), 1048-57, and WO 03/095677; Klebsiella pneumoniae NA sequences are disclosed in Journal of Microbiological Methods (2004), 58, 403- 411 and WO 03/095677; Enterococcus faecalis NA sequences are disclosed in Journal of Microbiological Methods (2004), 58, 403- 411; Enterococcus faecium NA sequences are disclosed in Journal of Microbiological Methods (2004), 58, 403- 411 and WO 03/095677; Escherichia coli NA sequences are disclosed in Journal of Microbiological Methods (2004), 58, 403- 411, J Clin Microbiol. (2004), 42(3), 1048-57 and WO 03/095677; Enterobacter cloacae NA sequences are disclosed in US 5,958,679. NA sequences of certain antibiotic resistance genes are known in the art, for example, beta lactamase SHV NA is known from J. Clin Microbiol (2001) 39, 3193-3196, J. Clin Microbiol (1998) 36, 3105-3110, J. Clin Microbiol (1999) 37, 4020-4027, US 2004/0002080, and US 6,242,223; beta lactamase GES-2 NA is known from International Journal of Antimicrobial Agents (2004) 24, 35-38 and /. Antimicrobial Chemotherapy (2002) 49 , 561-565; methicillin resistance MecA NA is known from J Clin Microbiol. (2002), 40(5), 1821-3, J Clin Microbiol. (1995), 33(11), 2864-7, J Clin Microbiol. 2000 Jun_38(6)_2429-33, WO02082086A2 and US 5,437,978; spA NA is known from J. Clin Microbiol (2003) 41, 5442-5448 and US 5,702,895; vanA NA is known from J. Clin. Microbiol. (1997), 703-707 and J. Clin. Microbiol. (2000) 3092-3095; vanB NA is known from J. Clin. Microbiol. (1997), 703-707 and /. Clin. Microbiol. (2000) 3092-3095; vanC NA is known from J. Clin. Microbiol. (1997), 703-707 and J. Clin. Microbiol. (2000) 3092- 3095; beta lactamase resistance TEM-IH NA is known from Antimicrobial Agents And Chemotherapy (2001), 2407-2413, US 2004/0002080 and US 6,242,223.
However, a problem in the art of NA detection is providing reliable primers or probes. Particularly where multiplex detection is employed, a problem is cross-reactions and false-positive or false-negative results. The present invention aims to overcome the problems of the art by providing methods, sequences and primers which are suited to specific and reliable single mode and multiplex NA detection. One embodiment of the present invention is a method of detecting one or more micro-organisms and/or one or more antibiotic resistance markers in a sample, comprising identifying the presence of distinct nucleic acid regions.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region of a micro-organism is in the 23S RNA gene.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region is identified using nucleic acid amplification.
Another embodiment of the present invention is a method as described above, wherein multiplex PCR is used to detect two or more distinct nucleic acid regions. Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region is identified using hybridisation.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterobacter cloacae, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 3 and 4 or SEQ ID NOs: 5 and 6.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterobacter cloacae, comprising the use of a hybridisation probe corresponding to a sequence represented by any of SEQ ID NOs: 3 to 6.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 1 or 2, and a microorganism is Enterobacter cloacae.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterococcus faecalis, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, or SEQ ID NOs: 15 and 11.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterococcus faecalis, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 9 to 15. Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 7 or 8, and a microorganism is Enterococcus faecalis.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterococcus faecium, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 18 and 19, SEQ ID NOs: 19 and 20, or SEQ ID NOs: 20 and 21.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Enterococcus faecium, comprising the use of a probe corresponding to the sequences represented by SEQ ID NOs: 19 to 21.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 16 or 17, and a microorganism is Enterococcus faecium.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Escherichia coli, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 24 and 25, SEQ ID NOs: 24 and 26, SEQ ID NOs: 27 and 29, or SEQ ID NOs: 28 and 29.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Escherichia coli, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 24 to 29.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 22 or 23, and a microorganism is Escherichia coli.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Klebsiella pneumoniae, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36 or SEQ ID NOs: 37 and 33.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Klebsiella pneumoniae, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 32 to 37.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 30 or 31, and a microorganism is Klebsiella pneumoniae.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Pseudomonas aeruginosa, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 40 and 41 or SEQ ID NOs: 40 and 42. Another embodiment of the present invention is a method as described above, wherein said micro-organism is Pseudomonas aeruginosa, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 40 to 42.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 38 or 39, and a micro-organism is Pseudomonas aeruginosa.
Another embodiment of the present invention is a method as described above, wherein a micro-organism is Staphylococcus aureus, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, SEQ ID NOs: 50 and 51.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Staphylococcus aureus, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 45 to 51. Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 43 or 44, and a micro-organism is Staphylococcus aureus.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Staphylococcus epidermidis, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, or SEQ ID NOs: 63 and 61.
Another embodiment of the present invention is a method as described above, wherein said micro-organism is Staphylococcus epidermidis, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 54 to 63.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 52 or 53, and a microorganism is Staphylococcus epidermidis. Another embodiment of the present invention is a method as described above, wherein said micro-organism is Candida albicans, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, or SEQ ID NOs: 70 and 71. Another embodiment of the present invention is a method as described above, wherein said micro-organism is Candida albicans, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 66 to 71.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 64 or 65, and a micro-organism is Candida albicans.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is blages-2, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 74 and 75 or SEQ ID NOs: 76 and 77.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is blages.2, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 74 to 77.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 72 or 73, and an antibiotic resistance marker is blages-2.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is blashv, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 80 and 81 or SEQ ID NOs: 82 and 83.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is blashv, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 80 to 83.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 78 or 79, and an antibiotic resistance marker is blas/,v.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is mecA, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 86 and 87 or SEQ ID NOs: 88 and 89.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is mecA, comprising the use of a probe corresponding to the sequences represented by SEQ ID NOs: 86 or 89. Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 84 or 85, and an antibiotic resistance marker is mecA.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is spA, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 92 and 93 or SEQ ID NOs: 94 and 95.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is spA, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 92 to 95.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 90 or 91, and an antibiotic resistance marker is Spa.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanA, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 98 and 99 or SEQ ID NOs: 100 and 101.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanA, comprising the use of a probe corresponding to the sequences represented by SEQ ID NOs: 98 to 101.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 96 or 97, and an antibiotic resistance marker is VanA.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanB, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 104 and 105 or SEQ ID NOs: 106 and 107.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanB, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 104 to 107.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 102 or 103, and an antibiotic resistance marker is VanB. Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanC, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 110 and 111 or SEQ ID NOs: 112 and 113. Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is VanC, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 110 to 113.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 108 or 109, and an antibiotic resistance marker is VanC.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is MDR-I, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 116 and 117 or SEQ ID NOs: 118 and 119. Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is MDR-I, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 116 to 119.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 114 or 115, and an antibiotic resistance marker is MDR-I.
Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is CDR- 1, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 122 and 123 or SEQ ID NOs: 124 and 125. Another embodiment of the present invention is a method as described above, wherein said antibiotic resistance marker is CDR- 1, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 122 to 125.
Another embodiment of the present invention is a method as described above, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 120 or 121, and an antibiotic resistance marker is CDR-I.
Another embodiment of the present invention is a container preloaded with one or more pairs of amplification primers, selected from the sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6,SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, SEQ ID NOs: 15 and 11,SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, SEQ ID NOs: 28 and 29, SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 33, SEQ ID NOs: 40 and 41, SEQ ID NOs: 40 and 42, SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, SEQ ID NOs: 50 and 51, SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, SEQ ID NOs: 63 and 61, SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, SEQ ID NOs: 70 and 71, SEQ ID NOs: 74 and 75, SEQ ID NOs: 76 and 77, SEQ ID NOs: 80 and 81, SEQ ID NOs: 82 and 83, SEQ ID NOs: 86 and 87, SEQ ID NOs: 88 and 89, SEQ ID NOs: 92 and 93, SEQ ID NOs: 94 and 95, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 104 and 105, SEQ ID NOs: 106 and 107, SEQ ID NOs: 110 and 111, SEQ ID NOs: 112 and 113, SEQ ID NOs: 116 and 117, SEQ ID NOs: 118 and 119, SEQ ID NOs: 122 and 123, and SEQ ID NOs: 124 and 125.
Another embodiment of the present invention is a container preloaded with one or more probes, selected from the sequences represented by any of SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
Another embodiment of the present invention is a kit comprising one or more pairs of amplification primers, selected from the sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6,SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, SEQ ID NOs: 15 and 11,SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, SEQ ID NOs: 28 and 29, SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 33, SEQ ID NOs: 40 and 41, SEQ ID NOs: 40 and 42, SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, SEQ ID NOs: 50 and 51, SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, SEQ ID NOs: 63 and 61, SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, SEQ ID NOs: 70 and 71, SEQ ID NOs: 74 and 75, SEQ ID NOs: 76 and 77, SEQ ID NOs: 80 and 81, SEQ ID NOs: 82 and 83, SEQ ID NOs: 86 and 87, SEQ ID NOs: 88 and 89, SEQ ID NOs: 92 and 93, SEQ ID NOs: 94 and 95, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 104 and 105, SEQ ID NOs: 106 and 107, SEQ ID NOs: 110 and 111, SEQ ID NOs: 112 and 113, SEQ ID NOs: 116 and 117, SEQ ID NOs: 118 and 119, SEQ ID NOs: 122 and 123, and SEQ ID NOs: 124 and 125.
Another embodiment of the present invention is a kit comprising one or more probes selected from the sequences represented by SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
Another embodiment of the present invention is a kit comprising one or more containers as described above.
Another embodiment of the present invention is a device comprising one or more pairs of amplification primers selected from the sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6,SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, SEQ ID NOs: 15 and 11,SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, SEQ ID NOs: 28 and 29, SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 33, SEQ ID NOs: 40 and 41, SEQ ID NOs: 40 and 42, SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, SEQ ID NOs: 50 and 51, SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, SEQ ID NOs: 63 and 61, SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, SEQ ID NOs: 70 and 71, SEQ ID NOs: 74 and 75, SEQ ID NOs: 76 and 77, SEQ ID NOs: 80 and 81, SEQ ID NOs: 82 and 83, SEQ ID NOs: 86 and 87, SEQ ID NOs: 88 and 89, SEQ ID NOs: 92 and 93, SEQ ID NOs: 94 and 95, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 104 and 105, SEQ ID NOs: 106 and 107, SEQ ID NOs: 110 and 111, SEQ ID NOs: 112 and 113, SEQ ID NOs: 116 and 117, SEQ ID NOs: 118 and 119, SEQ ID NOs: 122 and 123, SEQ ID NOs: 124 and 125. Another embodiment of the present invention is a device comprising one or more probes, selected from the sequences represented by SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
Another embodiment of the present invention is a use of a container, kit or device as described above for detecting one or more micro-organisms and/or one or more antibiotic resistance markers in a sample.
Another embodiment of the present invention is a composition comprising a probe selected from the sequences represented by: SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
Another embodiment of the present invention is a composition comprising two or more probes selected from the sequences represented by: SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.. Another embodiment of the present invention is a composition comprising a pair of amplification primers selected from the sequences represented by: SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, SEQ ID NOs: 51 and 52, SEQ ID NOs: 55 and 56, and SEQ ID NOs: 59 and 60.
Another embodiment of the present invention is a composition comprising two or more pairs of amplification primers selected from the sequences represented by: SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, SEQ ID NOs: 51 and 52, SEQ ID NOs: 55 and 56, and SEQ ID NOs: 59 and 60.
Another embodiment of the present invention is a sequence of 23S RNA gene selected from the sequences represented by SEQ ID NOs: 131 to 157. Another embodiment of the present invention is a sequence of antibiotic resistance marker selected from the sequences represented by SEQ ID NOs: 158 to 261.
Another embodiment of the present invention is a method as described above, wherein said sequence(s) represented by said SEQ ID NO(s) is (are) the complement(s) of said SEQ ID NO(s). Another embodiment of the present invention is a method as described above, wherein said sequence(s) represented by said SEQ ID NO(s) is (are) an homologous sequence(s) of said SEQ ID NO (s).
Another embodiment of the present invention is a container, kit, device or use as described above wherein said sequence(s) represented by said SEQ ID NO(s) is (are) the complement(s) of said SEQ ID NO(s).
Another embodiment of the present invention is a container, kit, device or use as described above wherein said sequence(s) represented by said SEQ ID NO(s) is (are) an homologous sequence(s) of said SEQ ID NO(s).
Another embodiment of the present invention is a composition as described above wherein said sequence(s) represented by said SEQ ID NO(s) is (are) the complement(s) of said SEQ ID NO(s).
Another embodiment of the present invention is a composition as described above, wherein a sequence represented by a SEQ ID NO is an homologous sequence of said SEQ ID NO. Another embodiment of the present invention is a sequence of 23S RNA gene as described above, wherein said sequence represented by said SEQ ID NO is the complement(s) of said SEQ ID NO.
Another embodiment of the present invention is a sequence of 23S RNA gene as described above, wherein said sequence represented by said SEQ ID NO is an homologous sequence of said SEQ ID NO.
Another embodiment of the present invention is a sequence of an antibiotic resistance marker as described above, wherein said sequence(s) represented by said SEQ ID NO(s) is(are) the complement(s) of said SEQ ID NO(s). Another embodiment of the present invention is a sequence of an antibiotic resistance marker as described above, wherein said sequence(s) represented by said SEQ ID NO(s) is(are) an homologous sequence(s) of said SEQ ID NO(s).
Figure 1: Sequences and alignments of 23S RNA sequences of microorganisms. Figure 2: Sequences and alignments of antibiotic resistance genes.
The present invention relates to sequences of 23S RNA genes of micro- organisms, and to antibiotic resistance genes and their use as templates for hybridisation and/or nucleic acid amplification reactions, and/or other identification methods in order to detect the presence of one or more micro-organisms and/or antibiotic resistance genes in a sample. The invention further relates to nucleic acid amplification primers and hybridisation probes suitable for amplification of and hybridisation to said sequences. The sample may be any sample of interest. It may be derived from animals
(e.g. human, agricultural livestock, domestic livestock, scientific livestock, zoological livestock) or non-animal (e.g. solid and liquid consumables, water systems, sewerage systems, soil, heating / cooling systems). Where the sample is human, it may be for example, blood, saliva, urine, faeces, any bodily fluid or tissue. The sample is any that warrants investigation, and is capable of providing template nucleic acid.
According to one embodiment of the present invention, species of microorganisms useful according to the invention are one or more of Staphylococcus aureus, Staphylococcus epidermidis, Enterobacter cloacae, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Enterococcus faecium, Klebsiella pneumoniae, and Candida albicans.
According to another embodiment of the present invention, antibiotic resistance markers useful according to the invention are one or more of mecA (methicillin resistance gene, confers resistance to B-lactams), vanA (vancomycin resistance gene A), vanB (vancomycin resistance gene B), vanC (vancomycin resistance gene C), blashv (beta- lactam resistance gene), blages-2 (beta-lactam resistance gene), spA (Staphylococcus-aureus protein A), MDR-I (multi-drug resistance gene-1 in Fungi), and CDR-I (multi-drug resistance gene-2 in Fungi).
According to the present invention, nucleic acid sequences of 23S RNA or antibiotic resistance markers serve as templates for sequence- specific nucleic acid detection methods. A detection method involves detecting the presence of one or more distinct regions i.e. a region of 23S RNA gene (including 23S RNA per se), or a region of an antibiotic marker sufficiently distinct to enable identification of a species or an antibiotic resistance marker by detecting the presence of the region. Detection may be by amplification of at least a portion the distinct region. Alternatively, or in addition, detection may be by the use of an anti-nucleic acid antibody or sequencing. Alternatively, or in addition, detection may be by hybridisation using a probe. Detection may occur when total nucleic acid from other species or taxonomical groups is present in the sample. The distinct regions may, for example,
- comprise sequence portions which are unconserved between species, - comprise sequence portions which are unconserved between antibiotic resistance markers,
- comprise an unconserved number of residues between conserved sequence portions, enabling identification based on product length of the amplification reaction,
- comprise certain physical properties of folding, or associated proteins particular to a species, permitting binding of primer or probe under certain conditions.
A distinct region includes homologous sequences in which one or more bases have been deleted, substituted and/or inserted. The number of substitutions, deletions and/or insertions permitted in an homologous sequence may be less than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 residues. Alternatively, the number is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of residues. An homologous sequence still permits identification of the distinct region based on some or all of the unchanged residues. A distinct region also includes the complement sequence of the distinct region.
Amplification Where nucleic acid amplification is used (e.g. PCR), primers pairs are of such sequence and length to provide amplification products (amplicons) only when one or more distinct regions of the species or resistance genes are present. Alternatively, the primers may provide a product of particular length or pattern for the species of interest, distinguishable from amplification products arising from the amplification of other sequences. Alternatively, or in addition, the amplification primers may provide a relative quantity of product, enabling identification of the species (e.g. one or more strong bands on a electrophoretic gel). Any method of matching the result of an amplification to the presence of the nucleic acid of interest is within the scope of the invention. Although nucleic acid amplification methods such as the PCR process are well known in the art (see U.S. Pat. Nos. 4,683,195 and 4,683,202 which are incorporated herein by reference) and although a variety of commercial vendors, such as Roche, Invitrogen, Qiagen, Promega sell PCR reagents and publish PCR protocols, some general PCR information is provided below for purposes of clarity. To begin the PCR process, the target nucleic acid in the sample is denatured
(assuming the sample nucleic acid is double- stranded). Denaturation is typically achieved by heating the samples up to about 950C.
Once the strands are separated, the next step in PCR involves hybridising the separated strands with primers that flank the target region or subsequence by lowering the temperature of the sample below the melting temperature TM. The primers are then extended to form complementary copies of the target strands by increasing the sample temperature up to the temperature for optimum extension (e.g. 70 to 75 deg C), and the cycle of denaturation, hybridisation, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid. Template-dependent extension of primers in PCR is catalysed by a polymerising agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of the appropriate salts, metal cations, and pH buffering system. Suitable polymerising agents are enzymes known to catalyse template-dependent DNA synthesis. For example, if the template is RNA, a suitable polymerising agent to convert the RNA into a complementary DNA
(cDNA) sequence is reverse transcriptase (RT), such as arian myeloblastosis virus RT. Once the target for amplification is DNA, suitable polymerases include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, and Taq polymerase, a heat stable DNA polymerase isolated from Thermus aquaticus. The latter enzyme, Taq DNA polymerase, is widely used in the amplification and sequencing of nucleic acids. The reaction conditions for using DNA polymerases are known in the art, and are described in, for example, the treatise Methods in Enzymology, and in Maniatis et al., Molecular Cloning: A Laboratory Manual. During the PCR process, the temperature is carefully controlled so that strand separation and primer annealing and extension occur in equilibrium. The control of temperature is typically achieved using dry heat generated from a thermocycler.
In the preferred embodiment of the PCR process, the reaction is catalysed by a thermostable DNA polymerase enzyme, such as Taq DNA polymerase, and carried out at an elevated temperature. The preferred temperature is one at which the enzyme is thermostable, and at which the nucleic acids are in an equilibrium of single and double strands, so that sufficient primer will anneal to template strands to allow a reasonable rate of polymerisation Strand separation is achieved by heating the reaction to a sufficiently high temperature for sufficient time to cause the denaturation of the duplex, but not to cause an irreversible denaturation of the polymerase.
The PCR method can be performed in a step-wise fashion, where after each step new reagents are added, or in a fashion where all of the reagents are added after a given number of steps. For example, if strand separation is induced by heat, and the polymerase is heat- sensitive, then the polymerase will have to be added after every round of strand separation. However, if, for example, a helicase is used for denaturation, or if a thermostable polymerase is used for extension, then all of the reagents may be added initially, or, alternatively, if molar ratios of reagents are of consequence to the reaction, the reagents may be replenished periodically as they are depleted by the synthetic reaction. Methods for detecting the presence, size and/or quantity of the PCR product are known in the art and include the use of electrophoresis, chromatography, capillary-zone electrophoresis, analytical centrifugation etc. Such method may be in combination with the use of labels (e.g. fluorescent, chemiluminescence, radioisotope, enzyme-labels (such as horse radish peroxidases or alkaline phosphatase), dye, antibody, etc). Detection may also be achieved by the use of hybridisation probes (mentioned below) either in solution or immobilised on a solid support or antibodies directed against the DNA to be detected.
According to one embodiment of the invention, the amplification is performed on or in a container. A container may be single sample or multiple-sample. Single sample containers include, tubes, vials, Eppendorf tubes etc., as known to the skilled person. Multi- sample containers include, but are not limited to multi-well plates, solid phase slides, solid phase membrane (e.g. nylon or nitrocellulose), microspheres, glass slides, microarrays, chips etc. Single sample containers may use the aforementioned substrates in a single sample mode. The multiple- sample containers permit, for example, several or a large number of PCRs to proceed simultaneously using a single thermal cycling block. The containers may be compatible with high-throughput screening or microarray devices. One or more pairs of primers or samples may be immobilised onto the solid phase. A container may be provided which already comprises one or more pairs of primers. Such preloaded containers may comprise combinations of primers for detection of specified micro-organisms and/or resistance genes. A preloaded container may comprise a combination of primers suited for detection of a limited combination of distinct regions which are of interest to the operator (e.g. for the detection of just E. coli and vanA or vanB or vanC). Several variations for the detection of particular combinations may be made available. Such preloaded containers may be available as part of a kit, or available separately. According to one aspect of the invention, two or more pairs of amplification primers are used to detect simultaneously the presence of two or more different species, or two or more different antibiotic resistance markers, or at least one species and at least one antibiotic resistance marker. The amplification products obtained thereby are sufficiently different in property to enable identification of the presence of said species and/or antibiotic resistance marker. The simultaneous amplifications may be performed under the same temperature cycling conditions, but in different wells or spaces (e.g. on a microarray having separate wells for different primer pairs). Optionally, the buffers may be the same for the different pairs of amplification primers. The different primer pairs are designed of certain sequences and length, to function under identical conditions of temperature and optionally buffer.
In another aspect of the invention, the amplification primers occupy the same well or space, i.e. all the primers are present in the same reaction (multiplexed). The multiplex mode involves the simultaneous amplification of different target regions using more than one set of amplification primer pairs. Therefore, conditions such as temperature and optionally buffers are identical for pairs of multiplex PCR primers. The primers are thus further designed to preclude cross-reaction between primer pairs, to have similar thermal melting points, and operate in identical buffer conditions.
It is preferred that all pairs for multiplexed PCR have Tms (hybdridisation melting temperatures) within 8 deg C of each other and that the average Tm is between 45 deg C and about 70 deg C. with preference for an average Tm of between 60 deg C and 66 deg C.
Simultaneous amplification enables a range of bacterial species and/or antibiotic resistance markers to be detected on a single microarray, or in a single reaction vessel, so permitting rapid, economic and accurate screening. The amplification primers may be completely complementary to the target i.e. there are no mismatches. It is also within the scope of the invention that a primer does not fully complement the target, but still allows amplification thereof. The primers may bind where there are one or more mismatches, deletions and/or insertions in the target template. The number of mismatches, deletions and/or insertions permitted in the target template may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues within the native complementary region, and which still allow amplification of the target region. Alternatively, the number may be less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of template residues within the native complementary region. Such values depend upon the length and composition of the primers as known by the skilled person. Preferably, the mismatches, deletions and/or insertions are restricted to sequences from the middle of the complementary region towards the 3' end of a template. The primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted. The number of substitutions, deletions and/or insertions permitted in a primer may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues within the native complementary region. Alternatively, the number is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of primer residues within the ends of native complementary region. Such values depend upon the length and composition of the probes as known by the skilled person. Preferably, the mismatches, deletions and/or insertions are restricted to sequences from the middle of the complementary region towards the 5' end of a primer. Furthermore, an amplification primer may be chemically modified, for example, with modified bases or backbones {e.g. phosphorothiates, alkylphosphorothiates, peptide nucleic acids, or may contain intercalating agents). Variations or modifications introduced may necessitate adaptations with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However, the eventual results of the amplification reaction will be essentially the same.
The introduction of deletions, substitutions, insertions or modifications may be advantageous in order to positively influence characteristics such as annealing kinetics, reversibility of annealing, biological stability of oligonucleotide molecules etc. Furthermore, an amplification primer may be extended in the 5' direction with one or more additional bases, modified bases, or chemical groups (e.g. tags). Such modifications are known to the skilled person and do not normally affect the amplification.
It is an aspect that identification comprises double amplification i.e. amplification of a region which encloses the distinct region, followed by the amplification of the distinct region e.g. by nested PCR. The product from the first reaction (optionally purified) may be applied as a template in the second reaction. Alternatively, the first reaction may be allowed to proceed for a limited number of cycles, before primers pertinent to the second reaction are added to the same reaction container. Such variations are known to the skilled person.
Hybridisation
Where a hybridisation probe is used, the probe may be of such sequence and length that hybridisation is indicated only when nucleic acid of a distinct region is present in the reaction. Methods and protocols to achieve selective binding of hybridisation probes are known to the skilled person. Alternatively, or in addition, the probe may provide a particular relative strength of signal to enable identification of the species against background or other hybridisation. Any method of matching the result of a hybridisation reaction to the presence of the nucleic acid of interest is within the scope of the invention. The methods and conditions for performing a hybridisation reaction are known in the art, and can be found, for example, in Molecular Cloning: A Laboratory Manual (Third Edition) (Joseph Sambrook, Peter MacCallum, David Russell, Cold Spring Habor Laboratory Press).
For designing probes with desired characteristics, the following useful guidelines known to the person skilled in the art can be applied.
Because the extent and specificity of hybridisation reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions are explained further herein.
The stability of the [probe: target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished, for example, by avoiding long AT-rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and %GC result in a Tm about 2 to 10 deg C higher than the temperature at which the final assay is be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability compared with A-T base pairs due to additional hydrogen bonding. Thus, hybridisation involving complementary nucleic acids of higher C-C content will be more stable at higher temperatures.
Conditions such as ionic strength and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that the degree of hybridisation will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridisation. Destabilisation of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridisation for probes of about 10 to 50 bases in length occurs approximately 5 deg C below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridise and can therefore result in reduced specificity.
It is desirable to have probes which hybridise only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form or will be indicated as weaker signal. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. The degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid.
Regions in the target DNA or RNA which are known to form strong internal structures inhibitory to hybridisation are less preferred. Likewise, probes with extensive self- complementarity should be avoided. Hybridisation is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand: It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridisation may be greatly increased. Computer programs are available to search for this type of interaction. However, in certain instances, it may not be possible to avoid this type of interaction.
Methods for detecting the presence of sequences may include, but are not limited to Southern blot, Northern blot, affinity chromatography and solid-phase assays. Methods may include the use of fluorescent markers, radioisotope markers, enzyme linked markers, dyes, antibodies, enzymes linked to the probe as understood by the person skilled in the art.
According to one embodiment of the invention, the analysis is performed on or in a container. A container may be single sample or multiple- sample. Single sample containers include, tubes, vials, Eppendorf tubes etc., as known to the skilled person. Multi- sample containers include, but are not limited to multi-well plates, solid-phase support, solid phase slides, solid phase membrane (e.g. nylon or nitrocellulose), porous structures, microspheres, glass slides, microarrays, chips etc. Single sample containers may use the aforementioned substrates in a single sample mode. Sample may be applied, for example, using an multiple applicator, soft lithography or microcontact printing, inkjet technology, etc. One or more probes or samples may be immobilised onto a container (e.g. onto a solid phase support). The multiple- sample solid supports permit, for example, several or a large number of hybridisations to proceed simultaneously using a single hybridisation oven or platform and/or single set of reagents. The solid support may be compatible with high-throughput screening or microarray devices. A container may be provided which already comprises one or more probes. Such preloaded containers may comprise combinations of probes for detection of specified micro-organisms and/or resistance genes. A preloaded container may comprise a combination of probes suited for detection of a limited combination of distinct regions which are of interest to the operator (e.g. for the detection of just E. coli and vanA or vanB or vanC). Several variations for the detection of particular combinations may be made available. Such preloaded containers may be available as part of a kit, or separately.
According to one aspect of the invention, two or more hybridisation probes are used to detect simultaneously the presence of two or more different species, or two or more different antibiotic resistance markers, or at least one species and at least one antibiotic resistance marker. The hybridisation products obtained thereby are sufficiently different in property to enable identification of the presence of said species and/or antibiotic resistance marker. The simultaneous hybridisation may be performed under the same temperature conditions, but in different wells or spaces (e.g. on a microarray having separate wells for different primer pairs). Optionally, the buffers may be the same for the different probes. The different probes are thus designed of certain sequences and length, to function under identical conditions of temperature and optionally buffer.
In another aspect of the invention, the hybridisation probes occupy the same well or space, i.e. all the probes are present in the same reaction. Therefore, conditions such as temperature and optionally buffers are identical for the probes. The probes are thus further designed to preclude cross-reaction, and to provide a result allowing the presence of two or more species, DNA resistance gene or both to be reliably identified.
Such simultaneous hybridisation enables detection of a range of bacterial species and/or antibiotic resistance markers on a single microarray, or in a single reaction, with the likelihood of false results from cross-binding minimized.
The hybridisation probes may be completely complementary to the target i.e. there are no mismatches. It is also within the scope of the invention that the probes do not fully complement the target, but still allow identification and discrimination thereof. The probe may bind when there are one or mismatches, deletions and/or insertions in the target template. The number of mismatches, deletions and/or insertions permitted in the target template may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues within the ends of the native complementary region. Alternatively, the number is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of template residues within the ends of the native complementary region. Such values depend upon the length and composition of the probes as known by the skilled person.
A sequence of a probe includes homologous sequences in which one or more bases have been deleted, substituted and/or inserted. The number of substitutions, deletions and/or insertions permitted in a probe may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues within the ends of the native complementary region. Alternatively, the number is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% of primer residues within the ends of native complementary region. Such values depend upon the length and composition of the probes as known by the skilled person. Furthermore, a probe may be chemically modified, for example, with modified bases or backbones {e.g. phosphorothiates, alkylphosphorothiates, peptide nucleic acids, or may contain intercalating agents). Variations or modifications introduced may necessitate adaptations with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However, the eventual results of the hybridisation will be essentially the same. The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridisation kinetics, reversibility of hybridisation, biological stability of oligonucleotide molecules etc.
A hybridisation probe according to the present invention is capable of annealing to a sequence of any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases of the distinct region, or the complement thereof.
It is an aspect that the template of hybridisation is an amplification product. That is to say nucleic acid enclosing a distinct region is first amplified and the hybridisation proceeds using the product of the amplification.
1. Enterobacter cloacae
According to one aspect of the invention a distinct region of Enterobacter cloacae 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 1 or 2) indicated in Tables 1 and 2. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 1 1251 ( 5 1 )
CGGTTTAAGCATGTAGGCGGAGGTTCCAGGTAAATCCGGTACCTTTTAAC GCTGAGGTGTGATGACGAGGCACTACGGTGCTGAAGTAACAAATGCCCTG CTTCCAGGAAAAGCCTCTAAGCATCAGGTAACAYSAAATCGTACCCCAAA CCGACACAGGTGGTCAGGTAGAGAATACCAAGGCGCTTGAGAGAACTCGG GTGAAGGAACTAGGCAAAATGGTGCCGTAACTTCGGGAGAAGGCACGCTG ATATGTAGGTGAAGCCCCTGCGGGTGGAGCTGAAATCAGTCGAAGATACC AGCTGGCTGCAACTGTTTATTAAAAACACAGCACTGTGCAAACACGAAAG TGGACGTATACGGTGTGACGCCTGCCCGGTGCCGGAAGGTTAATTGATGG GGTTAGCGGYAACGCGAAGCTCTTGATCGAAGCCCCGGTAAACGGCGGCC GTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCG ACCTGCACGAATGGCGTAATGATGGCCAGGCTGTCTCCACCCGAGACTCA GTGAAATTGAACTCGCTGTGAAGATGCAGTGTACCCGCGGCAAGACGGAA AGACCCCGTGAACCTTTACTATAGCTTGACACTGAACACTGGTCCTTGAT GTGTAGGATAGGTGGGAGGCTTTGAAGCGTGGACGCCAGTCTGCGTGGAG CCGTCCTTGAAATACCACCCTTTAATGGCTGGTGTTCTAACGTAGACCCG
TWAYCCGGGTTGCGGACAGTGTCTGGTGGGTAGTTTGACTGGGGCGGTCT
I 2050 (31) Table 1: Sequence of distinct region of 23S RNA gene of Enterobacter cloacae. An example of a substitution according to the invention is T1502C (underlined). W is a nucleotide with an adenine or thymine base.
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO:1. Preferably, a pair of amplification primers is capable of amplifying any region between residues 1251 and 2050 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1279 and 1998 (Table 2, SEQ ID NO: 2).
SEQ ID NO : 2
12 7 9 ( 5 1 )
GGTAAATCCGGTACCTTTTAACGCTGAGGTGTGATGACGAGGCACTACGG TGCTGAAGTAACAAATGCCCTGCTTCCAGGAAAAGCCTCTAAGCATCAGG TAACAYSAAATCGTACCCCAAACCGACACAGGTGGTCAGGTAGAGAATAC CAAGGCGCTTGAGAGAACTCGGGTGAAGGAACTAGGCAAAATGGTGCCGT AACTTCGGGAGAAGGCACGCTGATATGTAGGTGAAGCCCCTGCGGGTGGA GCTGAAATCAGTCGAAGATACCAGCTGGCTGCAACTGTTTATTAAAAACA CAGCACTGTGCAAACACGAAAGTGGACGTATACGGTGTGACGCCTGCCCG GTGCCGGAAGGTTAATTGATGGGGTTAGCGGYAACGCGAAGCTCTTGATC GAAGCCCCGGTAAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAA ATTCCTTGTCGGGTAAGTTCCGACCTGCACGAATGGCGTAATGATGGCCA GGCTGTCTCCACCCGAGACTCAGTGAAATTGAACTCGCTGTGAAGATGCA GTGTACCCGCGGCAAGACGGAAAGACCCCGTGAACCTTTACTATAGCTTG ACACTGAACACTGGTCCTTGATGTGTAGGATAGGTGGGAGGCTTTGAAGC GTGGACGCCAGTCTGCGTGGAGCCGTCCTTGAAATACCACCCTTTAATGG CTGGTGTTCTAACGTAGACC
1998 (31 Table 2: Sequence of distinct region of 23S RNA gene of Enterobacter cloacae. An example of a substitution according to the invention is T1502C (underlined).
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 1279 and 1998 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1279 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1998 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting Enterobacter cloacae comprise the sequences in Table 3. Combinations of forwards (F) and reverse (R) primers include SEQ ID NO: 3 (F) and SEQ ID NO: 4 (R); SEQ ID NO: 5 (F) and SEQ ID NO: 6 (R) as indicated in Table 3, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000026_0001
Table 3: Amplification primer examples for amplifying distinct region of 23S RNA gene of Enterobacter cloacae, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length. A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 1, or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 1279 and 1998 inclusive (SEQ ID NO: 2), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1279 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1998 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting Enterobacter cloacae comprise the sequences represented by SEQ ID NOs: 3 to 6, and the complements thereof.
Another aspect of the invention is a method for identifying Enterobacter cloacae by amplification of nucleic acid using primer pairs of Table 3, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 3 to 6. Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 3.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
2. Enterococcus faecalis
According to one aspect of the invention a distinct region of Enterococcus faecalis 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 7 or 8) indicated in Tables 4 and 5. According to another aspect of the invention, a distinct region is an homologous sequence of said SEQ ID NOs.
SEQ ID NO : 7
1259 ( 5 1 )
CATGCGATTGGAAGTGCATGTCCAAGCAATGAGTCTTGAGTAGAGTTAAATGCTTTACTC TTTAAGGACAAGTTGTGAYGGGGAGCGAAATAATAGTAGCGAAGTTCCTGATGTCACACT GCCAAGAAAAGCTTCTAGTGAGAAAACAACTGCCCGTACCGTAAACCGACACAGGTAGTC GAGGAGAGTATCCTAAGGTGAGCGAGCGAACTCTCGTTAAGGAACTCGGCAAAATGACCC CGTAACTTCGGGAGAAGGGGTGCTGACTTCGGTCAGCCGCAGTGAATAGGCCCAAGCGAC TGTTTATCAAAAACACAGGTCTCTGCAAAATCGTAAGATGAAGTATAGGGGCTGACGCCT GCCCGGTGCTGGAAGGTTAAGAGGATGGGTTAGCTTCGGCGAAGCTCAGAATTGAAGCCC CAGTAAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAG TTCCGACCCGCACGAAAGGCGTAACGATTTGGGCACTGTCTCAACGAGAGACTCGGTGAA ATTTTAGTACCTGTGAAGATGCAGGTTACCCGCGACAGGACGGAAAGACCCCATGGAGCT TTACTGTAGTTTGATATTGAGTGTTTGTACCACATGTACAGGATAGGTAGGAGCCGATGA GACCGGAACGCTAGTTTCGGAGGAGGCGCTGGTGGGATACTACCCTTGTGTTATGAACCC
1978 (31)
Table 4: Sequence of distinct region of 23S RNA gene of Enterococcus faecalis. Examples of substitutions according to the invention are Tl 320Y and/or Y1337C (underlined), where Y is a nucleotide with a pyrimidine base.
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 7. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1259 and 1978 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1268 and 1940 (Table 5, SEQ ID NO: 8).
SEQ ID NO :
12 68 ( 5 1 )
GGAAGTGCATGTCCAAGCAATGAGTCTTGAGTAGAGTTAAATGCTTTACTC
TTTAAGGACAAGTTGTGAYGGGGAGCGAAATAATAGTAGCGAAGTTCCTGATGTCACACT
GCCAAGAAAAGCTTCTAGTGAGAAAACAACTGCCCGTACCGTAAACCGACACAGGTAGTC
GAGGAGAGTATCCTAAGGTGAGCGAGCGAACTCTCGTTAAGGAACTCGGCAAAATGACCC
CGTAACTTCGGGAGAAGGGGTGCTGACTTCGGTCAGCCGCAGTGAATAGGCCCAAGCGAC
TGTTTATCAAAAACACAGGTCTCTGCAAAATCGTAAGATGAAGTATAGGGGCTGACGCCT
GCCCGGTGCTGGAAGGTTAAGAGGATGGGTTAGCTTCGGCGAAGCTCAGAATTGAAGCCC
CAGTAAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAG
TTCCGACCCGCACGAAAGGCGTAACGATTTGGGCACTGTCTCAACGAGAGACTCGGTGAA
ATTTTAGTACCTGTGAAGATGCAGGTTACCCGCGACAGGACGGAAAGACCCCATGGAGCT
TTACTGTAGTTTGATATTGAGTGTTTGTACCACATGTACAGGATAGGTAGGAGCCGATGA
GACCGGAACGCTAGTTTCGG
1940 (31 ) Table 5: Sequence of distinct region of 23S RNA gene of Enterococcus faecalis. Examples of substitutions according to the invention are Tl 320Y and/or Y1337C (underlined).
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 1268 and 1940 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1268 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1940 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting Enterococcus faecalis comprise the sequences in Table 6. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 9 (F) and 11 (R); SEQ ID NOs: 9 (F) and 12 (R); SEQ ID NOs: 13 (F) and 14 (R); SEQ ID NOs: 15 (F) and 12 (R); SEQ ID NOs: 15 (F) and 11 (R) as indicated in Table 6, though other primer pair combinations are possible given the similarity in melting temperatures. Such combination may be present in a composition.
Figure imgf000028_0001
Table 6: Amplification primer examples for amplifying distinct region of 23S RNA gene of Enterococcus faecalis, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:7, or the complement thereof. According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 1279 and 1998 inclusive (SEQ ID NO: 8), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1279 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1998 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting Enterococcus faecalis comprise the sequences represented by SEQ ID NOs: 9 to 15, and the complements thereof.
Another aspect of the invention is a method for identifying Enterococcus faecalis by amplification of nucleic acid using primers pairs of Table 6, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 9 to 15.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 6.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
3. Enterococcus faecium
According to one aspect of the invention a distinct region of Enterococcus faecium 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 16 or 17) indicated in Tables 7 and 8. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 1 6
1381 ( 5 1 )
GCCGAGAAAAGCTTCTAGTGAGAAAACAGCGGCCCGTACCGCAAACCGACACAGGTAGTC GAGGAGAGAATCCTAAGGTGAGCGAGAGAACTCTCGTTAAGGAACTCGGCAAAATGACCC CGTAACTTCGGGAGAAGGGGTGCTGATCATACGATCAGCCGCAGTGAATAGGCCCAAGCG
1560 (31 )
Table 7: Sequence of distinct region of 23S RNA gene of Enterococcus faecium A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 16. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1381 and 1560 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1392 and 1547 (Table 8, SEQ ID NO: 17).
SEQ ID NO : 1 7
13 92 ( 5 1 )
CTTCTAGTGAGAAAACAGCGGCCCGTACCGCAAACCGACACAGGTAGTC GAGGAGAGAATCCTAAGGTGAGCGAGAGAACTCTCGTTAAGGAACTCGGCAAAATGACCC CGTAACTTCGGGAGAAGGGGTGCTGATCATACGATCAGCCGCAGTGA
I 1547 (31)
Table 8: Sequence of distinct region of 23S RNA gene of Enterococcus faecium
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 1392 and 1547 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1392 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1547 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting Enterococcus faecium comprise the sequences in Table 9. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 18 and 19; SEQ ID NOs: 19 and 20; SEQ ID NOs: 20 and 21 as indicated in Table 9, though other primer pair combinations are possible given the similarity in melting temperatures.
Figure imgf000030_0001
Table 9: Amplification primer examples for amplifying distinct region of 23S
RNA gene of Enterococcus faecium, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 16, or the complement thereof. According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 1392 and 1547 inclusive (SEQ ID NO: 17), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1392 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1547 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting Enterococcus faecium comprise the sequences represented by any of SEQ ID NOs: 18 to 21, and the complements thereof.
Another aspect of the invention is a method for identifying Enterococcus faecium by amplification of nucleic acid using primers pairs of Table 9, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 18 to 21.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 9.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
4. Escherichia coli
According to one aspect of the invention a distinct region of Escherichia coli 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 22 or 23) indicated in Tables 10 and 11. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof. SEQ ID NO : 22
12 01 ( 5 1 )
GGGACGGAGAAGGCTATGTTGGCCGGGCGACGGTTGTCCCGGTTTAAGCGTGTAGGCTGG TTTTCCAGGCAAATCCGGAAAACCAAGGCTGAGGCGTGATGACGAGGCACTACGGTGCTG AAGCGACAAATGCCCTGCTTCCAGGAAAAGCCTCTAAGCATCAGGTAACATCAAATCGTA CCCCAAACCGACACAGGTGGTCAGGTAGAGAATACCAAGGCGCTTGAGAGAACTCGGGTG AAGGAACTAGGCAAAATGGTGCCGTAACTTCGGGAGAAGGCACGCTGATATGTAGGTGAA GCGACTTGCTCGTGGAGCTGAAATCAGTCGAAGATACCAGCTGGCTGCAACTGTTTATTA AAAACACAGCACTGTGCAAACACGAAAGTGGACGTATACGGTGTGACGCCTGCCCGGTGC CGGAAGGTTAATTGATGGGGTTAGCGGTAACGCGAAGCTCTTGATCGAAGCCCCGGTAAA CGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGAC 1740 ( 3 ' )
Table 10: Sequence of distinct region of 23S RNA gene of Escherichia coli.
An example of a deletion according to the invention is deletion of G1294 (underlined).
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 22. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1201 and 1740 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1265 and 1667 (Table 11, SEQ ID NO: 23).
SEQ ID NO : 23
12 65 ( 5 1 )
CCAGGCAAATCCGGAAAACCAAGGCTGAGGCGTGATGACGAGGCACTACGGTGCTG AAGCGACAAATGCCCTGCTTCCAGGAAAAGCCTCTAAGCATCAGGTAACATCAAATCGTA CCCCAAACCGACACAGGTGGTCAGGTAGAGAATACCAAGGCGCTTGAGAGAACTCGGGTG AAGGAACTAGGCAAAATGGTGCCGTAACTTCGGGAGAAGGCACGCTGATATGTAGGTGAA GCGACTTGCTCGTGGAGCTGAAATCAGTCGAAGATACCAGCTGGCTGCAACTGTTTATTA AAAACACAGCACTGTGCAAACACGAAAGTGGACGTATACGGTGTGACGCCTGCCCGGTGC CGGAAGGTTAATTGATGGGGTTAGCGGTAACGCGAAGCTCTTGATCG
I 1667 (31) Table 11 : Sequence of distinct region of 23S RNA gene of Escherichia coli.
An example of a deletion according to the invention is deletion of G1294 (underlined). A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 1265 and 1667 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1265 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1667 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting Escherichia coli comprise the sequences in Table 12. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 24 (F) and 25 (R); SEQ ID NOs: 24 (F) and 26 (R); SEQ ID NOs: 27 (F) and 29 (R); SEQ ID NOs: 28 (F) and 29 (R) as indicated in Table 12, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000033_0001
Table 12: Amplification primer examples for amplifying distinct region of
23S RNA gene of Escherichia coli, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:22, or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 1265 and 1667 inclusive (SEQ ID NO: 23), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1265 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1667 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting Escherichia coli comprise the sequences represented by SEQ ID NOs: 24 to 29, and the complements thereof.
Another aspect of the invention is a method for identifying Escherichia coli by amplification of nucleic acid using primers pairs of Table 12, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 24 to 29.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 12. Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
5. Klebsiella pneumoniae
According to one aspect of the invention a distinct region of Klebsiella pneumoniae 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 30 or 31) indicated in Tables 13 and 14. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 30 1251 ( 5 1 )
CGGTTGTCCCGGTTTAAGCATGTAGGCTGGTTRTCCAGGCAAATCCGGAT AATCAAGGCTGAGGTGTGATGACGAGGCACTACGGTGCTGAAGTAACAAA TGCCCTGCTTCCAGGAAAAGCCTCTAAGCATCAGGTAACATYAAATCGTA CCCCAAACCGACACAGGTGGTCAGGTAGAGAATACCAAGGCGCTTGAGAG AACTCGGGTGAAGGAACTAGGCAAAATGGTGCCGTAACTTCGGGAGAAGG CACGCTGGTGTGTAGGTGAAGYCCCTGCGGRTGGAGCTGAGACCAGTCGA AGATACCAGCTGGCTGCAACTGTTTATTAAAAACACAGCACTGTGCAAAC
I 1600 (3' )
Table 13: Sequence of distinct region of 23S RNA gene of Klebsiella pneumoniae, wherein R (underlined) is G or A and Y (underlined) is C or T. Example of a substitution according to the invention is C1354T (underlined).
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 30. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1251 and 1600 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1281and 1560 (Table 14, SEQ ID NO: 31).
SEQ ID NO : 31
12 81 ( 5 1 )
TTRTCCAGGCAAATCCGGAT
AATCAAGGCTGAGGTGTGATGACGAGGCACTACGGTGCTGAAGTAACAAA
TGCCCTGCTTCCAGGAAAAGCCTCTAAGCATCAGGTAACATYAAATCGTA
CCCCAAACCGACACAGGTGGTCAGGTAGAGAATACCAAGGCGCTTGAGAG
AACTCGGGTGAAGGAACTAGGCAAAATGGTGCCGTAACTTCGGGAGAAGG
CACGCTGGTGTGTAGGTGAAGYCCCTGCGGRTGGAGCTGAGACCAGTCGA
Figure imgf000035_0001
Table 14: Sequence of distinct region of 23S RNA gene of Klebsiella pneumoniae, wherein R (underlined) is any purine (G or A) and Y (underlined) is any pyrimidine (C or T). Example of a substitution according to the invention is C1354T (underlined).
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 1281 and 1560 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1281 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1560 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting Klebsiella pneumoniae comprise the sequences in Table 15. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 32 (F) and 34 (R); SEQ ID NOs: 32 (F) and 33 (R); SEQ ID NOs: 35 (F) and 36 (R); SEQ ID NOs: 37 (F) and 33 (R) as indicated in Table 12, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000035_0002
Table 15: Amplification primer examples for amplifying distinct region of 23S RNA gene of Klebsiella pneumoniae, length and temperature. Note R (underlined) is any purine (G or A) A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:30, or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 1281 and 1560 inclusive (SEQ ID NO: 31), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1281 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1560 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting Klebsiella pneumoniae comprise the sequences represented by any of SEQ ID NOs: 32 to 37, and the complements thereof.
Another aspect of the invention is a method for identifying Klebsiella pneumoniae by amplification of nucleic acid using primers pairs of Table 15, in the combination indicated or other suitable combination of forward and reverse primers.. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 32 to 37.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 15. Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
6. Pseudomonas aeruginosa
According to one aspect of the invention a distinct region of Pseudomonas aeruginosa 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 38 or 39) indicated in Tables 16 and 17. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 38
51 ( 5 ' )
TCATTGATTTTAGCGGAACGCTCTGGAAAGTGCGGCCATAGTGGGTGATA GCCCCGTACGCGAAAGGATCTTTGAAGTGAAATCGAGTAGGACGGAGCAC GAGAAACTTTGTCTGAACATGGGGGGACCATCCTCCAAGGCTAAATACTA CTGACTGACCGATAGTGAACCAGTACCGTGAGGGAAAGGCGAAAAGAACC CCGGAGAGGGGAGTGAAATAGAACCTGAAACCGTATGCGTACAAGCAGTG GGAGCCTACTTGTTAGGTGACTGCGTACCTTTTGTATAATGGGTCAGCGA CTTATATTCAGTGGCAAGCTTAATCGTATAGGGTAGGCGTAGCGAAAGCG AGTCTTAATAGGGCGTTTAGTCGCTGGGTATAGACCCGAAACCGGGCGAT CTATCCATGAGCAGGTTGAAGGTTAGGTAACACTGACTGGAGGACCGAAC CCACTCCCGTTGAAAAGGTAGGGGATGACTTGTGGATCGGAGTGAAAGGC TAATCAAGCTCGGAGATAGCTGGTTCTCCTCGAAAGCTATTTAGGTAGCG CCTCATGTATCACTCTGGGGGGTAGAGCACTGTTTCGGCTAGGGGGTCAT CCCGACTTACCAAACCGATGCAAACTCCGAATACCCAGAAGTGCCGAGCA TGGGAGACACACGGCGGGTGCTAACGTCCGTCGTGAAAAGGGAAACAACC
I 750 (3')
Table 16: Sequence of distinct region of 23S RNA gene of Pseudomonas aeruginosa. Example of a substitution according to the invention is T374C (underlined).
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 38. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 51 and 750 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 104 and 704 (Table 17, SEQ ID NO: 39).
SEQ ID NO : 39 104 ( 5 1 )
CCGTACGCGAAAGGATCTTTGAAGTGAAATCGAGTAGGACGGAGCAC
GAGAAACTTTGTCTGAACATGGGGGGACCATCCTCCAAGGCTAAATACTA
CTGACTGACCGATAGTGAACCAGTACCGTGAGGGAAAGGCGAAAAGAACC
CCGGAGAGGGGAGTGAAATAGAACCTGAAACCGTATGCGTACAAGCAGTG
GGAGCCTACTTGTTAGGTGACTGCGTACCTTTTGTATAATGGGTCAGCGA
CTTATATTCAGTGGCAAGCTTAATCGTATAGGGTAGGCGTAGCGAAAGCG
AGTCTTAATAGGGCGTTTAGTCGCTGGGTATAGACCCGAAACCGGGCGAT
CTATCCATGAGCAGGTTGAAGGTTAGGTAACACTGACTGGAGGACCGAAC
CCACTCCCGTTGAAAAGGTAGGGGATGACTTGTGGATCGGAGTGAAAGGC
TAATCAAGCTCGGAGATAGCTGGTTCTCCTCGAAAGCTATTTAGGTAGCG
CCTCATGTATCACTCTGGGGGGTAGAGCACTGTTTCGGCTAGGGGGTCAT
CCCGACTTACCAAACCGATGCAAACTCCGAATACCCAGAAGTGCCGAGCA
TGGG
704 (31)
Table 17: Sequence of distinct region of 23S RNA gene of Pseudomonas aeruginosa. Example of a substitution according to the invention is T374C (underlined).
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 104 and 704 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 104 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 704 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting Pseudomonas aeruginosa comprise the sequences in Table 18. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 40 (F) and 41 (R); SEQ ID NOs: 40 (F) and 42 (R) as indicated in Table 18, though other combinations are possible given the similarity in melting temperatures. Such combination may be present in a composition.
Figure imgf000038_0001
Table 18: Amplification primer examples for amplifying distinct region of 23S RNA gene of Pseudomonas aeruginosa, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:38, or the complement thereof. According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 104 and 704 inclusive (SEQ ID NO: 39), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 104 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 704 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting Pseudomonas aeruginosa comprise the sequences represented by any of SEQ ID NOs: 40 to 42, and the complements thereof.
Another aspect of the invention is a method for identifying Pseudomonas aeruginosa by amplification of nucleic acid using primers pairs of Table 18, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a sequence corresponding to any of SEQ ID NOs: 40 to 42.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 18.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 7. Staphylococcus aureus
According to one aspect of the invention a distinct region of Staphylococcus aureus 23S RNA gene comprises a nucleotide sequence (SEQ ID NO: 43 or 44) indicated in Tables 19 and 20. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 43
1021 ( 5 1 )
TAGGAGAGCGTTCTAAGGGCGTTGAAGCATGATCGTAAGGACATGTGGAGCGCTTAGAAG TGAGAATGCCGGTGTGAGTAGCGAAAGACGGGTGAGAATCCCGTCCACCGATTGACTAAG GTTTCCAGAGGAAGGCTCGTCCGCTCTGGGTTAGTCGGGTCCTAAGCTGAGGCCGACAGG CGTAGGCGATGGATAACAGGTTGATATTCCTGTACCACCTATAATCGTTTTAATCGATGG GGGGACGCAGTAGGATAGGCGAAGCGTGCGATTGGATTGCACGTCTAAGCAGTAAGGCTG
I 1320 (3' )
Table 19: Sequence of distinct region of 23S RNA gene of Staphylococcus aureus
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 43. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1021 and 1320 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1037 and 1263 (Table 20, SEQ ID NO: 44).
SEQ ID NO : 4 4 1037 ( 5 1 )
GGGCGTTGAAGCATGATCGTAAGGACATGTGGAGCGCTTAGAAG
TGAGAATGCCGGTGTGAGTAGCGAAAGACGGGTGAGAATCCCGTCCACCGATTGACTAAG
GTTTCCAGAGGAAGGCTCGTCCGCTCTGGGTTAGTCGGGTCCTAAGCTGAGGCCGACAGG
CGTAGGCGATGGATAACAGGTTGATATTCCTGTACCACCTATAATCGTTTTAATCGATGG
GGG
I
1263(3')
Table 20: Sequence of distinct region of 23S RNA gene of Staphylococcus aureus
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 1037 and 1263 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1037 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1263 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting Staphylococcus aureus comprise the sequences in Table 21. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 45 (F) and 46 (R); SEQ ID NOs: 48 (F) and 47 (R); SEQ ID NOs: 48 (F) and 49 (R); SEQ ID NOs: 48 (F) and 51 (R); SEQ ID NOs: 50 (F) and 51 (R) as indicated in Table 21, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000040_0001
Table 21: Amplification primer examples for amplifying distinct region of
23S RNA gene of Staphylococcus aureus, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:43, or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 1037 and 1263 inclusive (SEQ ID NO: 44), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1037 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1263 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting Staphylococcus aureus comprise the sequences represented by any of SEQ ID NOs: 45 to 51, and the complements thereof.
Another aspect of the invention is a method for identifying Staphylococcus aureus by amplification of nucleic acid using primers pairs of Table 21, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 45 to 51. Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 21.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
8. Staphylococcus epidermidis
According to one aspect of the invention a distinct region of Staphylococcus epidermidis 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 52 or 53) indicated in Tables 22 and 23. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 52
501 ( 5 1 )
CAAACTGCCCGCCTGACACTGTCTCCCACCACGATAAGTGGTGCGGGTTA GAAAGCCAACACAGCTAGGGTAGTATCCCACCAACGCCTCCACGTAAGCT AGCGCTCACGTTTCAAAGGCTCCTACCTATCCTGTACAAGCTGTGCCGAA TTTCAATATCAGGCTACAGTAAAGCTCCACGGGGTCTTTCCGTCCTGTCG CGGGTAACCTGCATCTTCACAGGTACTATGATTTCACCGAGTCTCTCGTT GAGACAGTGCCCAAATCGTTACGCCTTTCGTGCGGGTCGGAACTTACCCG ACAAGGAATTTCGCTACCTTAGGACCGTTATAGTTACGGCCGCCGTTTAC TGGGGCTTTGATTCGTAGCTTCGCAGAAGCTAACCACTCCTCTTAACCTT CCAGCACCGGGCAGGCGTCAGCCCCTATACATCACCTTACGGTTTAGCAG AGACCTGTGTTTTTGATAAACAGTCGCTTGGGCCTATTCACTGCGGCTCT TCTGGGCGTGAACCCTAAAGAGCACCCCTTCTCCCGAAGTTACGGGGTCA
I 1050 (3' )
Table 22: Sequence of distinct region of 23S RNA gene of Staphylococcus epidermidis. Example of a substitution according to the invention is T859C (underlined).
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 52 Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 501 and 1050 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1037 and 1263 (Table 23, SEQ ID NO: 53).
SEQ ID NO : 53
1037 ( 5 1 )
GCTAGGGTAGTATCCCACCAACGCCTCCACGTAAGCT
AGCGCTCACGTTTCAAAGGCTCCTACCTATCCTGTACAAGCTGTGCCGAA
TTTCAATATCAGGCTACAGTAAAGCTCCACGGGGTCTTTCCGTCCTGTCG
CGGGTAACCTGCATCTTCACAGGTACTATGATTTCACCGAGTCTCTCGTT
GAGACAGTGCCCAAATCGTTACGCCTTTCGTGCGGGTCGGAACTTACCCG
ACAAGGAATTTCGCTACCTTAGGACCGTTATAGTTACGGCCGCCGTTTAC
TGGGGCTTTGATTCGTAGCTTCGCAGAAGCTAACCACTCCTCTTAACCTT
CCAGCACCGGGCAGGCGTCAGCCCCTATACATCACCTTACGGTTTAGCAG
AGACCTGTGTTTTTGATAAACAGTCGCTTGGGCCTATTCACTGCGGCTCT
TCTGGGCGTGAACCCTAAAGAGCACCCCT
1263(3' )
Table 23: Sequence of distinct region of 23S RNA gene of Staphylococcus epidermidis. Example of a substitution according to the invention is T859C (underlined).
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 1037 and 1263 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 1037 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1263 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting Staphylococcus epidermidis comprise the sequences in Table 24. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 54 (F) and 55 (R); SEQ ID NOs: 54 (F) and 56 (R); SEQ ID NOs: 54 (F) and 57 (R); SEQ ID NOs: 58 (F) and 57 (R); SEQ ID NOs: 58 (F) and 59 (R); SEQ ID NOs: 58 (F) and 60 (R); SEQ ID NOs: 58 (F) and 61 (R); SEQ ID NOs: 58 (F) and 62 (R); SEQ ID NOs: 63 (F) and 59 (R); SEQ ID NOs: 63 (F) and 60 (R); SEQ ID NOs: 63 (F) and 61 (R) as indicated in Table 24, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000042_0001
Figure imgf000043_0001
Table 24: Amplification primer examples for amplifying distinct region of 23S RNA gene of Staphylococcus epidermidis, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:52, or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 1037 and 1263 inclusive (SEQ ID NO: 53), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 1037 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 1263 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting Staphylococcus epidermidis comprise the sequences represented by any of SEQ ID NOs: 54 to 63, and the complements thereof.
Another aspect of the invention is a method for identifying Staphylococcus epidermidis by amplification of nucleic acid using primers pairs of Table 24, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 54 to 63.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 24.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
9. Candida albicans
According to one aspect of the invention a distinct region of Candida albicans 23S RNA gene comprises a nucleotide sequence (SEQ ID NOs: 64 or 65) indicated in Tables 25 and 26. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 64
18 1 ( 5 1 )
TCCTTGGAACAGGACGTCACAGAGGGTGAGAATCCCGTGCGATGAGATGACCCGGGTCTG TGTAAAGTTCCTTYGACGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAAT TCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGA TGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCTT GAGATCAGACTTGGTATTTTGCATGYTGCTCTCTCGGGGGCGGCCGCTGCGGTTTACCGG GCCAGCATCGGTTTGGAGCGGCAGGATAATGGCGGAGGAATGTGGCACGGCTTCTGCTGT GTGTTATAGCCTCTGACGATACTGCCAGCCTAGACCGAGGACTGCGGTTTTTXXACCTAG GATGTTGGCATAATGATCTTAAGTCGCCCGTCTTGAAACACGGACCAAGGAGTCTAACGT CTATGCGAGTGTTTGGGTGTAAAACCCGTACGCGTAATGAAAGTGAACGAAGGTGGGGGC CCATTAGGGTGCACCATCGACCGATCCTGATGTGTTCGGATGGATTTGAGTAAGAGCATA
778 (31)
Table 25: Sequence of distinct region of 23S RNA gene of Candida albicans. Y is a nucleotide with a pyrimidine base. The nucleotides "XX" may both be absent, may be "TT" or may be a single nucleotide "A".
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 64. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 181 and 778 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 214 and 739 (Table 26, SEQ ID NO: 65). SEQ ID NO: 65
214 (51)
CCCGTGCGATGAGATGACCCGGGTCTG
TGTAAAGTTCCTTYGACGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAAT
TCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGA
TGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCTT
GAGATCAGACTTGGTATTTTGCATGYTGCTCTCTCGGGGGCGGCCGCTGCGGTTTACCGG
GCCAGCATCGGTTTGGAGCGGCAGGATAATGGCGGAGGAATGTGGCACGGCTTCTGCTGT
GTGTTATAGCCTCTGACGATACTGCCAGCCTAGACCGAGGACTGCGGTTTTTXXACCTAG
GATGTTGGCATAATGATCTTAAGTCGCCCGTCTTGAAACACGGACCAAGGAGTCTAACGT
CTATGCGAGTGTTTGGGTGTAAAACCCGTACGCGTAATGAAAGTGAACGAAGGTGGGGGC
CCATTAGGGTGCACCATCGAC
739 (31
Table 26: Sequence of distinct region of 23S RNA gene of Candida albicans Y is a nucleotide with a pyrimidine base. The nucleotides "XX" may both be absent, may be "TT" or may be a single nucleotide "A".
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 214 and 739 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 214 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 739 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive.
According to one aspect of the invention, amplification primers suitable for detecting Candida albicans comprise the sequences in Table 27. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 66 and 67; SEQ ID NOs: 68 and 69; SEQ ID NOs: 70 and 71 as indicated in Table 27, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000045_0001
Table 27 Amplification primer examples for amplifying distinct region of 23S RNA gene of Candida albicans, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 64, or the complement thereof. According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 214 and 739 inclusive (SEQ ID NO: 65), or complement thereof. It is within the scope of the invention, that the probes are capable of binding to the region between 214 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 739 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting Candida albicans comprise the sequences represented by any of SEQ ID NOs: 66 to 71, and the complements thereof.
Another aspect of the invention is a method for identifying Candida albicans by amplification of nucleic acid using primers pairs of Table 27, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 66 to 71.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 27.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
10. blages-2 (beta-lactam resistance gene)
According to one aspect of the invention a distinct region of a blages-2 gene comprises a nucleotide sequence (SEQ ID NOs: 72 or 73) indicated in Tables 28 and 29. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
Figure imgf000046_0001
AATTGACTCAGGCACCGAGCGGGGGGATCGAAAACTTTCATATGGGCCGG ACATGATCGTCRAATGGTCTCCTGCCACGGAGCGGTTTCTAGCATCGGGA CACATGACGGTTCTCGAGGCAGCGCAAGCTGCGGTGCAGCTTAGCGACAA TGGGGCTACTAACCTCTTACTGAGAGAAATTGGCGGACCTGCTGCAATGA CGCAGTATTTTCGTAAAATTGGCGACTCTGTGAGTCGGCTAGACCGGAAA GAGCCGGAGATGRGCGACAACACACCTGGCGACCTCAGAGATACAACTAC GCCTATTGCTATGGCACGTACTGTGGCTAAAGTCCTCTATGGCGGCGCAC TGACGTCCACCTCGACCCACACCATTGAGAGGTGGCTGATCGGAAACCAA ACGGGAGACGCGACACTACGAGCGGGTTTTCCTAAAGATTGGGTTGTTGG AGAGAAAACTGGTACCTGCGCCAACGGGGGCCGGAACGACATTGGTTTTT TTAAAGCCCAGGAGAGAGATTACGCTGTAGCGGTGTATACAACGGCCCCG AAACTATCGGCCGTAGAACGTGACGAATTAGTTGCCTCTGTCGGTCAAGT TAT
I
653(3' )
Table 28: Sequence of distinct region of blages.2 gene. R (underlined) is any purine (G or A). Examples of deletions according to the invention include deleting of any of Rl 12 and/or R313.
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 72. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 653 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 140 and 482 (Table 26, SEQ ID NO: 73).
SEQ ID NO : 73 14 0 ( 5 1 )
TAGCATCGGGACACATGACGGTTCTCGAGGCAGCGCAAGCTGCGGTGCAGCTTAGCGACAA
TGGGGCTACTAACCTCTTACTGAGAGAAATTGGCGGACCTGCTGCAATGA
CGCAGTATTTTCGTAAAATTGGCGACTCTGTGAGTCGGCTAGACCGGAAA
GAGCCGGAGATGRGCGACAACACACCTGGCGACCTCAGAGATACAACTAC
GCCTATTGCTATGGCACGTACTGTGGCTAAAGTCCTCTATGGCGGCGCAC
TGACGTCCACCTCGACCCACACCATTGAGAGGTGGCTGATCGGAAACCAA
ACGGGAGACGCGACACTACGAGCGGGTTTTCC 482 ( 3 ' ) Table 29: Sequence of distinct region of the blages.2 gene. Note R (underlined) is any purine (G or A). Examples of deletions according to the invention include the deleting any of Rl 12 and/or R313.
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 140 and 482 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 140 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 482 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting the blages.2 gene comprise the sequences in Table 30. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 74 (F) and 75 (R); SEQ ID NOs: 76 (F) and 77 (R) as indicated in Table 30, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000048_0001
Table 30: Amplification primer examples for amplifying a distinct region of the bla.ges-2 gene, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO:72, or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 140 and 482 inclusive (SEQ ID NO: 73), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between 140 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 482 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting the blages.2 gene comprise the sequences represented by any of SEQ ID NOs: 74 to 77, and the complements thereof. Another aspect of the invention is a method for identifying the blages-2 gene by amplification of nucleic acid using primers pairs of Table 30, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 74 to 77.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 30.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
11. blashv (beta-lactam resistance gene) According to one aspect of the invention a distinct region of a blashv gene comprises a nucleotide sequence (SEQ ID NO: 78 or 79) indicated in Tables 31 and 32. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 78 K 5 1 )
GTAGGCATGATAGAAATGGATCTGGCCAGCGGCCGCACGCTGACCGCCTG GCGCGCCGATGAACGCTTTCCCATGATGAGCACCTTTAAAGTAGTGCTCT GCGGCGCAGTGCTGGCGCGGGTGGATGCCGGTGACGAACAGCTGGAGCGA AAGATCCACTATCGCCAGCAGGATCTGGTGGACTACTCGCCGGTCAGCGA AAAACACCTTGCCGACGGCATGACGGTCGGCGAACTCTGYGCCGCCGCCA TTACCATGAGCGATAACAGCGCCGCCAATCTGCTGCTGGCCACCGTCGGC GGCCCCGCAGGATTGACTGCCTTTTTGCGCCAGATCGGCGACAACGTCAC CCGCCTTGACCGCTGGGAAACGGAACTGAATGAGGCGCTTCCCGGCGACG CCCGCGACACCACTACCCCGGCCAGCATGGCCGCGACCCTGCGCAAGCTG CTGACCAGCCAGCGTCTGAGCGCCCGTTCGCAACGGCAGCTGCTGCAGTG GATGGTGGACGATCGGGTCGCCGGACCGTTGATCCGCTCCGTGCTGCCGG CGGGCTGGTTTATCGCCGATAAGACCGGAGCTRGCGARCGGGGTGCGCGC GGGATTGTCGCCCTGCTTGGCCCGAATAACAAAGCAGAGCGCATTGTGGT GATTTATCTGCGGGATACS-CCGGCGAGCATGGCCGAGCGAAAT
I 693(3' )
Table 31: Sequence of distinct region of blashv gene. Note Y (underlined) is any pyrimidine (C or T), R (underlined) is any purine (A or G), S is a (C or G). Examples of deletions according to the invention includes the deletion of one or more of Y240, R583, R588 and S669. A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 78. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 693 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 149 and 350 (Table 32, SEQ ID NO: 79).
SEQ ID NO : 7 9
14 9 ( 5 ' )
GAAAGATCCACTATCGCCAGCAGGATCTGGTGGACTACTCGCCGGTCAGCGA AAAACACCTTGCCGACGGCATGACGGTCGGCGAACTCTGYGCCGCCGCCA TTACCATGAGCGATAACAGCGCCGCCAATCTGCTGCTGGCCACCGTCGGC GGCCCCGCAGGATTGACTGCCTTTTTGCGCCAGATCGGCGACAACGTCAC
I 350 ( 3 1 )
Table 32: Sequence of distinct region of the blashv gene. Note Y (underlined) is any pyrimidine (C or T). Note Y (underlined) is any pyrimidine (C or T), R (underlined) is any purine (A or G), S is a (C or G). Examples of deletions according to the invention includes the deletion of one or more of Y240, R583, R588 and S669.
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 149 and 350 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 149 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 350 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting the blas/,v gene comprise the sequences in Table 33. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 80 and 81; SEQ ID NOs: 82 and 83 as indicated in Table 33, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000050_0001
Table 33: Amplification primer examples for amplifying a distinct region of the blashv gene, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 78, or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 149 and 350 inclusive (SEQ ID NO: 79), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 149 (±10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 350 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting the blas/,v gene comprise the sequences represented by any of SEQ ID NOs: 80 to 83, and the complements thereof.
Another aspect of the invention is a method for identifying the blashv gene by amplification of nucleic acid using primers pairs of Table 33, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 80 to 83.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 33.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 12. mecA (methicillin resistance gene)
According to one aspect of the invention a distinct region of a mecA gene comprises a nucleotide sequence (SEQ ID NOs: 84 or 85) indicated in Tables 34 and 35. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 84 K 5 1 )
AAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTAT CCACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATG ACGTCTATCCATTTATGTATGGCATGAGTAACGAAGAATATAATAAATTA ACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGATTACAACTTC ACCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACA AAACATTAGACGATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAA AAAGATAAATCTTGGGGTGGTTACAACGTTACAAGATATGAAGTGGTAAA TGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCT TTGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATG AAAAAACTAGGTGTTGGTGAAGATATACCAAGTGATTATCCATTTTATAA TGCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTATTAGCTGATT CAGGTTACGGACAAGGTGAAATACTGATTAACCCAGTACAGATCCTTTCA ATCTATAGCGC
I 611 (3' )
Table 34: Sequence of distinct region of mecA gene. A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 84. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 611 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 184 and 484 (Table 35, SEQ ID NO: 85).
SEQ ID NO : 85
184 ( 5 1 )
AAACATTAGACGATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAA AAAGATAAATCTTGGGGTGGTTACAACGTTACAAGATATGAAGTGGTAAA TGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCT TTGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATG AAAAAACTAGGTGTTGGTGAAGATATACCAAGTG
I
484 (31)
Table 35: Sequence of distinct region of the mecA gene.
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 184 and 484 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 184 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 484 (+10, 9, 8, 7, 6, 5, 4, 3,
2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting the mecA gene comprise the sequences in Table 36.
Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 86 (F) and 87 (R); SEQ ID NOs: 88 (F) and 89 (R) as indicated in Table 36, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000052_0001
Table 36: Amplification primer examples for amplifying a distinct region of the mecA gene, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length. A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 84, or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 149 and 349 inclusive (SEQ ID NO: 85), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 184 (±10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 484 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting the mecA gene comprise the sequences represented by any of SEQ ID NOs: 86 to 89, and the complements thereof. Another aspect of the invention is a method for identifying the mecA gene by amplification of nucleic acid using primers pairs of Table 36, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 86 to 89.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 36.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
13. spA (Staphylococcus-aureus protein A)
According to one aspect of the invention a distinct region of a spA gene comprises a nucleotide sequence (SEQ ID NOs: 90 or 91) indicated in Tables 37 and 38.
According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 90
K 5 1 )
AAAACATTTATTCAATTCGTAAACTAGGTGTAGGTATTGCATCTGTAACT TTAGGTACATTACTTATATCTGGTGGCGTAACACCTGCTGCAAATGCTGC GCAACACGATGAAGCTCAACAAAATGCTTTTTATCAAGTS-TTAAATATGC CTAACTTAAAYGCTGATCAACGYAATGGTTTTATCCAAAGCCTTAAAGAT GATCCAAGCCAAAGTGCTAACGTTTTAGGTGAAGCTCAAAAACTTAATGA CTCTCAAGCTCCAAAAGCTGATGCGCAACAAAATAAS-TTCAACAAAGATC AACAAAGCGCCTTCTATGAAATCTTGAACATGCCTAACTTAAACGAAGHG CAACGYAAYGGYTTCATTCAAAGTCTTAAAGACGACYCAAGCCAAAGCAC TAACGTTTTAGGTGAAGCTAAAAAATTAAACGAATCTCAAGCACCGAAAG CTGAYAACAATTTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTGA
I 501 (31 )
Table 37: Sequence of distinct region of spA gene. Note S is (C or G), Y is pyrimidine (C or T), H is (A, C or T). Example of deletions according to the invention include the deletion of S140, Y161, Y173, S287, H349, Y356, Y359, Y362, Y387 and/or Y455. A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 90 Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 501 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 292 and 409 (Table 38, SEQ ID NO: 91).
SEQ ID NO : 91
292 ( 5 1 )
ACAAAGATCAACAAAGCGCCTTCTATGAAATCTTGAACATGCCTAACTTA AACGAAGHGCAACGYAAYGGYTTCATTCAAAGTCTTAAAGACGACYCAAG CCAAAGCACTAACGTTTT
I
409 (31)
Table 38: Sequence of distinct region of the spA gene. Note Y is pyrimidine (C or T), H is (A, C or T). Example of deletions according to the invention include the deletion of H349, Y356, Y359, Y362 and/or Y387.
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 292 and 409 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 292 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 409 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting the spA gene comprise the sequences in Table 39. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 92 (F) and 93 (R); SEQ ID NOs: 94 (F) and 95 (R) as indicated in Table 39, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000055_0001
Table 39: Amplification primer examples for amplifying a distinct region of the spA gene, length and melting temperature. . TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 90 or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 292 and 409 inclusive (SEQ ID NO: 46), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 292 (±10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 409 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting the spA gene comprise the sequences represented by any of SEQ ID NOs: 92 to 95, and the complements thereof. Another aspect of the invention is a method for identifying the spA gene by amplification of nucleic acid using primers pairs of Table 39, in the combinations indicated or other suitable combination of forward and reverse primers.. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 92 to 95.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 39.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 14. VanA (vancomycin resistance gene A)
According to one aspect of the invention a distinct region of a VanA gene comprises a nucleotide sequence (SEQ ID NOs: 96 or 97) indicated in Tables 40 and 41. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 96 K 5 1 )
AAAGTTGCAATACTGTTTGGGGGTTGCTCAGAGGAGCATGACGTATCGGT AAAATCTGCAATAGAGATAGCCGCTAACATTAATAAAGAAAAATACGAGC CGTTATACATTGGAATTACGAAATCTGGTGTATGGAAAATGTGCGAAAAA CCTTGCGCGGAATGGGAAAACGACAATTGCTATTCAGCTGTACTCTCGCC GGATAAAAAAATGCACGGATTACTTGTTAAAAAGAACCATGAATATGAAA TCAACCATGTTGATGTAGCATTTTCAGCTTTGCATGGCAAGTCAGGTGAA GATGGATCCATACAAGGTCTGTTTGAATTGTCCGGTATCCCTTTTGTAGG CTGCGATATTCAAAGCTCAGCAATTTGTATGGACAAATCGTTGACATACA TCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCTTTTGGGTTATTAAT AAAGATGATAGGCCGGTGGCAGCTACGTTTACCTATCCTGTTTTTGTTAA GCCGGCGCGTTCAGGCTCATCCTTCGGBGTGAAAAAAGTCAATAGCGCGG ACGAATTGGACTACGCAATTGAATCGGCAAGACAATATGACAGCAAAATC TTAATTGAGCAGGCTGTTTCGGGCTGTGAGGTCGGTTGTGCGGTATTGGG AAACAGTGCCGCGTTAGTTGTTGGCGAGGTGGACCAAATCAGGCTGCAGT ACGGAATCTTTCGTATTCATCAGGAAGTCGAGCCGGAAAAAGGCTCTGAA AACGCAGTTATAACCGTTCCCGCAGACCTTTCAGCAGAGGAGCGAGGACG GATACAGGAAACGGCAAAAAAAATATATAAAGCGCTCGGCTGTAGAGGTC TAGCCCGTGTGGATATGTTTTTACAAGATAACGGCCGCATTGTACTGAAC GAAGTCAATACTCTGCCCGGTTTCACGTCATACAGTCGTTATCC 944 ( 3 ' )
Table 40: Sequence of distinct region of VanA gene. Note B is (T, C or G).
Example of a deletion according to the invention includes the deletion of B528. A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 96 Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 944 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 138 and 641 (Table 41, SEQ ID NO: 97).
SEQ ID NO : 97 138 ( 5 1 )
AATGTGCGAAAAA
CCTTGCGCGGAATGGGAAAACGACAATTGCTATTCAGCTGTACTCTCGCC GGATAAAAAAATGCACGGATTACTTGTTAAAAAGAACCATGAATATGAAA TCAACCATGTTGATGTAGCATTTTCAGCTTTGCATGGCAAGTCAGGTGAA GATGGATCCATACAAGGTCTGTTTGAATTGTCCGGTATCCCTTTTGTAGG CTGCGATATTCAAAGCTCAGCAATTTGTATGGACAAATCGTTGACATACA TCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCTTTTGGGTTATTAAT AAAGATGATAGGCCGGTGGCAGCTACGTTTACCTATCCTGTTTTTGTTAA GCCGGCGCGTTCAGGCTCATCCTTCGGBGTGAAAAAAGTCAATAGCGCGG ACGAATTGGACTACGCAATTGAATCGGCAAGACAATATGACAGCAAAATC TTAATTGAGCAGGCTGTTTCGGGCTGTGAGGTCGGTTGTGC
64 1 ( 3 1 )
Table 41: Sequence of distinct region of the VanA gene. Note B is (T, C or G). Example of deletion according to the invention includes the deletion of B528.
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 138 and 641 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 138 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 641 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting the VanA gene comprise the sequences in Table 42. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 98 (F) and 99 (R); SEQ ID NOs: 100 (F) and 101 (R) as indicated in Table 42, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000057_0001
Table 42: Amplification primer examples for amplifying a distinct region of the VanA gene, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 96 or the complement thereof. According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 138 and 641 inclusive (SEQ ID NO: 97), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 138 (±10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 641 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting the VanA gene comprise the sequences represented by any of SEQ ID NOs: 98 to 101, and the complements thereof.
Another aspect of the invention is a method for identifying the VanA gene by amplification of nucleic acid using primers pairs of Table 42, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 98 to 101. Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 42.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
15. VanB (vancomycin resistance gene B)
According to one aspect of the invention a distinct region of a VanB gene comprises a nucleotide sequence (SEQ ID NOs: 102 or 103) indicated in Tables 43 and 44. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 102 K 5 1 )
ATCGGAATTACAAAAAACGGTGTATGGAAGCTATGCAAGAAGCCATGTAC GGAATGGGAAGCCGACAGTCTCCCCGCCATACTCTCCCCGGATAGGAAAA CGCATGGGCTGCTTGTCATGAAAGAAAGCGAATACGAAACACGGCGTATT GATGTGGCTTTCCCGGTTTTGCATGGCAAATGCGGGGAGGATGGTGCGAT ACAGGGGCTGTTTGTATTGTCTGGTATCCCCTATGTGGGCTGTGATATTC AAAGCTCCGCAGCTTGCATGGACAAATCACTGGCCTACATTCTTACAAAA AATGCGGGCATCGCCGTTCCCGAATTTCAAATGATTGATAAAGGTGACAA GCCGGAGGCGGGTGCGCTTACCTACCCTGTCTTTGTGAAGCCGGCACGGT CAGGTTCGTCCTTTGGCBTAACCAAAGTAAACGGTACGGAAGAACTTAAC GCTGCGATAGAAGCGGCAGGACAATATGATGGAAAAATCTTAATTGAGCA AGCGATTTCGGGCTGTGAGGTCGGGTGTGCGGTCATGGGRAACGAGGATG ATTTGATTGTCGGCGAAGTGGATCAAATCCGGCTGAGCCACGGTATCTTC CGCATCCATCAGGAAAACGAGCCGGAAAAAGGCTCAGAAAATGCGATGAT TACAGTTCCCGCAGACATTCCGGTCGAGGAACGAAATCGGGTGCARGAAA CGGCAAAGAAAGTATATCGGGTGCTTGGATGCAGAGGGCTT
Figure imgf000059_0001
Table 43: Sequence of distinct region of VanB gene. Note B is (T, C or G), and R is purine (G or A). Examples of deletions according to the invention include one of more of B418, R540 and R696.
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 102. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 741 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 126 and 574 (Table 44, SEQ ID NO: 103).
SEQ ID NO : 103 12 6 ( 5 ' )
AAGCGAATACGAAACACGGCGTATT
GATGTGGCTTTCCCGGTTTTGCATGGCAAATGCGGGGAGGATGGTGCGAT
ACAGGGGCTGTTTGTATTGTCTGGTATCCCCTATGTGGGCTGTGATATTC
AAAGCTCCGCAGCTTGCATGGACAAATCACTGGCCTACATTCTTACAAAA
AATGCGGGCATCGCCGTTCCCGAATTTCAAATGATTGATAAAGGTGACAA
GCCGGAGGCGGGTGCGCTTACCTACCCTGTCTTTGTGAAGCCGGCACGGT
CAGGTTCGTCCTTTGGCBTAACCAAAGTAAACGGTACGGAAGAACTTAAC
GCTGCGATAGAAGCGGCAGGACAATATGATGGAAAAATCTTAATTGAGCA
AGCGATTTCGGGCTGTGAGGTCGGGTGTGCGGTCATGGGRAACGAGGATG
ATTTGATTGTCGGCGAAGTGGATC
574 (31) Table 44: Sequence of distinct region of the VanB gene. Note B is (T, C or G), and R is purine (G or A). Examples of deletions according to the invention include one of more of B418 and R540.
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 126 and 574 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 126 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 574 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting the VanB gene comprise the sequences in Table 45. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 104 (F) and 105 (R); SEQ ID NOs: 106 (F) and 107 (R) as indicated in Table 45, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition. CODE SEQUENCE /LENGTH Tm (deg C) TYPE PAIR LEN
SEQID NO: 104 TCCCCTATGTGGGCTGTGAT / 20 62 F 105 445
SEQID NO: 105 GGAATGTCTGCGGGAACTGT / 20 62 R 104 445
SEQID NO: 106 AAGCGAATACGAAACACGGC / 20 60 F 107 449
SEQID NO: 107 GATCCACTTCGCCGACAATC / 20 62 R 106 449
Table 45: Amplification primer examples for amplifying a distinct region of the VanB gene, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 102 or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 126 and 574 inclusive (SEQ ID NO: 103), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 126 (±10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) and 574 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues) inclusive. According to an aspect of the invention, probes suitable for detecting the VanB gene comprise the sequences represented by SEQ ID NOs: 102 and 103, and the complements thereof. Another aspect of the invention is a method for identifying the VanB gene by amplification of nucleic acid using primers pairs of Table 45, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NOs: 104 to 107.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 45.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 16. VanC (vancomycin resistance gene C)
According to one aspect of the invention a distinct region of a VanC gene comprises a nucleotide sequence (SEQ ID NOs: 108 or 109) indicated in Tables 46 and 47. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 108 K 5 1 )
GCTTGATACTCAAAATCAACAGTCATCAATGCATTCTCTACGGCAAAGAA GTTTTCCTGACTCCCATTGAGTTCAAAATATTGCTTTATTTATTTGAGCA CCAAGGATCCGTCGTCTCTTCCGAAACACTTTTCGAAGCGGTTTGGAAAG AAAAATATTTAGATAACAATAATACTGTCATGGCACACATTGCTCGTTTA AGAGAAAAATTGCATGAAGAACCTCGTAAACCTAAATTAATCAAAACCGT ATGGGGGGTCGGCTATATCATTGAAAAATAGAAATCCTTTGATCCGAAAG CTCTTGACCCAATACTTCGTCACCACTGGAATCTTGCTGGCATTCCTTGT AATGATTCCATTAGTCATTCGCTTTATTGCCGGAACCCGGACTTGGTATG GAACGGAACCTATCTACTATATCTTACGTTTTTTTGCG
I
438 (31)
Table 46: Sequence of distinct region of VanC gene.
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 108 Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 438 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 27 and 407 (Table 47, SEQ ID NO: 109).
SEQ ID NO : 109 27 ( 5 ' )
CAATGCATTCTCTACGGCAAAGAA
GTTTTCCTGACTCCCATTGAGTTCAAAATATTGCTTTATTTATTTGAGCA CCAAGGATCCGTCGTCTCTTCCGAAACACTTTTCGAAGCGGTTTGGAAAG AAAAATATTTAGATAACAATAATACTGTCATGGCACACATTGCTCGTTTA AGAGAAAAATTGCATGAAGAACCTCGTAAACCTAAATTAATCAAAACCGT ATGGGGGGTCGGCTATATCATTGAAAAATAGAAATCCTTTGATCCGAAAG CTCTTGACCCAATACTTCGTCACCACTGGAATCTTGCTGGCATTCCTTGT AATGATTCCATTAGTCATTCGCTTTATTGCCGGAACCCGGACTTGGTATG GAACGGA I 407 (3' ) Table 47: Sequence of distinct region of the VanC gene. A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 27 and 407 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 27 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 407 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting the VanC gene comprise the sequences in Table 48. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 110 (F) and 111 (R); SEQ ID NOs: 112 (F) and 113 (R) as indicated in Table 48, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000062_0001
Table 48: Amplification primer examples for amplifying a distinct region of the VanC gene, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length.
A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 108 or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 27 and 407 inclusive (SEQ ID NO: 109), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 27 (±10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 407 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive. According to an aspect of the invention, probes suitable for detecting the VanC gene comprise the sequences represented by any of SEQ ID NOs: 110 to 113, and the complements thereof. Another aspect of the invention is a method for identifying the VanC gene by amplification of nucleic acid using primers pairs of Table 48, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NO: 110 to 113.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 48. Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above.
17. MDR-I
According to one aspect of the invention a distinct region of a MDR-I gene comprises a nucleotide sequence (SEQ ID NOs: 114 or 115) indicated in Tables 49 and 50. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO: 114
201 (51)
CCCTCAAAATTGGCCAACTTTACAAAAAGCATTTTTCATTTTCCAAATTT CATTTTTGACAACTTCAGTTTATATGGGATCAGCAGTTTATACCCCTGGT ATTGAAGAATTAATGCATGATTTTGGTATTGGAAGAGTCGTAGCTACATT ACCTTTAACATTATTTGTTATTGGTTATGGTGTTGGCCCATTGGTTTTCA GTCCGATGTCAGAAAATGCTATATTTGGTCGTACATCCATATATATCATA ACATTATTTTTATTTGTCATACTACAAATYCCCACTGCTTTGGTWAATAA TATTGCYGGTTTATGTATATTGAGATTCTTGGGTGGATTCTTTGCTAGTC CTTGTTTGGCYACTGGTGGTGCWAGTGTTGCTGATGTGGTTAAATTTTGG AATTTACCAGTTGGGTTAGCCGCTTGGAGTTTGGGTGCYGTTTGTGGTCC TAGTTTTGGTCCATTCTTTGGTTCAATTTTAACTGTCAAAGCCAGTTGGA GATGGACTTTTTGGTTCATGTGTATYATTTCTGGGTTTTCATTTGTTATG TTGTGTTTCACTTTACCTGAAACTTTTGGCAAAACATTATTRTATCGCAA GGCTAAAAGATTGAGAGCCATCACCGGTAACGACAGAATCACAAGTGAAG GAGAAATTGAAAATAGCAAAATGACAAGTCATGAATTGATCATTGATACA
I 850 (31)
Table 49: Sequence of distinct region of MDR-I gene. Y is any nucleotide with a pyrimidine base, R is any nucleotide with a purine base, W is any nucleotide with an adenine or thymine base.
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 108. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 201 and 850 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 275 and 834 (Table 50, SEQ ID NO: 115).
SEQ ID NO: 115
275 (51)
TGGGATCAGCAGTTTATACCCCTGGT
ATTGAAGAATTAATGCATGATTTTGGTATTGGAAGAGTCGTAGCTACATT ACCTTTAACATTATTTGTTATTGGTTATGGTGTTGGCCCATTGGTTTTCA GTCCGATGTCAGAAAATGCTATATTTGGTCGTACATCCATATATATCATA ACATTATTTTTATTTGTCATACTACAAATYCCCACTGCTTTGGTWAATAA TATTGCYGGTTTATGTATATTGAGATTCTTGGGTGGATTCTTTGCTAGTC CTTGTTTGGCYACTGGTGGTGCWAGTGTTGCTGATGTGGTTAAATTTTGG AATTTACCAGTTGGGTTAGCCGCTTGGAGTTTGGGTGCYGTTTGTGGTCC TAGTTTTGGTCCATTCTTTGGTTCAATTTTAACTGTCAAAGCCAGTTGGA GATGGACTTTTTGGTTCATGTGTATYATTTCTGGGTTTTCATTTGTTATG TTGTGTTTCACTTTACCTGAAACTTTTGGCAAAACATTATTRTATCGCAA GGCTAAAAGATTGAGAGCCATCACCGGTAACGAC
I 834 (31)
Table 50: Sequence of distinct region of the MDR-I gene.
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 275 and 834 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 275 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 834 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting the MDR-I gene comprise the sequences in Table 51. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 116 (F) and 117 (R); SEQ ID NOs: 118 (F) and 119 (R) as indicated in Table 51, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000064_0001
Table 51: Amplification primer examples for amplifying a distinct region of the MDR-I gene, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length. A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 114 or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 275 and 834 inclusive (SEQ ID NO: 115), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 275 (±10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 834 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive. According to an aspect of the invention, probes suitable for detecting the MDR-I gene comprise the sequences represented by any of SEQ ID NOs: 116 to 119, and the complements thereof. Another aspect of the invention is a method for identifying the MDR-I gene by amplification of nucleic acid using primers pairs of Table 51, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NO: 116 to 119.
Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 51.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. 18. CDR l
According to one aspect of the invention a distinct region of a CDR l gene comprises a nucleotide sequence (SEQ ID NOs: 120 or 121) indicated in Tables 52 and 53. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
SEQ ID NO : 120
1 ( 5 1 )
TCTTTTTCTATTGGTTAATGTGTRTTTGGTGTACATTTGTTATGTCCCAT TTGTTTAGATCCATTGGTGCTGTTTCAACATCTATTKCTGGTGCYATGAC YCCTGCTACYGTGTTGTTATTGGCTATGGTTATTTAYACTGGGTTCGTTA TCCCAACTCCAAGTATGTTGGGTTGGTCWMGATGGATTAATTAYATYAAY CCTGTTGGTTATGTGTTYGAAKCS-CTYATGGTTAATGARTTCCAYGGTCG TGAATTCCAATGTGCTCAATATGTTCCAAGTGGYCCAGGTTWTGAAAATR TATCACGTTCRAATCAAGTGTGTACTGCAGTKGGGTCTRTTCCAGGTAAT GAAATGGTTAGTGGTACCAATTATTTGGCTGGTGCTT
387 ( 3 1 )
Table 52: Sequence of distinct region of CDR l gene. Y is any nucleotide with a pyrimidine base, R is any nucleotide with a purine base, K is any nucleotide with a thymine or guanine base, W is any nucleotide with an adenine or thymine base, M is any nucleotide with a cytosine or adenine base, S is any nucleotide with a cytosine or guanine base.
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of SEQ ID NO: 120. Preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 1 and 387 inclusive. Even more preferably, a pair of amplification primers is a pair capable of amplifying any region between residues 111 and 178 (Table 53, SEQ ID NO: 121).
SEQ ID NO : 121
111 ( 5 1 )
GTGTTGTTATTGGCTATGGTTATTTAYACTGGGTTCGTTA TCCCAACTCCAAGTATGTTGGGTTGGT
I
178 (31)
Table 53: Sequence of distinct region of the CDR-I gene. Sequence of distinct region of CDR-I gene. Y is any nucleotide with a pyrimidine base.
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying the region between residues 111 and 178 inclusive. It is within the scope of the invention, that the primers are capable of amplifying the region between residues 111 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 178 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive. According to a preferred aspect of the invention, amplification primers suitable for detecting the CDR l gene comprise the sequences in Table 54. Combinations of forwards (F) and reverse (R) primers include SEQ ID NOs: 122 (F) and 123 (R); SEQ ID NOs: 124 (F) and 125 (R) as indicated in Table 54, though other primer pair combinations are possible given the similarity of melting temperatures. Such combination may be present in a composition.
Figure imgf000066_0001
Figure imgf000067_0001
Table 54: Amplification primer examples for amplifying a distinct region of the CDR-I gene, length and melting temperature. TYPE is either forward (F) or reverse (R) primer, PAIR is a paired primer SEQ ID NO. for amplification, LEN is the amplification product length. A hybridisation probe according to one aspect of the present invention is capable of annealing to SEQ ID NO: 120 or the complement thereof.
According to one embodiment of the invention, hybridisation probe is capable of hybridising to the region between residues 111 and 178 inclusive (SEQ ID NO: 121), or complement thereof. It is within the scope of the invention that the probes are capable of binding to the region between residues 111 (±10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) and 178 (+10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue) inclusive. According to an aspect of the invention, probes suitable for detecting the MDR-I gene comprise the sequences represented by any of SEQ ID NOs: 122 to 125, and the complements thereof.
Another aspect of the invention is a method for identifying the CDR-I gene by amplification of nucleic acid using primers pairs of Table 54, in the combination indicated or other suitable combination of forward and reverse primers. A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification; according to one embodiment of the invention, such hybridisation probe comprises a suitable sequence corresponding to any of SEQ ID NO: 122 to 125. Another aspect of the invention is an oligonucleotide (primer or probe) corresponding to a sequence indicated in Table 54.
Homologous sequences of the above mentioned distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. Figure 1
The Figure shows an alignment of sequences 23S RNA genes of each of Staphylococcus aureus, Staphylococcus epidermidis, Enterobacter cloacae, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Enterococcus faecium, Klebsiella pneumoniae, and Candida albicans, and an indication of identity (marked with *).
According to one aspect of the invention a distinct region of a 23S RNA gene of a micro-organism comprises a nucleotide sequence (SEQ ID NOs: 131 to 157) indicated in Figure 1. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. One aspect of the present invention is nucleotide acid corresponding to a sequence represented by any of SEQ ID NOs: 131 to 157 indicated in Figure 1, or complement thereof. According to one aspect of the invention a distinct region of a 23S RNA gene of a micro-organism comprises a nucleotide sequence indicated in Figure 1, corresponding to a distinct region represented by any of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), 64 and 65 (Candida albicans).
According to one aspect of the invention a distinct region of a 23S RNA gene of a micro-organism comprises a nucleotide sequence indicated in Figure 1, obtainable using a pair of amplification primers specific to said micro-organism. Said amplification primer are mentioned above, and are, depending on the micro-organism: Enterobacter cloacae: SEQ ID NOs: 3 and 4, or SEQ ID NOs: 5 and 6,
Enterococcus faecalis: SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12 or SEQ ID NOs: 15 and 11,
Enterococcus faecium: SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, or SEQ ID NOs: 20 and 21, Escherichia coli: SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID
NOs: 27 and 29, or SEQ ID NOs: 28 and 29,
Klebsiella pneumoniae: SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, or SEQ ID NOs: 37 and 33,
Pseudomonas aeruginosa: SEQ ID NOs: 40 and 41 or SEQ ID NOs: 40 and 42, Staphylococcus aureus: SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47,
SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, or SEQ ID NOs: 50 and 51,
Staphylococcus epidermidis: SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, or SEQ ID NOs: 63 and 61,
Candida albicans: SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, or SEQ ID NOs: 70 and 71.
According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof. A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of a SEQ ID NO in Figure 1, a complement thereof, or an homologous sequence of said region or complement.
A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying a region of a sequence listed in Figure 1 corresponding to a distinct region represented by any of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), 64 and 65 (Candida albicans).
It is within the scope of the invention, that the primers are capable of amplifying the aforementioned corresponding region in Figure 1, +10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues from either or both ends.
A hybridisation probe according to the present invention is capable of hybridising to a region of a sequence listed in Figure 1, corresponding to a distinct region, or complement thereof, represented by any of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), 64 and 65 (Candida albicans). It is within the scope of the invention that the probes are capable of binding to the aforementioned corresponding sequences of Figure 1, +10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues from either or both ends.
Another aspect of the invention is a method for identifying a distinct region by amplification of nucleic acid using a pair of primers directed towards a region of a sequence in Figure 1 corresponding to a distinct region, or complement thereof, represented by any of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), 64 and 65 (Candida albicans). A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification.
Homologous sequences of the above mentioned regions corresponding to distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. Figure 2
The Figure shows an alignment of sequenced genes of each of mecA, vanA, vanB, vanC, blashv, blages-2, spA, MDR-I, and CDR-I, together with a consensus sequence.
According to one aspect of the invention a distinct region of an antibiotic resistance gene of a comprises a nucleotide sequence (SEQ ID NOs: 158 to 261) indicated in Figure 2. According to another aspect of the invention, a distinct region is a complement of said SEQ ID NOs. One aspect of the present invention is nucleotide acid corresponding to a sequence represented by any of SEQ ID NOs: 158 to 261 indicated in Figure 2, or complement thereof.
According to one aspect of the invention a distinct region of an antibiotic resistance gene comprises a nucleotide sequence indicated in Figure 2, corresponding to a distinct region represented by any of SEQ ID NOs: 72 or 73 (blages-2), 78 or 79 (blashv), 84 or 85 (mecA), 90 or 91 (spA), 96 or 97 (vanA), 102 or 103 (vanB), 108 or 109 (vanC), 114 or 115 (MDR-I), 120 or 121 (CDR-I).
According to one aspect of the invention a distinct region of an antibiotic resistance gene comprises a nucleotide sequence indicated in Figure 2, obtainable using a pair of amplification primers specific to said micro-organism. Said amplification primer are mentioned above, and are, depending on the marker: blages-2 marker: SEQ ID NOs: 74 and 75, or SEQ ID NOs: 76 and 77, blashv marker: SEQ ID NOs: 80 and 81, or SEQ ID NOs: 82 and 83, mecA marker: SEQ ID NOs: 86 and 87, or SEQ ID NOs: 88 and 89, spA marker: SEQ ID NOs: 92 and 93, or SEQ ID NOs: 94 and 95, VanA marker: SEQ ID NOs: 98 and 99, or SEQ ID NOs: 100 and 101,
VanB marker: SEQ ID NOs: 104 and 105, or SEQ ID NOs: 106 and 107, VanC marker: SEQ ID NOs: 110 and 111, or SEQ ID NOs: 112 and 113, MDR-I marker: SEQ ID NOs: 116 and 117, or SEQ ID NOs: 118 and 119, and CDR l marker: SEQ ID NOs: 122 and 123, or SEQ ID NOs: 124 and 125. According to another aspect of the invention, a distinct region is an homologous sequence of the distinct region or complement thereof.
A pair of amplification primers according to one embodiment of the invention is a pair capable of amplifying any region of at least 30 bases of a SEQ ID NO in Figure 2, a complement thereof, or an homologous sequence of said region or complement. A pair of amplification primers according to another embodiment of the invention, is a pair capable of amplifying a region of a sequence listed in Figure 2 corresponding to a distinct region represented by any of SEQ ID NOs: 72 or 73 (blages-2), 78 or 79 (blashv), 84 or 85 (mecA), 90 or 91 (spA), 96 or 97 (vanA), 102 or 103 (vαnB), 108 or 109 (yanC), 114 or 115 (MDR-I), 120 or 121 (CDR-I).
It is within the scope of the invention, that the primers are capable of amplifying the aforemention corresponding region in Figure 2, +10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues from either or both ends.
A hybridisation probe according to the present invention is capable of hybridising to a region of a sequence listed in Figure 2, corresponding to a distinct region, or complement thereof, represented by any of SEQ ID NOs: 72 or 73 (blages-2), 78 or 79 (blashv), 84 or 85 (mecA), 90 or 91 (spA), 96 or 97 (vanA), 102 or 103 (vαnB), 108 or 109 (vanC), 114 or 115 (MDR-I), 120 or 121 (CDR-I).
It is within the scope of the invention that the probes are capable of binding to the aforementioned corresponding sequences of Figure 2, +10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues from either or both ends.
Another aspect of the invention is a method for identifying a distinct region by amplification of nucleic acid using a pair of primers directed towards a region of a sequence in Figure 2 corresponding to a distinct region, or complement thereof, represented by any of SEQ ID NOs: 72 or 73 (blages-2), 78 or 79 (blashv), 84 or 85 (mecA), 90 or 91 (spA), 96 or 97 (vanA), 102 or 103 (vαnB), 108 or 109 (vanC), 114 or 115 (MDR-I), 120 or 121 (CDR-I). A further aspect of the invention is a subsequent detection step using one or more hybridisation probes specific for the product of the amplification.
Homologous sequences of the above mentioned regions corresponding to distinct regions, amplification primers and hybridisation probes are within the scope of the invention. The distinct regions, probes and primers include homologous sequences in which one or more bases have been deleted, substituted and/or inserted as mentioned above. Combinations
As mentioned above, the present invention also relates to the simultaneous detection of two or more (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) distinct regions of nucleic acid. One embodiment of the invention is a method for detection of two or more (e.g. 3, 4, 5, 6, 7, 8, 9 or more) of Staphylococcus aureus, Staphylococcus epidermidis, Enterobacter cloacae, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Enterococcus faecium, Klebsiella pneumoniae and Candida albicans by detecting nucleic acid corresponding to distinct regions of 23S RNA therein.
Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting nucleic acid corresponding to two or more {e.g. at least 3, 4, 5, 6, 7, 8, or 9) of:
SEQ ID NOs: 1 or 2 (Enterobacter cloacae),
SEQ ID NOs: 7 or 8 (Enterococcus faecalis),
SEQ ID NOs: 16 or 17 (Enterococcus faecium),
SEQ ID NOs: 22 or 23 (Escherichia coli), SEQ ID NOs: 30 or 31 (Klebsiella pneumoniae),
SEQ ID NOs: 38 or 39 (Pseudomonas aeruginosa),
SEQ ID NOs: 43 or 44 (Staphylococcus aureus),
SEQ ID NOs: 52 or 53 (Staphylococcus epidermidis), and
SEQ ID NOs: 64 and 65 (Candida albicans). Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) of nucleic acid sequences listed in Figure 1, each sequence corresponding to the micro-organism for detection.
Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting at least two (e.g. at least 3, 4, 5, 6, 7, 8, or 9) regions of a nucleic acid listed in Figure 1, each region corresponding a distinct region of SEQ ID NOs: 1 or 2 (Enterobacter cloacae), 7 or 8 (Enterococcus faecalis), 16 or 17 (Enterococcus faecium), 22 or 23 (Escherichia coli), 30 or 31 (Klebsiella pneumoniae), 38 or 39 (Pseudomonas aeruginosa), 43 or 44 (Staphylococcus aureus), 52 or 53 (Staphylococcus epidermidis), or 64 and 65 (Candida albicans).
Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by using two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) primer pairs, wherein the two or more organisms for detection and the primer pairs are selected from the following, preferably, though not necessarily, one primer pair selected for one micro-organism:
Enterobacter cloacae: SEQ ID NOs: 3 and 4, or SEQ ID NOs: 5 and 6,
Enterococcus faecalis: SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12 or SEQ ID NOs: 15 and 11, Enterococcus faecium: SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, or SEQ ID NOs: 20 and 21,
Escherichia colt SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, or SEQ ID NOs: 28 and 29, Klebsiella pneumoniae: SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33,
SEQ ID NOs: 35 and 36, or SEQ ID NOs: 37 and 33,
Pseudomonas aeruginosa: SEQ ID NOs: 40 and 41 or SEQ ID NOs: 40 and 42,
Staphylococcus aureus: SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, or SEQ ID NOs: 50 and 51, Staphylococcus epidermidis: SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and
56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, or SEQ ID NOs: 63 and 61,
Candida albicans: SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, or SEQ ID NOs: 70 and 71.
Another embodiment of the present invention is a composition comprising two or more {e.g. at least 3, 4, 5, 6, 7, 8, or 9) of the following primer pairs:
Enterobacter cloacae: SEQ ID NOs: 3 and 4,
Enterococcus faecalis: SEQ ID NOs: 9 and 12, Enterococcus faecium: SEQ ID NOs: 18 and 19,
Escherichia coli: SEQ ID NOs: 24 and 25,
Klebsiella pneumoniae: SEQ ID NOs: 32 and 33,
Pseudomonas aeruginosa: SEQ ID NOs: 40 and 42,
Staphylococcus aureus: SEQ ID NOs: 48 and 49, Staphylococcus epidermidis: SEQ ID NOs: 54 and 55,
Candida albicans: SEQ ID NOs: 66 and 67.
Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by using two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) single probes corresponding to said two or more organisms for detection below, preferably, though not necessarily, one probe selected for one micro-organism:
Enterobacter cloacae: any of SEQ ID NOs: 3 to 6,
Enterococcus faecalis: any of SEQ ID NOs: 9 to 15,
Enterococcus faecium: any of SEQ ID NOs: 18 to 21,
Escherichia coli: any of SEQ ID NOs: 24 to 29, Klebsiella pneumoniae: any of SEQ ID NOs: 32 to37,
Pseudomonas aeruginosa: any of SEQ ID NOs: 40 to 42,
Staphylococcus aureus: any of SEQ ID NOs: 45 to 51,
Staphylococcus epidermidis: any of SEQ ID NOs: 54 to 63, Candida albicans: SEQ ID NOs: any of 66 to 71.
Another embodiment of the present invention is a composition comprising two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) of the following probes, preferably, though not necessarily, one probe selected for one micro-organism:
Enterobacter cloacae: SEQ ID NOs: 3 or 4, Enterococcus faecalis: SEQ ID NOs: 9 or 12,
Enterococcus faecium: SEQ ID NOs: 18 or 19,
Escherichia colt SEQ ID NOs: 24 or 25,
Klebsiella pneumoniae: SEQ ID NOs: 32 or 33,
Pseudomonas aeruginosa: SEQ ID NOs: 40 or 42, Staphylococcus aureus: SEQ ID NOs: 48 or 49,
Staphylococcus epidermidis: SEQ ID NOs: 54 or 55,
Candida albicans: SEQ ID NOs: 66 or 67.
Another embodiment of the invention is a method for detecting two or more (e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10) of the antibiotic resistance markers mecA, SpA, vanA, vanB, vanC, blashv, blages-2, MDR-I, CDR-I by detecting nucleic acid corresponding to distinct regions therein.
Another embodiment of the invention is a method for identifying at least two antibiotic resistance markers in a sample by detecting nucleic acid corresponding to two or more (e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10) of SEQ ID NOs: 72 or 73 (blages-2 marker),
SEQ ID NOs: 78 or 79 (blashv marker),
SEQ ID NOs: 84 or 85 (mecA marker),
SEQ ID NOs: 90 or 91 (spA marker),
SEQ ID NOs: 96 or 97 (VanA marker), SEQ ID NOs: 102 or 103 (VanB marker),
SEQ ID NOs: 108 or 109 (VanC marker)
SEQ ID NOs: 114 or 115 (MDR-I marker)
SEQ ID NOs: 120 or 121 (CDR-I marker) Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting two or more ((e.g. at least 3, 4, 5, 6, 7, 8, or 9) of nucleic acid sequences listed in Figure 2, each sequence corresponding to the micro-organism for detection. Another embodiment of the invention is a method for identifying at least two micro-organisms in a sample by detecting at least two (e.g. at least 3, 4, 5, 6, 7, 8, or 9) regions of a nucleic acid listed in Figure 1, each region corresponding a distinct region of SEQ ID NOs: 72 or 73 (blages-2 marker), SEQ ID NOs: 78 or 79 (blashv marker), SEQ ID NOs: 84 or 85 (mecA marker), SEQ ID NOs: 90 or 91 (spA marker), SEQ ID NOs: 96 or 97 (VanA marker), SEQ ID NOs: 102 or 103 (VanB marker), SEQ ID NOs: 108 or 109 (VanC marker), SEQ ID NOs: 114 or 115 (MDR-I marker), or SEQ ID NOs: 120 or 121 (CDR-I marker).
Another embodiment of the invention is a method for identifying at least two antibiotic resistance markers in a sample by using two or more (e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10) primer pairs wherein the antibiotic resistance markers for detection and the primer pairs are selected from the following preferably, though not necessarily, one primer pair selected for one marker: blages-2 marker: SEQ ID NOs: 74 and 75, or SEQ ID NOs: 76 and 77, blashv marker: SEQ ID NOs: 80 and 81, or SEQ ID NOs: 82 and 83, mecA marker: SEQ ID NOs: 86 and 87, or SEQ ID NOs: 88 and 89, spA marker: SEQ ID NOs: 92 and 93, or SEQ ID NOs: 94 and 95,
VanA marker: SEQ ID NOs: 98 and 99, or SEQ ID NOs: 100 and 101,
VanB marker: SEQ ID NOs: 104 and 105, or SEQ ID NOs: 106 and 107,
VanC marker: SEQ ID NOs: 110 and 111, or SEQ ID NOs: 112 and 113, MDR-I marker: SEQ ID NOs: 116 and 117, or SEQ ID NOs: 118 and 119, and
CDR l marker: SEQ ID NOs: 122 and 123, or SEQ ID NOs: 124 and 125.
Another embodiment of the invention is a composition comprising two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) of the following primer pairs: blages-2 marker: SEQ ID NOs: 76 and 77, blashv marker: SEQ ID NOs: 80 and 81 , mecA marker: SEQ ID NOs: 88 and 89, spA marker: SEQ ID NOs: 92 and 93,
VanA marker: SEQ ID NOs: 100 and 101,
VanB marker: SEQ ID NOs: 106 and 107, VanC marker: SEQ ID NOs: 112 and 113,
MDR-I marker: SEQ ID NOs: 116 and 117,
CDR l marker: SEQ ID NOs: 124 and 125.
Another embodiment of the invention is a method for identifying at least two antibiotic resistance markers in a sample by using two or more (e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10) probes wherein the antibiotic resistance markers for detection and the probes are selected from the following, preferably, though not necessarily, one probe selected for one micro-organism: blages-2 marker: any of SEQ ID NOs: 74 to 77, blashv marker: any of SEQ ID NOs: 80 to 83, mecA marker: any of SEQ ID NOs: 86 to 89, spA marker: any of SEQ ID NOs: 92 to 95,
VanA marker: any of SEQ ID NOs: 98 to 101,
VanB marker: any of SEQ ID NOs: 104 to 107, VanC marker: any of SEQ ID NOs: 110 to 113,
MDR-I marker: any of SEQ ID NOs: 116 to 119,
CDR l marker: any of SEQ ID NOs: 122 to 125.
Another embodiment of the present invention is composition comprising two or more (e.g. at least 3, 4, 5, 6, 7, 8, or 9) of the following probes, preferably, though not necessarily, one probe selected for one marker: blages-2 marker: SEQ ID NOs: 76 or 77, blashv marker: SEQ ID NOs: 80 or 81, mecA marker: SEQ ID NOs: 88 or 89, spA marker: SEQ ID NOs: 92 or 93, VanA marker: SEQ ID NOs: 100 or 101,
VanB marker: SEQ ID NOs: 106 or 107,
VanC marker: SEQ ID NOs: 112 or 113,
MDR-I marker: SEQ ID NOs: 116 or 117,
CDR l marker: SEQ ID NOs: 124 or 125. Another embodiment of the invention is a method for detecting at least one
(e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) of the antibiotic resistance markers mecA, SpA, vanA, vanB, vanC, blashv, blages-2, MDR-I and CDR-I and at least one (e.g. 2, 3, 4, 5, 6, 7, 8 or 9 or more) of the micro-organisms Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans by detecting nucleic acid corresponding to distinct regions therein.
Another embodiment of the invention is a method for identifying at least one micro-organism and at least one antibiotic resistance marker in a sample, by detecting the distinct regions corresponding to the SEQ ID NOs mentioned above.
Another embodiment of the invention is a method for identifying at least one micro-organism and at least one antibiotic resistance marker in a sample, by using two or more of the primer pairs or single probes corresponding to the SEQ ID NOs mentioned above. Another embodiment of the invention is a method for identifying one microorganism and at least one antibiotic resistance marker in a sample, by using one of the primer pairs or single probes corresponding to the SEQ ID NOs mentioned above relating to 23S RNA and two or more {e.g. 3, 4, 5, 6, 7, 8, 9 or 10 or more) primer pairs or single probes corresponding to the SEQ ID NOs mentioned above relating to antibiotic resistance genes. Another embodiment of the present invention is a composition comprising one or more {e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) of the following primer pairs:
Enterobacter cloacae: SEQ ID NOs: 3 and 4,
Enterococcus faecalis: SEQ ID NOs: 9 and 12,
Enterococcus faecium: SEQ ID NOs: 18 and 19, Escherichia colt SEQ ID NOs: 24 and 25,
Klebsiella pneumoniae: SEQ ID NOs: 32 and 33,
Pseudomonas aeruginosa: SEQ ID NOs: 40 and 42,
Staphylococcus aureus: SEQ ID NOs: 48 and 49,
Staphylococcus epidermidis: SEQ ID NOs: 54 and 55, Candida albicans: SEQ ID NOs: 66 and 67, and one of more {e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) of the following primer pairs: blages-2 marker: SEQ ID NOs: 76 and 77, blashv marker: SEQ ID NOs: 80 and 81, mecA marker: SEQ ID NOs: 88 and 89, spA marker: SEQ ID NOs: 92 and 93,
VanA marker: SEQ ID NOs: 100 and 101,
VanB marker: SEQ ID NOs: 106 and 107,
VanC marker: SEQ ID NOs: 112 and 113, MDR-I marker: SEQ ID NOs: 116 and 117,
CDR l marker: SEQ ID NOs: 124 and 125.
Another embodiment of the present invention is a composition comprising one or more (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) of the following probes, preferably, though not necessarily, one probe selected for one micro-organism:
Enterobacter cloacae: SEQ ID NOs: 3 or 4,
Enterococcus faecalis: SEQ ID NOs: 9 or 12,
Enterococcus faecium: SEQ ID NOs: 18 or 19,
Escherichia colϊ. SEQ ID NOs: 24 or 25, Klebsiella pneumoniae: SEQ ID NOs: 32 or 33,
Pseudomonas aeruginosa: SEQ ID NOs: 40 or 42,
Staphylococcus aureus: SEQ ID NOs: 48 or 49,
Staphylococcus epidermidis: SEQ ID NOs: 54 or 55,
Candida albicans: SEQ ID NOs: 66 or 67. and one of more {e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) of the following probes, preferably though not necessarily, one probe selected for one marker: blages-2 marker: SEQ ID NOs: 76 or 77, blashv marker: SEQ ID NOs: 80 or 81, mecA marker: SEQ ID NOs: 88 or 89, spA marker: SEQ ID NOs: 92 or 93,
VanA marker: SEQ ID NOs: 100 or 101,
VanB marker: SEQ ID NOs: 106 or 107,
VanC marker: SEQ ID NOs: 112 or 113,
MDR-I marker: SEQ ID NOs: 116 or 117, CDR l marker: SEQ ID NOs: 124 or 125.
A composition according to the present invention may be a solution, a mixture, an admixture, or may constitute the components in or on a container or device of the invention.
Another embodiment of the present invention is a container comprising two or more (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) primer pairs as defined in the above method and composition embodiments.
Another embodiment of the present invention is a container comprising two or more probes (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) as defined in the above method and composition embodiments. Another embodiment of the present invention is a kit comprising two or more (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) primer pairs as defined in the above method and composition embodiments.
Another embodiment of the present invention is a kit comprising two or more probes (e.g. at least 2, 3, 4, 5, 6, 7, 8, or 9) as defined in the above method and composition embodiments.
According to an aspect of the invention, a method detects the presence of one or more antibiotic resistance genes a micro-organism. According to another aspect of the invention, a method detects the presence of one or more antibiotic resistance genes a micro- organism and also the micro-organism.
The primers advantageously permit simultaneous or multiplexed PCR of template sequences in a single reaction, without the formation of primer dimer or cross reactions. Furthermore, the length of a product is particular to a species or antibiotic resistance marker. This allows the product of an amplification reaction to be separated, for example, by electrophoresis, and identification of several genes by evaluating the length attributed to one or more bands.
Multiplex detection of target sequences not only has the benefit of increasing throughput, but also allows differential diagnosis and monitoring of therapy in clinical applications. For example, the disease of bacteraemia and septicaemia is caused by one or more different pathogens and the therapy strongly depends on the detection of the involved bacteria and involved antibiotic resistant bacterial strains, which will guide the application of the right antibiotics. Therefore, for a proper and effective therapy it is essential to very specific and sensitive detection of the presence, absence or amount of specific NA sequences of one or more pathogens and their antibiotic resistant species, - the so called panel of pathogens and antibiotic resistant species. It is well known to the person skilled in the art, that the treatment of bacteraemia and septicaemia will only be successful in a very narrow timeframe after the outbreak of the disease. Furthermore, it is also well known to the person skilled in the art, that the earlier a disease is detected, the more successful and the faster therapy will work. Furthermore it is also well known to the person skilled in the art, that the detection of pathogenicity as a whole is not only restricted to panels containing bacteria and antibiotic resistant bacterial strains, but also to panels combining fungal strains, virus strains, proteins, and/ or haptens alone or in combination with bacteria and antibiotic resistant bacterial strains. Products
The present invention includes products comprising one or more primers or probes, suitable for use in a device enabling identification of the distinct regions mentioned herein. Such products include, for example, - one or more containers (e.g. microarray or multi-sample container) preloaded with one or more pairs of amplification primers. A multi-sample container allows simultaneous detection of distinct regions either in the same reaction or as separate reactions,
- a kit comprising one or more pairs of primers and optionally buffers, reagents and containers for performing amplification reactions, - a kit comprising one or more containers and optionally buffers, reagents for performing amplification reactions,
- a device comprising one or more pairs of primers for performing amplification reactions.
- one or more containers (e.g. microarray or multi-sample consumable) preloaded with one or more preloaded with one or more probes. A multi-sample container allows simultaneous detection of distinct regions either in the same reaction or as separate reactions, ,
- a kit comprising one or more probes and optionally buffers, reagents and containers for performing hybridisation, - a kit comprising one or more containers and optionally buffers, reagents and containers for performing hybridisation,
- a device comprising one or more probes for performing hybridisation.
- a composition comprising one or more primer pairs as mentioned herein. a composition comprising one or more probes as mentioned herein.
EXAMPLES
The following examples are intended to illustrate the various methods and compounds of the invention
Example 1: Sample preparation 200 μl of patient' s blood was pipetted into a vessel containing glass beads, acid-washed (Sigma- Aldrich) with a diameter 106 μm and finer. On a commerical IKA MS2 Minishaker the suspension was vortexed for 1 to 3 min at ambient temperature. Example 2: Nucleic acid extraction and purification The extraction and purification of nucleic acid was performed on a modified commercial biorobot EZl (Qiagen). The vortexed blood sample was incubated with lysostaphin at 370C for 10 min. In a following reaction step the nucleic acid was immobilized on magnetic beads. By actuating the magnetic beads via external magnetic fields in different washing and rinsing solutions, the immobilized nucleic acid will be freed from cell debris and other cell proteins. In a final elution step the nucleic acid will be separated from the magnetic particles and then mixed in a separate vessel with a multiplex PCR Mastermix (Qiagen). After mixing, the solution will be distributed to strip vessels with multiplex primer pairs and human control primer pairs. Example 3 : Multiplex PCR
Multiplex PCR was performed on a commercial thermal cycler, e.g. Perkin Elmer 9700, with a Qiagen Multiplex PCR kit. Following the universal multiplex cycling protocol, a first initial activation step was performed at 950C for 15 min. It followed a 3 step cycling with denaturing at 940C for 30 sec, annealing at 610C for 90 sec, and extension at 720C for 90 sec. The cycle number was between 30 and 45, dependend on the sensitivity requirements. A final extension at 720C for 10 min finished the amplification.
Example 4: Detection
The detection of the amplified nucleic acid was performed on a DNA 1000 kit. The chip, which is part of the kit, was read out with a 2100 bioanalyzer. Kits and Analyzer were purchased from Agilent Technologies.
According to the manufacturer' s recommendation the chip was prepared and loaded with the references and amplified nucleic acids and then inserted in the 2100 bioanalyzer. After an analysis run time of 30 min, proprietary software running on a PC read out and analyzed the data from the bioanalyzer.

Claims

CLAIMS:
1. Method of detecting one or more micro-organisms and/or one or more antibiotic resistance markers in a sample, comprising identifying the presence of distinct nucleic acid regions.
2. Method according to claim 1, wherein said distinct nucleic acid region of a micro-organism is in the 23S RNA gene.
3. Method according to claims 1 or 2, wherein said distinct nucleic acid region is identified using nucleic acid amplification.
4. Method according to claims 2 or 3, wherein multiplex PCR is used to detect two or more distinct nucleic acid regions.
5. Method according to claims 1 or 2, wherein said distinct nucleic acid region is identified using hybridisation.
6. Method according to claims 3 or 4, wherein said micro-organism is Enterobacter cloacae, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 3 and 4 or SEQ ID NOs: 5 and 6.
7. Method according to claim 5, wherein said micro-organism is Enterobacter cloacae, comprising the use of a hybridisation probe corresponding to a sequence represented by any of SEQ ID NOs: 3 to 6.
8. Method according to any of claims 1 to 5, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 1 or 2, and a micro-organism is Enterobacter cloacae.
9. Method according to claims 3 or 4, wherein said micro-organism is
Enterococcus faecalis, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, or SEQ ID NOs: 15 and 11.
10. Method according to claim 5, wherein said micro-organism is Enterococcus faecalis, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 9 to 15.
11. Method according to any of claims 1 to 5 wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 7 or 8, and a micro-organism is Enterococcus faecalis.
12. Method according to claims 3 or 4, wherein said micro-organism is Enterococcus faecium, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 18 and 19, SEQ ID NOs: 19 and 20, or SEQ ID NOs: 20 and 21.
13. Method according to claim 5, wherein said micro-organism is Enterococcus faecium, comprising the use of a probe corresponding to the sequences represented by SEQ ID NOs: 19 to 21.
14. Method according to any of claims 1 to 5 wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 16 or 17, and a micro-organism is Enterococcus faecium.
15. Method according to claims 3 or 4, wherein said micro-organism is
Escherichia coli, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 24 and 25, SEQ ID NOs: 24 and 26, SEQ ID NOs: 27 and 29, or SEQ ID NOs: 28 and 29.
16. Method according to claim 5, wherein said micro-organism is Escherichia coli, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 24 to 29.
17. Method according to any of claims 1 to 5 wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 22 or 23, and a micro-organism is Escherichia coli.
18. Method according to claims 3 or 4, wherein said micro-organism is Klebsiella pneumoniae, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36 or SEQ ID NOs: 37 and 33.
19. Method according to claim 5, wherein said micro-organism is Klebsiella pneumoniae, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 32 to 37.
20. Method according to any of claims 1 to 5 wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 30 or 31, and a micro-organism is Klebsiella pneumoniae.
21. Method according to claims 3 or 4, wherein said micro-organism is Pseudomonas aeruginosa, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 40 and 41 or SEQ ID NOs: 40 and 42.
22. Method according to claim 5, wherein said micro-organism is Pseudomonas aeruginosa, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 40 to 42.
23. Method according to any of claims 1 to 5 wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 38 or 39, and a microorganism is Pseudomonas aeruginosa.
24. Method according to claims 3 or 4, wherein a micro-organism is Staphylococcus aureus, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, SEQ ID NOs: 50 and 51.
25. Method according to claim 5, wherein said micro-organism is Staphylococcus aureus, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 45 to 51.
26. Method according to any of claims 1 to 5 wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 43 or 44, and a microorganism is Staphylococcus aureus.
27. Method according to claims 3 or 4, wherein said micro-organism is Staphylococcus epidermidis, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, or SEQ ID NOs: 63 and 61.
28. Method according to claim 5, wherein said micro-organism is Staphylococcus epidermidis, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 54 to 63.
29. Method according to any of claims 1 to 5 wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 52 or 53, and a micro-organism is Staphylococcus epidermidis.
30. Method according to claims 3 or 4, wherein said micro-organism is Candida albicans, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, or SEQ ID NOs: 70 and 71.
31. Method according to claim 5, wherein said micro-organism is Candida albicans, comprising the use of a probe corresponding to a sequence represented by any of SEQ ID NOs: 66 to 71.
32. Method according to any of claims 1 to 5 wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 64 or 65, and a microorganism is Candida albicans.
33. Method according to claims 3 or 4, wherein said antibiotic resistance marker is blaSeS-2, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 74 and 75 or SEQ ID NOs: 76 and 77.
34. Method according to claim 5, wherein said antibiotic resistance marker is blaSeS-2, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 74 to 77.
35. Method according to any of claims 1, 3 to 5, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 72 or 73, and an antibiotic resistance marker is blages.2.
36. Method according to claims 3 or 4, wherein said antibiotic resistance marker is blashv, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 80 and 81 or SEQ ID NOs: 82 and 83.
37. Method according to claim 5, wherein said antibiotic resistance marker is blashv, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 80 to 83.
38. Method according to any of claims 1, 3 to 5, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 78 or 79, and an antibiotic resistance marker is blashv.
39. Method according to claims 3 or 4, wherein said antibiotic resistance marker is mecA, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 86 and 87 or SEQ ID NOs: 88 and 89.
40. Method according to claim 5, wherein said antibiotic resistance marker is mecA, comprising the use of a probe corresponding to the sequences represented by SEQ ID NOs: 86 or 89.
41. Method according to any of claims 1, 3 to 5 wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 84 or 85, and an antibiotic resistance marker is mecA.
42. Method according to claims 3 or 4, wherein said antibiotic resistance marker is spA, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 92 and 93 or SEQ ID NOs: 94 and 95.
43. Method according to claim 5, wherein said antibiotic resistance marker is spA, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 92 to 95.
44. Method according to any of claims 1, 3 to 5, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 90 or 91, and an antibiotic resistance marker is Spa.
45. Method according to claims 3 or 4, wherein said antibiotic resistance marker is VanA, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 98 and 99 or SEQ ID NOs: 100 and 101.
46. Method according to claim 5, wherein said antibiotic resistance marker is VanA, comprising the use of a probe corresponding to the sequences represented by SEQ ID NOs: 98 to 101.
47. Method according to any of claims 1, 3 to 5, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 96 or 97, and an antibiotic resistance marker is VanA.
48. Method according to claims 3 or 4, wherein said antibiotic resistance marker is VanB, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 104 and 105 or SEQ ID NOs: 106 and 107.
49. Method according to claim 5, wherein said antibiotic resistance marker is VanB, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 104 to 107.
50. Method according to any of claims 1, 3 to 5, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 102 or 103, and an antibiotic resistance marker is VanB.
51. Method according to claims 3 or 4, wherein said antibiotic resistance marker is VanC, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 110 and 111 or SEQ ID NOs: 112 and 113.
52. Method according to claim 5, wherein said antibiotic resistance marker is VanC, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 110 to 113.
53. Method according to any of claims 1, 3 to 5, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 108 or 109, and an antibiotic resistance marker is VanC.
54. Method according to claims 3 or 4, wherein said antibiotic resistance marker is MDR-I, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 116 and 117 or SEQ ID NOs: 118 and 119.
55. Method according to claim 5, wherein said antibiotic resistance marker is
MDR-I, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 116 to 119.
56. Method according to any of claims 1, 3 to 5, wherein said distinct nucleic acid region corresponds to SEQ ID NOs: 114 or 115, and an antibiotic resistance marker is MDR- 1.
57. Method according to claims 3 or 4, wherein said antibiotic resistance marker is CDR-I, comprising the use of a pair of amplification primers corresponding to the sequences represented by SEQ ID NOs: 122 and 123 or SEQ ID NOs: 124 and 125.
58. Method according to claim 5, wherein said antibiotic resistance marker is CDR l, comprising the use of a probe corresponding to the sequences represented by any of SEQ ID NOs: 122 to 125.
59. Method according to any of claims 1, 3 to 5, wherein said distinct nucleic acid region corresponds to a sequence represented by SEQ ID NOs: 120 or 121, and an antibiotic resistance marker is CDR-I.
60. A container preloaded with one or more pairs of amplification primers, selected from the sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6,SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, SEQ ID NOs: 15 and 11,SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, SEQ ID NOs: 28 and 29, SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 33, SEQ ID NOs: 40 and 41, SEQ ID NOs: 40 and 42, SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, SEQ ID NOs: 50 and 51, SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, SEQ ID NOs: 63 and 61, SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, SEQ ID NOs: 70 and 71, SEQ ID NOs: 74 and 75, SEQ ID NOs: 76 and 77, SEQ ID NOs: 80 and 81, SEQ ID NOs: 82 and 83, SEQ ID NOs: 86 and 87, SEQ ID NOs: 88 and 89, SEQ ID NOs: 92 and 93, SEQ ID NOs: 94 and 95, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 104 and 105, SEQ ID NOs: 106 and 107, SEQ ID NOs: 110 and 111, SEQ ID NOs: 112 and 113, SEQ ID NOs: 116 and 117, SEQ ID NOs: 118 and 119, SEQ ID NOs: 122 and 123, and SEQ ID NOs: 124 and 125.
61. A container preloaded with one or more probes, selected from the sequences represented by any of SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
62. A kit comprising one or more pairs of amplification primers, selected from the sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6,SEQ ID NOs: 9 and
10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, SEQ ID NOs: 15 and 11,SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, SEQ ID NOs: 28 and 29, SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 33, SEQ ID NOs: 40 and 41, SEQ ID NOs: 40 and 42, SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, SEQ ID NOs: 50 and 51, SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, SEQ ID NOs: 63 and 61, SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, SEQ ID NOs: 70 and 71, SEQ ID NOs: 74 and 75, SEQ ID NOs: 76 and 77, SEQ ID NOs: 80 and 81, SEQ ID NOs: 82 and 83, SEQ ID NOs: 86 and 87, SEQ ID NOs: 88 and 89, SEQ ID NOs: 92 and 93, SEQ ID NOs: 94 and 95, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 104 and 105, SEQ ID NOs: 106 and 107, SEQ ID NOs: 110 and 111, SEQ ID NOs: 112 and 113, SEQ ID NOs: 116 and 117, SEQ ID NOs: 118 and 119, SEQ ID NOs: 122 and 123, and SEQ ID NOs: 124 and 125.
63. A kit comprising one or more probes selected from the sequences represented by SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
64. A kit comprising one or more containers according to claims 60 or 61.
65. A device comprising one or more pairs of amplification primers selected from the sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6,SEQ ID NOs: 9 and 10, SEQ ID NOs: 9 and 11, SEQ ID NOs: 9 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 12, SEQ ID NOs: 15 and 11,SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 24 and 26, SEQ ID NOs: 24 and 25, SEQ ID NOs: 27 and 29, SEQ ID NOs: 28 and 29, SEQ ID NOs: 32 and 34, SEQ ID NOs: 32 and 33, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 33, SEQ ID NOs: 40 and 41, SEQ ID NOs: 40 and 42, SEQ ID NOs: 45 and 46, SEQ ID NOs: 48 and 47, SEQ ID NOs: 48 and 49, SEQ ID NOs: 48 and 51, SEQ ID NOs: 50 and 51, SEQ ID NOs: 54 and 55, SEQ ID NOs: 54 and 56, SEQ ID NOs: 54 and 57, SEQ ID NOs: 58 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 58 and 60, SEQ ID NOs: 58 and 61, SEQ ID NOs: 58 and 62, SEQ ID NOs: 63 and 59, SEQ ID NOs: 63 and 60, SEQ ID NOs: 63 and 61, SEQ ID NOs: 66 and 67, SEQ ID NOs: 68 and 69, SEQ ID NOs: 70 and 71, SEQ ID NOs: 74 and 75, SEQ ID NOs: 76 and 77, SEQ ID NOs: 80 and 81, SEQ ID NOs: 82 and 83, SEQ ID NOs: 86 and 87, SEQ ID NOs: 88 and 89, SEQ ID NOs: 92 and 93, SEQ ID NOs: 94 and 95, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 104 and 105, SEQ ID NOs: 106 and 107, SEQ ID NOs: 110 and 111, SEQ ID NOs: 112 and 113, SEQ ID NOs: 116 and 117, SEQ ID NOs: 118 and 119, SEQ ID NOs: 122 and 123, SEQ ID NOs: 124 and 125.
66. A device comprising one or more probes, selected from the sequences represented by SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
67. Use of a container, kit or device according to any of claims 60 to 66 for detecting one or more micro-organisms and/or one or more antibiotic resistance markers in a sample.
68. Composition comprising a probe selected from the sequences represented by: SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125.
69. Composition comprising two or more probes selected from the sequences represented by: SEQ ID NOs: 3 to 6, SEQ ID NOs: 9 to 15, SEQ ID NOs: 18 to 21, SEQ ID NOs: 24 to 29, SEQ ID NOs: 32 to 37, SEQ ID NOs: 40 to 42, SEQ ID NOs: 45 to 51, SEQ ID NOs: 54 to 63, SEQ ID NOs: 66 to 71, SEQ ID NOs: 74 to 77, SEQ ID NOs: 80 to 83, SEQ ID NOs: 86 to 89, SEQ ID NOs: 92 to 95, SEQ ID NOs: 98 to 101, SEQ ID NOs: 104 to 107, SEQ ID NOs: 110 to 113, SEQ ID NOs: 116 to 119, and SEQ ID NOs: 122 to 125..
70. Composition comprising a pair of amplification primers selected from the sequences represented by: SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, SEQ ID NOs: 51 and 52, SEQ ID NOs: 55 and 56, and SEQ ID NOs: 59 and 60.
71. Composition comprising two or more pairs of amplification primers selected from the sequences represented by: SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, SEQ ID NOs: 51 and 52, SEQ ID NOs: 55 and 56, and SEQ ID NOs: 59 and 60.
72. Sequence of 23S RNA gene selected from the sequences represented by SEQ
ID NOs: 131 to 157.
73. Sequence of antibiotic resistance marker selected from the sequences represented by SEQ ID NOs: 158 to 261.
74. A method according to any of claims 6 to 59, wherein said sequence(s) represented by said SEQ ID NO(s) is (are) the complement(s) of said SEQ ID NO(s).
75. A method according to any of claims 6 to 59 and 74, wherein said sequence(s) representedby said SEQ ID NO(s) is (are) an homologous sequence(s) of said SEQ ID NO (s).
76. A container, kit, device or use according to any of claims 60 to 67 wherein said sequence(s) represented by said SEQ ID NO(s) is (are) the complement(s) of said SEQ ID NO(s).
77. A container, kit, device or use according to any of claims 60 to 67 and 76 wherein said sequence(s) represented by said SEQ ID NO(s) is (are) an homologous sequence(s) of said SEQ ID NO(s).
78. A composition according to claims 68 to 71 wherein said sequence(s) represented by said SEQ ID NO(s) is (are) the complement(s) of said SEQ ID NO(s).
79. A composition according to any of claims 68 to 71, and 78 wherein a sequence represented by a SEQ ID NO is an homologous sequence of said SEQ ID NO.
80. Sequence of 23S RNA gene according to claim 72 wherein said sequence represented by said SEQ ID NO is the complement(s) of said SEQ ID NO.
81. Sequence of 23S RNA gene according to claims 72 or 80 wherein said sequence represented by said SEQ ID NO is an homologous sequence of said SEQ ID NO.
82. Sequence of an antibiotic resistance marker according to claim 73 wherein said sequence(s) represented by said SEQ ID NO(s) is(are) the complement(s) of said SEQ ID NO(s).
83. Sequence of an antibiotic resistance marker according to claims 73 or 82 wherein said sequence(s) represented by said SEQ ID NO(s) is(are) an homologous sequence(s) of said SEQ ID NO(s).
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