WO2007011367A2 - Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16s ribosomal rna gene and identification of bacterial species by amplicon sequencing - Google Patents

Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16s ribosomal rna gene and identification of bacterial species by amplicon sequencing Download PDF

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Publication number
WO2007011367A2
WO2007011367A2 PCT/US2005/029296 US2005029296W WO2007011367A2 WO 2007011367 A2 WO2007011367 A2 WO 2007011367A2 US 2005029296 W US2005029296 W US 2005029296W WO 2007011367 A2 WO2007011367 A2 WO 2007011367A2
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primer
class
pcr
primers
bacteria
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PCT/US2005/029296
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French (fr)
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WO2007011367A3 (en
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Luc Montagnier
Claude Lavallee
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Luc Montagnier
Claude Lavallee
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Publication of WO2007011367A3 publication Critical patent/WO2007011367A3/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

Definitions

  • the present invention relates to the field of PCR methods, and specific primers therefore, as well as their use in the identification of any type of bacteria, and in particular RNA forms of bacteria.
  • biological fluids for therapeutic human application such as plasmas, albumin, live vaccines, stem cells requires that they are absolutely devoid of bacterial contamination. It has been found that filtering, and possibly other traditional methods, may fail to eliminate all forms of organisms, leading to possible contamination or experimental artifacts.
  • Devices are also provided for the in vitro detection of mycoplasmas in biological fluid by means of a reagent which is specific for the mycoplasma group without being specific for particular species within said group.
  • Devices for testing mycoplasma sensitivity to antibiotics are also described.
  • 20030134343 ; 20030134310; 20030134301; 20030134295; 20030134293; 20030134275; 20030131377; 20030131375; 20030129746; 20030129724; 20030129702; 20030129687; 20030129667; 20030129631; 20030129599; 20030129589; 20030129577; 20030129202; 20030125539; 20030125524; 20030125269; 20030125258; 20030125248; 20030124673; and 20030124613.
  • the present invention allows improved detection of such bacterial contaminants, including unconventional forms such as filtering forms (nanoforms), nanobacteria, and L-forms.
  • MOLL primers have been designed initially to detect mollicutes (mycoplasma) species based on the conserved regions of the 16s ribosomal RNA gene. In fact they can also detect gram positive bacteria
  • BACT primers will detect gram positive and gram negative bacteria.
  • the sequence of MOLL primers is included in the degenerated sequence of the BACT primers.
  • Bact Outer Primer (antisense) CCCGRGAACGTATTCACSG
  • Bact Inner Primer (sense) CCCGRGAGGCWGCAGTRRGGAAT
  • the GNEG set of primers is specific of gram negative bacteria. It differs from the Moll 16 out S by a single nucleotide 1) Gneg Outer Primer (sense) (RAYGGGTGAGTAAYGYMT),
  • the present invention therefore provides a method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first amplified genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.
  • Nanoforms are a stable, low metabolic rate form of bacteria, which may be related to pathology, which have characteristic DNA which is generally undetectable by PCR or nested PCR. However, these organisms do have characteristic RNA, and therefore these can be detected by nested RT-PCR. Likewise, because these are now detectable according to the present invention, it is therefore possible to monitor and optimize treatments directed toward clearing these from infected subjects.
  • Nanoforms are involved in human pathology, and further that these low metabolic organisms are involved in a constellation of chronic human diseases. Further, it is believed that some of these Nanoforms may be subcellular, that is, incomplete, and therefore may require association with other Nanoform, or other organisms or cells, for replication or reconstitution as a complete DNA bacterial form. Preliminary evidence suggests that the genetic material within a single Nanoforms is insufficient to reconstitute the entirety of a related bacterial (DNA) form, and therefore that multiple Nanoforms may be required in order to be self- replicating for the complete organism.
  • DNA related bacterial
  • Nanofoms may infect a single cell, together constituting a complete genome for the associated DNA bacteria.
  • Reverse transcriptase activity for example, due to retroviruses, endogenous retroviral sequences, DNA pol I activity, etc., may be sufficiently active to generate the bacterial genome.
  • Nanoforms may be biologically associated with retroviruses, such as HIV, which would therefore increase their likelihood of replication, since they would then carry their own reverse transcriptase, and potentially account for replication of sub-cellular fragments.
  • retroviruses such as HIV
  • the retroviruses may be passengers within the Nanoforms, and the Nanoforms represent an infectious particle for the virus.
  • Nanoforms retain conserved sequences of 16S rRNA, and therefore may be targeted on this basis, for example by tetracycline analog antibiotics, especially administered over extended durations.
  • oligonucleotide primers used were:
  • Expected lengths of amplicons are -1,200 bp and -450 bp with the outer and inner primers, respectively.
  • the primers employed were formulated as equal amounts of each primer within the class identified by the sequence.
  • Cell line supernatants 400 ⁇ l
  • human plasma 200-400 ⁇ l
  • human peripheral blood mononuclear cells PBMC, 3-10 millions cells
  • lOmMTris pH7.4, 1OmM EDTA, 15OmM NaCl, 0.4% SDS, and lO ⁇ g Proteinase K at 60°C for 1 h.
  • Nucleic samples were extracted three times with one volume of phenol/chloroform and one time with chloroform and precipitated by addition of 1/10 volume of 3M sodium acetate and two volumes of ethanol at -60°C for 1 h. Samples were centrifuged 30 min. and the nucleic acid pellets were washed with 70% cold ethanol and solubilized in 10 mM Tris-HCI, pH 8.0. These preparations were stored at -6O 0 C.
  • PCR reaction mix (50 ⁇ l) consisted of 5mM MgCl2, 5OmM Tris, pH 8.0, 15mM (NH4)2SO4, lOmM B-Mercaptoethanol, 500 ⁇ M dATP, dCTP, dGTP, and DTTP, 0.025% BSA, l ⁇ M of each outer primer, 1 U Taq polymerase (Roche Molecular biochemicals, Laval, Canada), and 5-10 ⁇ l nucleic acid sample.
  • the denaturation, annealing, and elongation temperatures and times used were 95°C for 30 s, 42 0 C for 30 s, and 78°C for 2 m, respectively, for 42 cycles.
  • the products were kept at 78°C for 10 m.
  • One ⁇ l of the PCR product was subjected to a second round PCR with the set of inner primers. Denaturation, annealing, and elongation temperatures and times used were 95°C for 30 s, 47°C for 30 s, and 78°C for 1 m, respectively, for 42 cycles, followed by a single incubation at 78 0 C for 10 m.
  • lO ⁇ l of PCR product was analyzed by gel electrophoresis using 1.5% agarose, stained with ethidium bromide, visualized under ultraviolet light and photographed. Visible bands with appropriate size were cut and sequenced using the inner primers (DNA Landmarks, St-Jean sur Ie Richelieu, Canada). Sequence homology search was performed using the BLAST program of the NTH web site.
  • RT-PCR reaction mix 50 ⁇ l was composed of 5mM MgCl2, 50 mM
  • Tris pH 8.0, 15 mM (NH4)2SO4, 10 mM B-Mercaptoethanol, 500 ⁇ M dATP, dCTP, dGTP, and
  • DTTP 0.025% BSA, l ⁇ M of each outer primer, and Titan enzyme mix (Roche Molecular biochemicals, Laval, Canada), and 5 ⁇ l nucleic acid sample.
  • the reverse transcription step was performed at 42°C for 30 m.
  • the first and second rounds PCR were performed as described above.
  • Second round PCR (nested-PCR): all amplicon ( ⁇ 450bp) sequences related to bacterial 16S ribosomal RNA gene.
  • RNA sequences such as the 16S rRNA
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase PCR
  • the present invention therefore provides a sensitive and specific method for detecting bacterial forms, which may be called “Nanoforms", even when traditional methods fail. This therefore allows diagnosis of pathogens previously unrecognized, and monitoring of treatment thereof.

Abstract

A method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.

Description

SENSITIVE DETECTION OF BACTERIA
BY IMPROVED NESTED POLYMERASE CHAIN REACTION
TARGETING THE 16S RIBOSOMAL RNA GENE
AND H)ENTEFICATION OF BACTERIAL SPECEES
BY AMPLICON SEQUENCING
FEELD OF THE INVENTION
The present invention relates to the field of PCR methods, and specific primers therefore, as well as their use in the identification of any type of bacteria, and in particular RNA forms of bacteria.
BACKGROUND OF THE INVENTION
The use of biological fluids for therapeutic human application such as plasmas, albumin, live vaccines, stem cells requires that they are absolutely devoid of bacterial contamination. It has been found that filtering, and possibly other traditional methods, may fail to eliminate all forms of organisms, leading to possible contamination or experimental artifacts.
It is believed that certain pathologies are associated with bacteria or bacterial forms which are difficult to detect, and which may pass through nano-porous barriers. This leads to possible errors in diagnosis or causation, and which may lead to erroneous treatment and impede prevention.
See, Hopert, Anne, UphofF, Cord C, Wirth, Manfred, Hauser, Hansjorg, and Drexler, Hans G., "Specificity and sensitivity of Polymerase Chain Reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines", J. Immunological Methods, 164(1993):91-100, expressly incorporated herein by reference.
Kajander, E.O., et al., "Comparison of Staphylococci and novel Bacteria-Like Particles from blood", ZbI. Bakt. Suppl. 26, 1994, expressly incorporated herein by reference.
Akerman, Kari K., "Scanning Electron Microscopy of Nanobacteria - Novel Biofϊlm Producing Organisms in Blood", Scanning Vol. 15, Suppl. Ill (1993), expressly incorporated herein by reference.
Figure imgf000003_0001
expressly incorporated herein by reference.
Cifticioglu, Neva, et al., "Apoptotic effect of nanobacteria on cultured mammalian cells", MoI. Biol. Cell. Suppl., Vol. 7 (1996):517a
Cifticioglu, Neva, et al., "A new potential threat in antigen and antibody products: Nanobacteria", Vaccines 97, Brown et al. Ed., Cold Spring Harbor Laboratory Press, New York, 1997, expressly incorporated herein by reference.
Baseman, Joel B., et al., "Mycoplasmas: Sophisticated, Reemerging, and Burdened by their Notoriety", EID Vol. 3, N° 1 www, cdc.gov/ncidod/EID/vol3 no l/baseman,htm. expressly incorporated herein by reference.
Relman, David A., "Detection and Identification of Previously Unrecognized Microbial
Pathogens", EID Vol. 4, N° 3 www.cdc.gov/ncidod/EID/vol4no3/relman.htm. expressly incorporated herein by reference.
Mattman, Lida H., Cell Wall Deficient Forms-Stealth Pathogens, 2nd Ed., CRC Press (1993), expressly incorporated herein by reference.
U.S. Pat. No. 5,688,646, expressly incorporated herein by reference, describes novel mycoplasmas which are prominent in patients who are thought to be suffering from AIDS.
Devices are also provided for the in vitro detection of mycoplasmas in biological fluid by means of a reagent which is specific for the mycoplasma group without being specific for particular species within said group. Devices for testing mycoplasma sensitivity to antibiotics are also described.
See the following U.S. Patents and Published Patent Applications, each of which and cited references is expressly incorporated herein by reference in their entirety: 6,562,611; 6,558,902; 6,555,357; 6,555,338; 6,537,774; 6,492,113; 6,489,139; 6,489,110; 6,458,572; 6,458,535; 6,448,037; 6,432,703; 6,410,286; 6,399,343; 6,372,424; 6,361,965; 6,353,093; 6,350,600; 6,348,582; 6,348,328; 6,348,314; 6,346,397; 6,340,564; 6,331,411; 6,326,172; 6,312,932; 6,309,866; 6,303,771; 6,294,652; 6,294,357; 6,287,807; 6,287,804;
6,277,595; 6,274,719; 6,274,361; 6,270,762; 6,268,177; 6,261,802; 6,261,769; 6,255,075;
6,251,631; 6,251,629; 6,248,721; 6,245,891; 6,245,542; 6,238,900; 6,238,887; 6,238,882;
6,228,625; 6,228,584; 6,210,880; 6,204,014; 6,197,549; 6,197,300; 6,194,170; 6,190,881; 6,168,797; 6,165,992; 6,165,991; 6,165,764; 6,162,619; 6,162,618; 6,156,537; 6,146,863;
6,146,846; 6,140,079; 6,140,061; 6,111,074; 6,110,723; 6,110,685; 6,090,543; 6,060,294;
6,001,567; 5,994,111; 5,985,557; 5,882,643; 5,851,764; 5,843,669; 5,843,654; 5,795,976;
5,789,217; 5,786,197; 5,776,750; 5,763,246; 5,750,387; 5,688,646; 6,562,957; 6,545,140;
6,531,148; 6,495,661; 6,475,990; 6,451,601; 6,403,564; 6,399,373; 6,368,600; 6,339,151; 6,277,830; 6,258,778; 6,248,329; 6,245,750; 6,022,687; 6,593,086; 6,583,266; 6,573,068;
6,569,647; 6,562,611; 6,558,953; 6,558,909; 6,558,905; 6,558,902; 6,555,357; 6,548,633;
6,531,648; 6,524,795; 6,521,426; 6,518,020; 6,503,747; 6,495,325; 6,458,584; 6,458,535;
6,444,876; 6,436,399; 6,432,649; 6,423,499; 6,403,093; 6,399,307; 6,387,617; 6,372,424;
6,348,314; 6,329,138; 6,312,922; 6,312,903; 6,306,653; 6,300,091; 6,300,072; 6,287,779; 6,268,142; 6,261,773; 6,252,130; 6,251,607; 6,248,519; 6,221,582; 6,214,982; 6,214,548;
6,194,145; 6,180,339; 6,103,468; 6,090,543; 6,033,858; 6,025,132; 6,001,567; 6,001,564;
5,994,066; 5,985,557; 5,976,805; 5,958,693; 5,948,888; 5,948,634; 5,942,391; 5,939,262;
5,922,538; 5,919,625; 5,912,117; 5,907,085; 5,876,924; 5,843,669; 5,830,670; 5,795,976;
5,763,169; 5,747,257; 5,744,306; 5,734,086; 5,707,802; 5,693,467; 5,688,669; 5,677,124; 5,660,981; 5,658,749; 5,656,740; 5,643,723; 5,593,836; 5,589,570; 5,518,901; 5,310,874;
20030124545; 20030118992; 20030108873; 20030082614; 20030065156; 20030059771;
20030054338; 20030044864; 20030044796; 20030027286; 20030017532; 20020162123;
20020160447; 20020150963; 20020146790; 20020146751; 20020128437; 20020127596;
20020123047; 20020120116; 20020119520; 20020119510; 20020119454; 20020115075; 20020106776; 20020102700; 20020098544; 20020091237; 20020082234; 20020058799;
20020052472; 20020048789; 20020025516; 20020004581; 20020004580; 20010027183;
20010023064; 20010020010; 20010016334; 20010014670; 20010010912; 20030145347;
20030144490; 20030144234; 20030143684; 20030143671; 20030143618; 20030143600;
20030143593; 20030143577; 20030143553; 20030143537; 20030143534; 20030143531; 20030143241; 20030143219; 20030143202; 20030139590; 20030138925; 20030138854;
20030138816; 20030138803; 20030138798; 20030138783; 20030138772; 20030135879;
20030134343; 20030134310; 20030134301; 20030134295; 20030134293; 20030134275; 20030131377; 20030131375; 20030129746; 20030129724; 20030129702; 20030129687; 20030129667; 20030129631; 20030129599; 20030129589; 20030129577; 20030129202; 20030125539; 20030125524; 20030125269; 20030125258; 20030125248; 20030124673; and 20030124613.
SUMMARY OF THE INVENTION
The present invention allows improved detection of such bacterial contaminants, including unconventional forms such as filtering forms (nanoforms), nanobacteria, and L-forms.
Its principal applications are the:
Detection of very low levels of mycoplasma contamination of cell lines and biological fluids
Identification of latent bacterial infections in various pathologies - Detection of live forms passing through filters having a pore size of between 100 and
20nm Direct detection of RNA-containing subunits of bacteria.
Different primers have been designed: - MOLL primers have been designed initially to detect mollicutes (mycoplasma) species based on the conserved regions of the 16s ribosomal RNA gene. In fact they can also detect gram positive bacteria
1) Moll Outer Primer (sense) (AAYGGGTGAGTAACACGT),
2) Moll Outer Primer (antisense) (CCCGAGAACGTATTCACCG) 3) Moll Inner Primer (sense) (CTACGGGAGGCAGCAGTA)
4) Moll Inner Primer (antisense) (GTATCTAATCCTRTTTGCTCCCCA) BACT primers will detect gram positive and gram negative bacteria. The sequence of MOLL primers is included in the degenerated sequence of the BACT primers. 1) Bact Outer Primer (antisense) (CCCGRGAACGTATTCACSG), 2) Bact Inner Primer (sense) (CTACGGGAGGCWGCAGTRRGGAAT),
3) Bact Inner Primer (antisense) (WGGGTATCTAATCCTRTTTGMTCCCCW)
The GNEG set of primers is specific of gram negative bacteria. It differs from the Moll 16 out S by a single nucleotide 1) Gneg Outer Primer (sense) (RAYGGGTGAGTAAYGYMT), The present invention therefore provides a method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first amplified genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.
It is believed that the Nanoforms are a stable, low metabolic rate form of bacteria, which may be related to pathology, which have characteristic DNA which is generally undetectable by PCR or nested PCR. However, these organisms do have characteristic RNA, and therefore these can be detected by nested RT-PCR. Likewise, because these are now detectable according to the present invention, it is therefore possible to monitor and optimize treatments directed toward clearing these from infected subjects.
It is believed that Nanoforms are involved in human pathology, and further that these low metabolic organisms are involved in a constellation of chronic human diseases. Further, it is believed that some of these Nanoforms may be subcellular, that is, incomplete, and therefore may require association with other Nanoform, or other organisms or cells, for replication or reconstitution as a complete DNA bacterial form. Preliminary evidence suggests that the genetic material within a single Nanoforms is insufficient to reconstitute the entirety of a related bacterial (DNA) form, and therefore that multiple Nanoforms may be required in order to be self- replicating for the complete organism.
For example, multiple Nanofoms may infect a single cell, together constituting a complete genome for the associated DNA bacteria. Reverse transcriptase activity, for example, due to retroviruses, endogenous retroviral sequences, DNA pol I activity, etc., may be sufficiently active to generate the bacterial genome.
These Nanoforms may be biologically associated with retroviruses, such as HIV, which would therefore increase their likelihood of replication, since they would then carry their own reverse transcriptase, and potentially account for replication of sub-cellular fragments. The retroviruses may be passengers within the Nanoforms, and the Nanoforms represent an infectious particle for the virus.
The present invention reveals that the Nanoforms retain conserved sequences of 16S rRNA, and therefore may be targeted on this basis, for example by tetracycline analog antibiotics, especially administered over extended durations.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Material and methods
Oligonucleotide primers.
The oligonucleotide primers used were:
1) Moll Outer Primer (sense) (AAYGGGTGAGTAACACGT), 2) Gneg Outer Primer (sense) (RAYGGGTGAGT AAYGYMT),
3) Bact Outer Primer (antisense) (CCCGRGAACGTATTCACSG),
4) Bact Inner Primer (sense) (CTACGGGAGGCWGCAGTRRGGAAT), and
5) Bact Inner Primer (antisense) (WGGGTATCTAATCCTRTTTGMTCCCCW) where R= G or A, S= G or C, W = A or T, M = A or C, and Y = T or C.
Expected lengths of amplicons are -1,200 bp and -450 bp with the outer and inner primers, respectively. The primers employed were formulated as equal amounts of each primer within the class identified by the sequence.
Nucleic acid preparation and PCR/RT-PCR.
Cell line supernatants (400 μl), human plasma (200-400μl) and human peripheral blood mononuclear cells (PBMC, 3-10 millions cells) were lyzed with lOmMTris, pH7.4, 1OmM EDTA, 15OmM NaCl, 0.4% SDS, and lOμg Proteinase K at 60°C for 1 h. Nucleic samples were extracted three times with one volume of phenol/chloroform and one time with chloroform and precipitated by addition of 1/10 volume of 3M sodium acetate and two volumes of ethanol at -60°C for 1 h. Samples were centrifuged 30 min. and the nucleic acid pellets were washed with 70% cold ethanol and solubilized in 10 mM Tris-HCI, pH 8.0. These preparations were stored at -6O0C.
PCR reaction mix (50μl) consisted of 5mM MgCl2, 5OmM Tris, pH 8.0, 15mM (NH4)2SO4, lOmM B-Mercaptoethanol, 500μM dATP, dCTP, dGTP, and DTTP, 0.025% BSA, lμM of each outer primer, 1 U Taq polymerase (Roche Molecular biochemicals, Laval, Canada), and 5-10 μl nucleic acid sample. For the first round PCR, the denaturation, annealing, and elongation temperatures and times used were 95°C for 30 s, 420C for 30 s, and 78°C for 2 m, respectively, for 42 cycles. After the final cycle, the products were kept at 78°C for 10 m. One μl of the PCR product was subjected to a second round PCR with the set of inner primers. Denaturation, annealing, and elongation temperatures and times used were 95°C for 30 s, 47°C for 30 s, and 78°C for 1 m, respectively, for 42 cycles, followed by a single incubation at 780C for 10 m. After the first and second round PCR, lOμl of PCR product was analyzed by gel electrophoresis using 1.5% agarose, stained with ethidium bromide, visualized under ultraviolet light and photographed. Visible bands with appropriate size were cut and sequenced using the inner primers (DNA Landmarks, St-Jean sur Ie Richelieu, Canada). Sequence homology search was performed using the BLAST program of the NTH web site.
Samples negative for the appropriate band by PCR were subjected to a first round RT-PCR followed by a second round PCR. RT-PCR reaction mix (50μl) consisted of 5mM MgCl2, 50 mM
Tris, pH 8.0, 15 mM (NH4)2SO4, 10 mM B-Mercaptoethanol, 500μM dATP, dCTP, dGTP, and
DTTP, 0.025% BSA, lμM of each outer primer, and Titan enzyme mix (Roche Molecular biochemicals, Laval, Canada), and 5 μl nucleic acid sample. The reverse transcription step was performed at 42°C for 30 m. The first and second rounds PCR were performed as described above.
The precautions addressed elsewhere (Kwok and Higuchi, 1989) were followed to minimize the risk of false-positive results caused by the carry-over of previously amplified DNA. For example, extraction of nucleic acids and preparation of PCR/RT-PCR mix were performed under a sterile flow bench, only aliquoted reagents and filter tips were used, and negative controls were incorporated into each run. Results
All Samples:
First round PCR / RT-PCR: no detection of expected amplicon (~l,200bp).
Second round PCR (nested-PCR): all amplicon (~450bp) sequences related to bacterial 16S ribosomal RNA gene.
Patients' lymphocytes (18 samples):
No 450bp amplicon detected by nested-PCR from first round PCR.
All samples positive (450bp) by nested-PCR from RT-PCR.
Therefore, bacteria are in an "RNA state", and are referred to herein as "Nanoforms".
Samples of Patients' Plasma:
11 samples/12 positive for 450bp amplicon by nested-PCR from first round PCR.
Discussion
Cell wall deficient pathogenic microorganisms, which may be mycoplasma, so-called L- forms, or potentially other types, are difficult to detect. Therefore, their involvement in pathology may be vastly under-reported.
It has been found, however, that these organisms have a well-conserved RNA sequences, such as the 16S rRNA, even when corresponding DNA or RNA is undetectable by a traditional polymerase chain reaction (PCR) or reverse transcriptase PCR (RT-PCR) method, which may be detected by nested RT-PCR amplification, using primers according to the present invention.
The present invention therefore provides a sensitive and specific method for detecting bacterial forms, which may be called "Nanoforms", even when traditional methods fail. This therefore allows diagnosis of pathogens previously unrecognized, and monitoring of treatment thereof.
What is claimed is:

Claims

CLAIMS:
1. A method for amplifying genetic sequences, comprising the steps of:
(1) first, reverse transcribing RNA to DNA, and conducting PCR using at least one outer primer having a sequence within the class: (AAYGGGTGAGTAACACGT) for Gram positive bacteria or,
(RAYGGGTGAGTAAYGYMT) for Gram negative bacteria, and at least one primer having a sequence within the class: (CCCGRGAACGTATTCACSG),
(2) second, conducting nested PCR, using inner primers, comprising at least one primer having a sequence within the class:
(CTACGGGAGGCWGCAGTRRGGAAT), and at least one primer having a sequence within the class: (WGGGTATCTAATCCTRTTTGMTCCCCW), wherein R= G or A, S= G or C, W = A or T, M = A or C, and Y = T or C.
2. The method according to claim 1 further comprising the step of identifying an organism corresponding to an amplicon resulting from said nested PCR step.
3. The method according to claim 1, wherein said PCR steps employ all primers having sequences within the specified classes, except optionally excluding primers within either the Gram positive primer or Gram negative primer classes.
4. A method for identifying an organism, comprising: (a) reverse transcribing RNA material to DNA; (b) conducting PCR using sense and antisense primers for a first highly conserved genetic sequence generic for the class of organism;
(c) conducting nested PCR using sense and antisense primers for a second highly conserved genetic sequence, within the first highly conserved genetic sequence; and
(d) identifying the organism based on unconserved amplified sequences linked to the conserved sequences.
5. The method according to claim 4, wherein said first and second highly conserved genetic sequences each comprise a part of a 16S ribosomal KNA sequence.
6. The method according to claim 4, wherein at least one of the primers are part of a degenerate set, further comprising the step of employing a plurality of primers within the class definition of the degenerate set.
7. A kit, comprising a unit amount of each of the following combinations of primer sequences, substantially absent interfering DNA primer sequences, sufficient for a PCR identification of a bacteria in a biological sample:
(a) at least one of:
(AAYGGGTGAGTAACACGT) and (RAYGGGTGAGT AAYGYMT), and (CCCGRGAACGTATTCACSG); and
(b) (CTACGGGAGGCWGCAGTRRGGAAT), and (WGGGTATCTAATCCTRTTTGMTCCCCW), wherein R= G or A, S= G or C, W = A or T, M = A or C, and Y = T or C.
8. The kit according to claim 7, wherein said component (a) further comprises a reverse transcriptase (RNA-dependent, DNA polymerase) activity, and deoxynucleotide triphosphates.
9. The kit according to claim 7, wherein said component (b) further comprises temperature resistant, DNA-dependent DNA polymersase activity, and deoxynucleotide triphosphates.
10. The kit according to claim 7, wherein at least one of said component (a) and (b) comprises 5mM MgC12, 50 mM Tris, pH 8.0, 15 mM (NH4)2SO4, 10 niM B-Mercaptoethanol, 500μM dATP, dCTP, dGTP, and dTTP, 0.025% BSA, lμM of each primer, a reverse transcriptase (RNA dependent DNA polymerase) activity, and a temperature resistant, DNA- dependent DNA polymerase activity.
11. The kit according to claim 7, wherein said component (a) comprises: all primer sequences falling within the class of at least one of: (AAYGGGTGAGTAACACGT) and (RAYGGGTGAGTAAYGYMT); and all primer sequences falling within the class: (CCCGRGAACGTATTCACSG).
12. The kit according to claim 11, wherein said component (b) comprises: all primer sequences falling within the class:
(CTACGGGAGGCWGCAGTRRGGAAT); and all primer sequences falling within the class:
(WGGGTATCTAATCCTRTTTGMTCCCCW).
13. The kit according to claim 17, wherein said component (b) comprises all primer sequences falling within the class:
(CTACGGGAGGCWGCAGTRRGGAAT); and all primer sequences falling within the class: (WGGGTATCTAATCCTRTTTGMTCCCCW).
PCT/US2005/029296 2004-08-20 2005-08-17 Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16s ribosomal rna gene and identification of bacterial species by amplicon sequencing WO2007011367A2 (en)

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