CA1192120A - Method and reagent combination for the diagnosis of microorganisms - Google Patents

Method and reagent combination for the diagnosis of microorganisms

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Publication number
CA1192120A
CA1192120A CA000413539A CA413539A CA1192120A CA 1192120 A CA1192120 A CA 1192120A CA 000413539 A CA000413539 A CA 000413539A CA 413539 A CA413539 A CA 413539A CA 1192120 A CA1192120 A CA 1192120A
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nucleic acid
fragment
reagent
sample
labelled
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French (fr)
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Tuula M. Ranki
Hans E. Soderlund
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Sangtec Medical AB
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Orion Yhtyma Oy
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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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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    • 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
    • C12Q1/6813—Hybridisation assays
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    • 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
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    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/804—Radioisotope, e.g. radioimmunoassay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/808—Automated or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/811—Test for named disease, body condition or organ function
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/823—Immunogenic carrier or carrier per se

Abstract

ABSTRACT OF THE DISCLOSURE:
A microbial diagnostic method based on the hybridization of nucleic acids on a solid carrier, and a reagent combination for carrying out the method. The method which can be used to identify all the microbes or microbial groups from a single sample containing nucleic acids of the microbes or microbial groups to be diagnosed, after having first rendered said nucleic acids single-stranded, basically consists in adding to the sample two nucleic acid reagents for each microbe or microbial group to be diagnosed. One of these nucleic acid reagents consists of a nucleic acid fragment from the same microbe or microbial group, which is attached to a solid carrier in a single stranded form. The other reagent consists of a completely different nucleic acid fragment from the same microbe or microbial group, which is labelled with a given marker. The nucleic acid fragment attached to the carrier is selected to hybridize to a complementary single-stranded nucleic acid from the sample to form a hybrid on the carrier. This hybrid becomes labelled when the labelled nucleic acid fragment anneals to it. Of course, the nucleic acid reagents are selected not to hybridize to each other. The labelled hybrid attached to the carrier can be measured using established methods.

Description

The present invention rela-tes to a me-thod Eor the cletection of microorganisms and more particularly the diag-nosis oE microbes, based on sandwlch hybridization of nucleic acids to a solid carrier. The inven-tion also rela-tes -to a combina-tiorl of reagen-ts tha-t can be used for carrying out -this method.
In traditional microbial diagnostics, the presence of a microbe in a given sample is demonstrated by isolation o~ the microbe in question. ~fter enrichment cultivation, the microbe is identified either on the basis of its bio-chemical properties, or using immunological methods. This type o-f diagnosis requires that the microbe in the sample is viable. Identification by isolation is moreover a laborious method, which may require from 4 to 6 weeks in the case of viruses.
The object of this inven-tion is -to provide a diag-nostic method in which the presence of a microbe in a sample is demonstrated by iden-tification of i-ts genetic material, namely its nucleic acid, using a sensitive and specific nucleic acid hybridization technique. In i-tself, nucleic acid hybridization is an old and well known method for in-vestigating -the iden-tity of nucleic acids. Complementary nucleic acid strands have the ability to form a tight double-stranded struc-ture according to the rules of base pairing, and the resulting hybrid can be separated from -the residual single-stranded nucleic acid.
Some methods based on the identiEication of nucleic acid(s) have already been applied -to microbial diagnostics.
By way of example, enterotoxigenic Escherichia coli have been identified from faecal samples by colony hybridization using the gene for toxin production as a probe. Positive hybridi-zation is demonstrated by autoradiography (Moseley, S.L. et al., J~ Infect. Dis. (1980) 1~2, 892 - 898). Colony hybridi-zation is based on a me~hod ori~inally developed by Grunstein -1- ~

and Hogness ~Proc. Natl. Acacl. Sci. USA (1975) 72, 3961 -3965). Hybridizatlon has also been used as a method -to distinguish between Herpes simplex virus type 1 and type 2 (Brautigam, A.R. et al. J. Clin. Microbiol (1980) 12, 226-234). However, this method cannot be used for rapid diay-nostics as it requires typing of the virus after enrichment cultiva-tion. In this method, the double-stranded hybrid is separa-ted from the fraction of nucleic acids remaining single-s-tranded in the solution by affinity chromatography.
It has already been disclosed that DNA from cells infected with Epstein-Barr virus can be fixed onto filters after appropriate pretreatment. The nucleic acid is identi-fied by hybridizing the filters with a radioactive probe and positive hybridiæation is detected by autoradiography (Brandsma, I. and Miller, K. (1980) Proc. Natl. Acad. Sci.
USA 77, 6851 - 6855).
The method according to -the present invention is based on a sandwich hybridization technique such as -the one used by Dunn, A.R. and Hassell to map transcripts in cells infected with a virus ((1977) Cell 12, 23 - 36). This method advantageously simplifies handling of the sample and detection of the hybrid. For this reason, this method is particularly suitable for diagnostic use.
In the method according to the invention, all the desired microbes or microbial groups can be iclentified in one step from one single sample, which contains denaturated single-stranded nucleic acid strands of the microbes, without sample division.
The method requires two nucleic acid reagents for each microbe or group of microbes to be identified. The reagents are two separate nucleic acid fragments derived from the genome of each microbe to bè identified, which have no se~uences in common but preferably are situated close to each other in the genome. The reagents can be prepared directly from the microbial genomes or by using established recombinant DNA

techniques. One of the -two nuclelc acid fragmen-ts, is in single-stranded form and is fi~ed to a solid carrier, preferably a nitrocellulose filter, after having been denaturated. The other fragmen-ts which is also in single-s-tranded form, is labelled with a suitable marker.
When these nucleic acid reagen-ts, namely -two different nucleic acid fragmen-ts for each microbe or micro-bial group -to be identified, are placed in con-tact with the slngle stranded nucleic acid strands of -the microbes to be identified in the sample, the nucleic acid strands anneal to the complementary nucleic acid fragments on the solid carrier. The hybrids thus formed on the carrier become labelled after annealing to the labelled complementary nucleic acid fragmen-ts. The labelled nucleic acid fragments do not alone hybridize to the nucleic acid fragments at-tached to the carrier, but only -to the correc-t single s-tranded nucleic acids originating from the sample. Thus only those carriers to which the complementary nucleic acids from the sample have hybridized become labelled. These carriers are easily washed and the marker detected and meas-ured by es-tablished methods~
The method according to the inven-tion can be used for the identification of all DNA- or RNA- containing organisms, such as viruses, bacteria, fungi and yeasts~
This method has the specific advantage of permitting iden-tification of all the bacteria and viruses possibly in a single sample at the same time, regardless of whether the microbes contain DNA or RNA. By suitable combination of reagents, it is possible to develop kits such that each microbe to be identified has its own specific solid carrier and labelled nucleic acid reagent. All the carriers included in -the kit can be added to the sample simultaneously, along with the labelled nucleic acid reagents. When hybridization has taken place, the solid carriers are washed and -their r~

labelling is measured. The on]y carrier to become labelled is the one which contains sequences complementary -to the microbial genome presen-t in the investigated sarnple.
The method and combination of reagents accordiny to the presen-t invetioll can be used in medical microbiology, ve-terinary microbiology, food hygiene investigations and microbial diagnosis of plant diseases. Sui-table sample materials can be, for example, animal- and plant tissue homogenates, patient secretions such as blood, faeces and nasal~ and urethral mucous.
The method according to -the invention is suffi-ciently sensitive to detect the microbe levels normally present in clinical samples. Preliminary enrichment of the microbe present in the sample by cultivation is of course possible before the identification test and in some cases would be essen-tial. The method is also suitable for the investigation of samples from which the microbe can no longer be cultivated but which con-tain considerable amounts of microbial debris (eg. after the commencement of antibiotic treatment), or when cultivation of the microbe is particu-larly laborious and difficult (eg. anaerobic bacteria, which are present in large numbers in suppurative samples in the case of infections caused by anaerobes).
The method according to the invention can be used for -the diagnosis of the following diseases, usiny the following combinations of reagents (or kits), whose com-ponents may of course be used separately.

Respiratory infections: 0 a) Bacteria: ~-haemolytic streptococci (A-group), Haemophilus influenzae, Pneumococci, _ycoplasma pneumoniae, mycobacteria b) Viruses: Influenza A, Influenza B, Parainfluenza 1-3 Respiratory syncytial virus, adenoviruses, corona viruses, rhinoviruses Diarrohoeas a) Bac-teria: salmonellae, shigellae, Yersinia enterocolitica, enterotoxigenic, E. coli, Clos-tridium difficile, campylobacteria b) Viruses rotaviruses, parvoviruses, adenoviruses, entero-viruses Veneral diseases-a) sacteria: Neisseria ~onorrhoeae, Treponema pallldum, Chlamydia trachomatis b) Viruses: Herpes simplex-virus c) Yeasts: Candida albicans d) Protozoa: Trichomonas vaginalis _epsis:
a) Bacteria: ~-haemolytic strep-tococci (~-group), pneumo-cocci, enterobacteria as a single group - _ood hygiene-.

a~ Bacteria: Salmonella and Clos-t~d ~ _ Depending on the choice of reagents, the specificity of the test can be limited to a defined microbial group (e.g.
salmonella bacteria) or to a wider group e.g. enterobacteria-ceae. In the latter case, the identifying reagents is choosen from the area of a common gene.
The nucleic acid reagents required in the sandwich hybridization technique used in the method according to the invention can be produced by recombinant D~A technology. The production of the reagents used in example 1 given hereinafter and the test procedure followed in this example 1 will now be described, by way of example.

2i~
Reagents Adenovirus type 2 (strain deposi-ted a-t KTI,, i.c.
the Public ~-leall~h Labo:ratory of l-lelsinki, Finland) was cultiva-ted and purified and its DNA was isola-ted (Pet-terson, U. and Sambrook, J. (1973) J. Mol. Biol. 73, 125-130) this DN~ will be reerred to hereinafter as Ad2-DNA). The DNA
was digested with BamHI-restriction enzyme (BRL, i.e.
Bethesda Research Laboratories), to cut the DNA into four reproducable fragments. Two of -these four fragments were inserted -to the BamHI-site of the vector plasmid pBR 322 (BRL) with the aid of T4-ligase (BRL). The fragments were no-t separated before ligation, but the insert added to the plasmid was in each case identified only after cloning.
Subsequently the bacterial host (E. coli HB101 (K12) gal , pro , leu , hrs , hrm , recA, strr, F ) obtained from KTL., was transformed with -the plasmid DNA composed of recombinant plasmids, i.e. molecules which had accepted fragments of the adenovirus-DNA (Cohen, S.N. e-t al. (1972) Proc~ Natl.
Acad. Sci. USA _, 2110-211~). The transformed bacterial clones which most probably contained the recombinant plasmid, were chosen.
In practice, ampicillin and tetracyclin resistance is transfered to the bacterium by -the pBR322-plasmid (Bolivar F. et al. (1977) Gene 2, 95-113). Bacteria contain-ing recombinant plasmids are however sensitive to tetra-cycline, because the BamHI-restric-tion site is within -the tetracyclin gene and the foreign DNA inserting into this region destroys the gene.
The inser-tion of the plasmid was characterized after plasmid enrichment by determination of the size of the restriction fragments after BamHI digestion, using agarose gel electrophoresis. The adjacent BamHI D- and C-fragments of the Ad2-DNA (based on the gene map) were chosen as re-agents ~Soderlund, H. et al. (1976) Cell 7, 585-593). The v desired recombinarlt plasmids, Ad2C-pBR322, KTI. no. E231 and Ad2D-pBR322, KTL no. E11230, were cul-tivated and purified as has been described in -the litera-ture (Clewell, D.B. and llelinski, D.R. (1969) Proc. Natl. Acad. Sci. USA 62, 1159-1166).
The recombinant plasmid Ad2D-pBR322 was used as the reagen-t attached to the carrier. I-t was not necessary to remove the plasmid se~uences, because the sample did no-t contain pBR322-sequences. However, for radioactive labelling of the other reagen-t, the nucleic acid was separated from pBR322-DNA after BamHI-diges-tion with -the aid of agarose gel electrophoresis. The C-fragmen-t was isolated from LGT-agarose (Marine Colloids, Inc.) by phenol extraction or electro-elution (Wieslander, L. (1979) Anal. Biochem. 98, 305-309) and concentrated by ethanol precipitation.
It is particularly expedien-t to subclone the nucleic acid fragment chosen for labelling in a separate vector, in order to avoid -the hybridization background re-sulting from the direct hybridization of the residual plasmid se~uences with the carrier, which may con-taminate the labelled nucleic acid reagent. The single-stranded DNA-phage M13 mp7 (BRL), -to which DNA fragments obtained by BamHI digestion can easily be transferred could be used as an optimal vec-tor (Messing, J. et al. (1981) Nucleic Acids Res. 9, 309-323).
Attachment of DNA to -the carxier The recombinant plasmid Ad2D-pBR322 was denatured to a single stranded form and nicked randomly at several sites by treatment with 0.2 N NaOH (5 min. 100C), whereafter the DNA was chllled and, immediately prior to transfert to a carrier, neutralized and pipetted to the transfer-t solution, 4 x SSC medium on ice (SSC = 0.15 M NaCl, 0.015 M Na-citrate).
The carrier consisted of a nitrocellulose filter (Schleicher and Schull sA85 ni-trocellulose) that was thoroughly wetted in 4 x SSC solution for abou-t 2 h before the application of DNA. The DNA was a-ttached -to the filter in a dilute solu-tion (0.5-1.0 ~Ig/ml) by sucking the so]ution through the fil-ter under a weak vacuum. The filter is capable of absorb-ing DNA up to about 180 ~g/cm2 (Kafatos, F.C. et al. (1979) Nucleic Acids Res. 7, 154:L-1552). The DNA-concentrations were ranging from 0.5 ~g DNA/2.5 cm diameter o Eilter to 1.0 ~g DNA/0.7 cm diameter of filter. After DNA-fil-tration, the il-ters were washed in 4 x SSC, dried at room temperature and finally baked in a vacuum oven at 80C Eor 2 h, after which the DNA on the filters remained stable and the filters were able to be stored or long periods at room temperature (Southern, E.M. (1975) J~ Mol. Biol. 98, 503-5:L7).

Labelling of the radioactive nucleic acid fragmen-t The radioactive marker used was the 125I-isotope.
This isotope can be detected using ~f-counters, which are available in most large labora-tory uni-ts. The half-life of the isotope is 60 days, for which reason the u-tilization period of 125I-labelled reagents is about 4 months.

Nick-translation labelling _ The principle of this method is -to displace one of the nucleotides in the nucleic acid with a radioactive onte, upon which the whole DNA molecule becomes labelled.
This can be carried out according to the method published by Rigby, P.W.J. et al. (J. Mol Biol. (1977) 113, 237-251). In the reaction the DNA becomes labelled when the solution contains 125I-labelled deo~ynucleoside triphosphate as sub-strate, in this case 125I-dCTP (Radiochemical Centre, Amersham: ~1500 Ci/mmol). Under optimal conditions a specific ac-tivi-ty of 109 cpm/~g DNA can be obtained. The labelled DNA is purified from nucleotides remaining in -the reaction mixture by simple gel filtration, eg. using BioGel P30 (BioRad).

Other labelling methods The single-s-tranded nucleic acid reagent produced in M13 mp7-phage can be labelled by chemical iodination, in which a reactive 1~5I is added covalently to the nucleic acid (Commerford, S.L. (1971) Biochemistry 10, 1993-2000, Orosz, J.M. and We-tmur, J.G. (1974) Biochemistry 13, 5467-5473). Alterna-tively, the nucleic acid can be made radio-active by endlabelling with radioactive nucleo-tides by terminal transEerase (Roychoudhury, R. and Wu, R. (1980) Meth. Enzymol. 65, 43-62).
The above described preparation o reagents relates to microbes of which the genetic material is in the form of DNA. In the case of RNA viruses the cloning of genome frag-ments takes place af-ter a DNA copy (cDNA) of the virus RNA
has been made with the aid of reverse transcriptase, followed by DNA~polymerase to copy the second DNA strand. Thereafter the DNA is cloned as described above (Salse-, W. (1979) in Genetic Engineering, Ed. A.M. Chakrabar-ty, CRC Press, pp.
53-81)-~ he most suitable cloning method is chosen depend-ing on the microbe used. The hosts as well as -the vectors can vary. Possibilities include ~-phage as vector, other plasmids, cosmids, cloning eg. in Bacillus subtilis bacteria, etc. (Recombinant DNA, Benchmarck Papers in Microbiology, Vol. 15, ~ds. K.J. Denniston and L.W. En~vist, Dowden, Hutchinson and Ross, Inc. (1981); Ish-Horowicz, D. and Burke, J.F. (1981) Nucleic Acids Res. 9, 2989-2998).
Pe,rformance of the tes-t Sample treatment In accordance wi-th the invention, -the microbial nucleic acid to be investigated mus-t be released from within -the microbe i-tself and also from the infected cells, after which i-t mus-t be denatured to -the s:inyle-s-tranded form.
Virus genomes can be liberated by treating the sample material with 1 % sodlum dodecylsulpha-te ~SDS) and destroyiny the proteins protec-ting the genome by proteinase D-treatment (1 mg/ml, 37C, 60 min). Bacterial samples must in addition be broken down using lysozyme- and EDTA-treatment.
If the sample contains large quantities of viscous, high-molecular weight cellular-DNA, this must be sheared at a few sites in order to reduce i-ts viscosity, eg. by sonica--tion or by passiny the sample a few times through a fine needle.
Hybridization Hybridization may take place in 50 ~ formamide (deionized, stored at -20C), in 4 x SSC Denhardt solution (Denhardt, D.T. 1966 Biochem. Biophys. Res. Commun. 23, 641-646) containing 1 ~ SDS and 0.5 mg/ml DNA (salmon sperm or calf thymus) at 37C and usually overnight for 16-20 hours. The filters chosen for the test are incubated in a suitable vessel, to which the hybridization mixture is added and the hybridization is started. The hybridiza-tion mixture contains the pretrea-ted sample to which is added the radio-active nucleic acid reagent(s). The sample and reagents are denaturated together by boiling for 5 minutes followed by quick cooling at 0C. The hybridization mixture also contains concentrated formamide-, SSC- and Denhardt-solutions, with are pipetted to the dena-tured and cooled nucleic acid mixture.
After mixing, -the hybridization mixture is pipetted to the filters in the hybridization vessel. After hybridization the filters are carefully washed and counted individually in the y-counter.
The invention will be better understood with refer-ence to the following non restrictive examples.

Example 1 Detec _ n of adenovirus by sandwich hybr ization me hod (Table 1) Tlle detai.ls of this test are given in Table 1.
The sandwich hybridization method can detect virus-DNA from a solution, but the viral genome can equally be well detected from infected cells.
The hybridization back~round was measured in a tube containing only -the filter and the labelled nucleic aci.d reagen-t, without the samp]e. The background results from the pBR322 sequences occurring in the labelled nucleic acid reagent. These sequences hybridize dlrectly with the filter without the sample mediating it. Filters containing calf thymus and no DNA were used in the -test as controls, indicating one hand the specificity of hybridization and on the other hand the level of the nonspecific background arising eg. from insufficient washing.
In the table 1, the background due to the reagents was subtracted from the cpm-values hybridized to the filters.
Table I
Adenovirus test Sample Fil-ters (cpm) _ _ _ Adeno 1) Calf thymus 2) Blank 3) .
Adenovirus type 2-DNA
(BRL) (500 ng) 9000 49 HeLa-cells (6 x 105) lnfected with adenoviruc 8200 -Filters:
1) Ad2D-pBR322-plasmid, 2 ~g
2) Calf thymus DNA 1 ~g (Boehringer Mannheim) ~3~
3) Bland (no DNA) Labelled nucleic acid reagent:
Ad2-BamHI C-fragmen-t, purified, specific aetivity 90 x 106 cpm/~lg (200000 cpm 5I/reaction) Hybridization:
50 ~ formamide, 4 x SSC
Denhardt solution, con-taining 0,5 mg/ml salmon sperm DNA and 1 % SDS/ 37C, 16 h Washing:
0,1 x SSC, room temperature, 40 min Samples -Adenovirus type 2 DNA (BRL) Infection with type 2 adenovirus -took place in HeLa-cells. The cells were then disrupted by treatment with 1 % SDS, followed by digestion with 1 mg/ml proteinase-K-enzyme (Sigma) for 30 min 37C. Before denaturation the sample was passed through a fine needle. The values appearing in table 1 were eorrected by subtraction of the reagent background, obtained by carrying out a similar hybridization but without sample.

Example 2 Deteetion of an RNA-virus with the aid of sandwich hybridiza-tion (Table 2) The modes RN~-virus used was the Semliki Forest virus (prototype strain, obtained from the London School of Hygiene and Tropical Medieine), of which the genome is single-stranded RNA. Using -the virus genome as a template, cDNA was produced, which was cloned into the pstI site of pBR322 plasmid as described by Garo~f et al. (Proc. Na-tl.
Acad. Sci. (1980) USA 77 6376-6380). The recombinant plasmid thus obtained is pKTH312 KTL no. EH 232. The insert of this plasmid originating from the virus genome is about ,f~

1~00 nucleotides lony and is from the struc-tural protein area, approximately from nucleo-tide 200 to nucleo-tide 1600 when numbering is started Erom the heginniny of the struc-tural genes (Garoff, H. et al. 1980). For -the produc-tion of reagent the whole recombinan-t plasrnid pKr~1312 was linear-ized wi-th EcoR:[ restriction enzyme (BRL) as -the sequence originating from the Semliki Fores-t virus does not contain recognition sites for the EcoRI-enzyme, and the linearized plasmid was cut into -two fragments using XhoI-enzyme (BRL).
The restriction site of the latter was located with -the Serrlliki Forest virus sequence. The larger EcoRI-XhoI-fragment A (about 3900 base pairs) was attached to the filter and the smaller fragment B (abou-t 1850 base pairs) was labelled with 125I using the nick translation techni~ue.
Both free Semliki Forest virus and virus-infected cells were used as samples in this tes-t. In both cases the virus-specific nucleic acids of the sample were composed en-tirely of RNA.
Table 2 De-tection of Semliki Forest virus wi-th the aid of the sand--wich hybr dization me-thod Sample Filters (cpm) , _ _ Semliki Calf Blank 3) Forest thymus 2) virus 1) __ _ Semliki Forest virus 30 ~g 3340 _ 33 ~ells infected with Semliki Forest virus (5 x 105) 2698 8 10 Non-infected cells 10 5 8 Filters:

:~ ~3~

1) EcoRI-XhoI-fragment A (1.2 llg) of -the pK'L'11312 plasmid 2) Calf -thymus DNA 1 ~Ig 3) Blank (no ])NA) Labelled nucleic acid reagen-ts:
_.
EcoRI-~hoI-Eragment B of the plasmicl pKT~1312, specific activity 90 x 106 cpm/llg NDA (200000 cpm 5I/
reaction).
Hybridization:
As described in Table 1 Washing:
As described in Table 1 Samp_:
Semliki Forest vlrus (30 ,ug) was disrupted with SDS before -the test. The infected cells were handled as described in Table ]. The infection with Semliki Forest virus was carried out in BHK-21 cells.
The values given in table 2 were correc-ted by substraction of the reagen-t bac]cground, obtained from a similar hybridization without sample.
- Example 3 Detection of the viral RNA messenger of a virus sample by sandwich hybridization (Table 3) I`he filter hybridization reagen-ts were produced from SV40-virus DNA (BRL) by cutting the DNA into two parts using PstI-enzyme (Boehringer Mannheim) as described by Lebowitz and Weissman (Curr. Topics in Microbiol. Immunol.
87, 43-172) and the fragments were isolated and purified by agarose gel electrophoreses. Fragmen-t A (4000 base pairs) was radioactively labelled with 125I by nick translation and fragmen-t B (1220 base pairs) was attached to the filter.
The DNA fragments were chosen so that each con-tained areas coding for both early and la-te messengers.
Thus fragment B contains abou-t 700 bases from the structural v protein gene VPl and over 600 bases Erom the gene for early messengers. Because the DNA of SV40 virus is in ,itself a covalen-tly closecl ring, i-t cannot be detected by -the test before linearizati,on. Therefore, when infected cells are used as sample, i-t is possible -to test how wall -the method is adaptable to the detection of RNA copies of the viral genome. As can be seen from the resul-ts yiven in Table 3, the tes-t is excellently suited to the investigation of infected cells. The table also demonstrates that the same reagen-ts can be used -to investigate both the viral DNA and mRNA made from it.
Table 3 Detection of SV40-virus by the sandwich hybridization techniclue Sample Fil-ters (cpm) Test 1 SV40 1) Calf thymus 2) SV40 viral DNA (50 ng) (linearized) 20061 159 104 No sample _ _ _ Test 2 CVl-cells infec-ted wi-th SV40-vi rus 40 h after infection (106 cells) 30814 294 580 jNon-infected cells Filters~
1) The shorter fragment PstI B (0.2 ~g) of the circular SV40-virus DNA digested with PstI-restriction enzyme 2) Calf thymus DNA 1 ~,g 3) Blank (no DNA) Labelled nucleic acid reagent:
The longer Ps-kI A-fragrnent of the SV40-virus DNA, specific activity 28 x 106 cpm/l.lg DN~ (200000 cpm I/
reaction) IIybridization:
As described in Table 1 The hybridization time is 40 h Washing:
As descxibed in Table 1 _m~
SV40-virus DNA (BRL) was linearized with EcoRI
restriction enzyme (BRL). CV:L-cells (Biomedical Centre, Upsala University) were infected with SV40-virus (obtained from Janice Y. Chou and Robert G. Martini, NIH, Be-thesda) and the cells were harves-ted ~0 h after infecti.on. Treat-ment of the sample was as described in Table 1~
The values presented in the table were corrected by substraction of the reagen-t background, obtained from a similar hybridization carried out without sample.
Example 4 Detection of Bacillus amyloliquefaciens by sandwich hybridization (Table. 4) The reagents were fragments of the ~--amylase gene of B. amyloliquefaciens E18 (Technical Research Centre of Finland, VTT), which were isolated for the purpose of this test from the recombinant plasmid pKTI-I10 (Palva, I., et al.
(1981) Gene, 15 43-51) by treatment with restric-tion enzyme and subsequent agarose gel electrophoresis. The fragments used for this test were the ClaI-EcoRI fragment (460 base pairs) (ClaI Boehringer Mannheim) and the EcoRI-BamHI fragment (1500 base pairs). The EcoRI-BamHI fragment was attached -to the fil-ter and the ClaI-EcoRI fragmen-t was labelled with 125I by nick translation.
As can be seen from Table 4 the B. amylol~
aciens in a sample was iden-tifiable by sandwich hyhridiza--tion on the basi.s o:E the single ~-amylase gene. E. coli gave a negative result in this test ~indistinguishable frorn the hackground).
Table 4 Bacterial diagnostics b~ sandwich hybridization _ Sample Filters (cmp) ~-amylase 1) Calf thymus 2) Blank 3) __ __ _ pKTH10-plasmid-DNA
(linearized) 1 ~Ig 5773 47 No sample _ _ _ R. co:Li HB101 (109) _ _ Bacillus amylolique-faciens (3 x~ 10 ) 3377 _ Bacillus am lolique-faciens (10~) 2871 I ~ _ _ Filters:
1) EcoRI-BamHI fragment of the ~-amylase gene from plasmid pKTH10, 0.35 ~g 2) Calf thymus DNA, 1 ~Ig .
3) Blank (no DNA) Labelled nucleic acid reagent:
The ClaI-EcoRI fragment of the ~ amylase gene from plasmid pKTH10, specific activity 35 x 106 cpm~g (200000 cpm I/reaction) Hybridizatio :
As described in Table 1.
Washing:

l.,~C~

As described in Table 1 Bacterlal samples were -treated with lysozyme (67 llg/m]) for 30 min at 37C; 5 mM EDTA was added -to E. coli samples, too. ~fter -the treatment SDS was added to all the the samples (final concentration 12 ~), which were than passed twice through a fine needle to reduce their viscosity before being denatured by boiling as described in the tex-t relating to handling of samples.
The values appearing in the table have been corrected by substraction of the reagent background, obtained from a similar hybridiza-tion without sample.

Examp e 5 _ ample oE a reagent combination ]cit using the sandwich _~bridi~ation method according to the invention (Table 5) The samples investigated in this test were cells infected by -three viruses (adenovirus, SV40 virus and Herpex simplex virus) and a sample containing Bacillus amylolique faciens bacteria. The following reagents were all simulta-neously added to each sample:
5 filters, each containing one type of DNA from SV40 virus, adenovirus, Bacillus amyloliquefaciens ~-amylase gene and calf thymus;
a filter containing no DNA a-t all; and 200000 cpm of each of the following labelled nucleic acid reagents: SV40 virus-, adenovirus- and ~-amylase gene DNA-reagent.
This example showed that it is possible, without division or dukytuib of the sample, to investigate simulta-neously a suitable series of microbes by adding a suitable combination of reagents to the sample. The sample may contain bo-th viral and bacterial nucleic acid. The ilters can be recognized by a sign ~marker -tags), which identifies t~2~

the sequence it contains and which microbe was attached/
hybridized to i-t. The slgrls can be numbers or letters, e.g. 1 or SV40 2 or ~d etc. or other marker such as * for SV40 or Q Eor l~D or o for Bacillus.
Table 5 ~Cit based on the sandwich hybridiza-tion technique Sample Filters (cpm) SV40 1) Adeno 2) ~Y~a~lase CalEBlank 5) 3) thymus 4) _ _ _ Cells infected with SV40 virus (106)18390 2 13 22 31 Cells infected with adenovirus type 2 (6 x 105) _ 8750 5 13 Cells inEected with Her~ex simpleY~ virus _ _ _ 5 13 Bacillus lol 2 0 an~y ique15 8 6500 16 5 Non-infected cells _ _ F i l t_:
1 ) As in Table 3 2 5 2 ) As in Table 3 ) As in Table 4
4) Calf thymus DNA, 1 ,ug
5 ) Blank (no DNA) Labelled nucleic acid reagents:
SV40 virus as in Table 3 Adenovirus as in Table 1 c~-amylase gene as in Table 4 Hybridization:
As in Table 1 _ ] g _ 2~
Washing:
As in Table 1 Samples-.

Cell samples infected with SV40 virus and adeno--virus have been described in Tables 3 and 1, respec-tively.
106 Vero cells were infected wi-th l-lerpes simplex virus type 1. The cells were harves-ted 20 h post infection as cytopathic effect could be observed. The sample was treated as described for adenovirus infected cells (Table 1).
Bacillus am loliquefaciens sam le-_ Y p .
As in Table 4 The values given in table 5 were corrected by substraction of the reagent background, obtained by carrying out a similar hybridization without sample.
Example 6 -Detection of Escherichia coli by sandwich hybridization (Table 6) The reagents were prepared from the ompA-gene (outer membrane protein A-gene) of Escherichia coli.
The hybrid plasmids pKTH40 and pKTH45, used as starting material, were prepared from the pTU100 plasmid described by Henning e-t al. (1979) Proc. Natl. Acad. Sci.
USA 76, 4360-4364.
The plasmid pKTH45 (deposited at KTL or National Public Health Institute, Helsin]ci No..... ), used as a filter reagent, was composed of 740 base pairs from 5' -terminal end of the ompA-gene inserted into the pBR322-plasmid.
The plasmid pKTH40 contains 300 base pairs from the 3' -terminal end of the ompA-gene and the immediately following 1700 base pairs from the genome of E. coli. The pKTH40 plasmid was cleaved with the BamHI restriction enzyme to receive the DNA fragment of E. coli, which contains the 1700 base pairs, as mentioned above. ~his fragment was -transferred to -the single-stranded bac-teriophage M13mp7 according to the methods described by Messing et al. (1981), Nucl. ~cids Res. 9, 309-321, Heiaecker e-t al. (lg80), Gene 10, 69-73, and Gardner et al. (1981), Nucl. Acids Res.
9, 2871-2888. The recombination-phage mKTH1207 (deposited at KTL no. ...... ) was labelled with 125I-iso-tope as de-scribed on page 9 under the heading Other labellincl methods~
and was used as a probe in the sandwich hybridization method.
DNA from disrupted E. coli cells, as well as isolated, purified DNA from E. coli were detected by sand-wich hybridization as shown in table 6.
Table 6 Detection of Escherichia coli by sandwich hybridization Sample Fil-ters (cpm) _ ompA 1) Calf thymus 2) Blank 3) E. coli K12 HB101 DNA
a) 2 x 107 282 _ E. coli K12 HB101 DNA
a) 2 x 108 2206 _ E. coli K12 HB101 Cel]s b) 2 x 107 1113 _ E coli K12 HB101 Cells bj 2 x 108 2327 12 a) number of DNA-moleeules, b) number of cells Fil-ters:
1) pKI'H45 plasmid 1,088 ~g (2 x 10 1 molecules) 2) Calf thymus DNA 1,08~ ~g 3) Blank (no DNA) Labelled nueleie aeid reacJents:
mKTH1207, specific ac-tivi-ty 8 x 10 cpm/~g DNA (200000 .~.t~ 2~
cpm/reaction) Hybridi~a-tion:
4 x SSC, 1 x Denhardt solution wi-thout BSA (bovi.ne serum albumin), 0,25 ~ SDS, 200 ~g/ml ~lerring sperm DNA, 17,5 h~ -~65C
Washing:
As described in table 1 Samples:
E. coli K12 Hsl01 - DNA was isolated according to the Marmur-method described by Marmur (1961) J. Mol. Biol.
3, 208-218. DNA was denatura-ted at 7 mM NaOH, 100C, 5 min.
The cells were treated with lysozyme (500 ~g/ml), EDTA (70 mM -~37C, 30 min), SDS (0,25 ~ 65C) and the free DNA was denatura-ted by boiling at 1~ mM NaOE-I, ~100C, 5 mln).
The values presented in the table have been corrected by substraction of -the reagen-t background obtained from a similar hybridization without sample.

Claims (18)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A microbial diagnostic method, based on sand-which hybridization of nucleic acids on a solid carrier, in which a pair of nucleic acid reagents derived from a given microbe or microbial group and not capable of hybridizing with each other, is used for the identification of said given microbe or microbial group in a given sample, one of said nucleic acid reagents being a single stranded nucleic acid fragment attached to a solid carrier, the other of said nucleic acid reagents being another single stranded nucleic acid fragment labelled with a suitable marker, said method being characterized in that all the microbes or microbial groups in one single, undivided sample are identified simulta-neously by contacting each pair of nucleic acid reagents in a single step with the nucleic acids contained in the sample after said nucleic acid corresponding to the microbe or microbial group to be identified has been rendered single stranded, whereby a given nucleic acid of the sample hybridizes to the complementary nucleic acid fragment annealed to the solid carrier and the carrier-attached hybrid becomes labelled due to the subsequent hybridization of the complementary labelled nucleic acid fragment to the nucleic acid of the sample, after which the carrier-attached label is measured.
2. A microbial diagnostic reagent combination comprising at least one pair of nucleic acid reagents for each microbe or microbial group to be identified in a sample, said reagents consisting of two nucleic acid fragments not capable of hybridizing with each other, said fragments being derived from the same microbe or microbial group and produced either directly from the microbial genome or by using recombinant DNA techniques, said group- or species- specific nucleic acid fragments being rendered single-stranded, one of them being attached to a solid carrier while the other is labelled with a suitable marker.
3. A reagent combination according to claim 2 for the identification of adenovirus, wherein the nucleic acid reagent attached to the carrier is a adenovirus recombinant plasmid Ad2DpBR322 and the labelled nucleic acid reagent is an adenovirus Ad2-BamHI C-fragment.
4. A reagent combination according to claim 2 for the identification of the Semliki Forest virus, wherein the solid carrier attached nucleic acid reagent is a EcoRI-XhoI
fragment A of pKTH312 plasmid and the labelled nucleic acid reagent is a EcoRI-XhoI fragment B of pKTH312 plasmid.
5. A reagent combination according to claim 2 for the identification of SV40 virus, wherein the solid carrier attached nucleic acid reagent is a PstI B fragment of SV40 virus and the labelled nucleic acid reagent is a PstI A
fragment of SV40 virus.
6. A reagent combination according to claim 2 for the identification of Bacillus amyloliquefaciens, wherein the solid carrier attached nucleic acid reagent is a EcoRI-BamHI fragment of the .alpha.-amylase gene of the Bacillus amyl-oliquefaciens plasmid pKTH10 and the labelled nucleic acid reagent is a ClaI-EcoRI fragment of .alpha.-amylase gene of Bacillus amyloliquefaciens plasmid pKTH10.
7. A microbial diagnostic reagent combination for the identification of different DNA- and RNA viruses and bacteria selected in the group consisting of adenovirus, S-40 virus and Bacillus amyloliquefaciens, in one single sample containing nucleic acids originating from said different viruses and bacteria, comprising a first set of reagents con-sisting of Ad2DpBR322 recombinant plasmid of adenovirus, PstI B fragment of SV40 virus and EcoRT-BamHI fragment of the .alpha.-amylase gene of the Bacillus amyloliquefaciens plasmid pKTH10, each attached to a carrier, and a second set of reagents consisting of Ad2-BamHI C fragment of adenovirus PstI A fragment of SV40 virus and ClaI-EcoRI fragment of the .alpha.-amylase gene of the Bacillus amyloliquefaciens plasmid pKTH10, each labelled with a marker.
8. A reagent combination according to claim 2 for the identification of E. coli, wherein the solid carrier attached nucleic acid reagent is a plasmid pKTH45 and the labelled nucleic acid reagent is a recombination-phage mKTH1207.
9. A method for identifying nucleic acids by a one-step sandwich hybridization test, said method compris-ing the steps of:
(a) rendering the nucleic acids in the sample to be identified single-stranded;
(b) allowing said single-stranded nucleic acids of the sample to hybridize simultaneously in a single step with a combination of at least one pair of nucleic acid reagents, said reagents having been purified, the first nucleic acid reagent of said pair comprising a single-stranded fragment of nucleic acid affixed to a solid carrier, the second nucleic acid reagent of said pair comprising a single-stranded fragment of nucleic acid labeled with a detectable label, said first and second nucleic acid reagents being capable of forming hybrid molecules by complementary base pairing with given sequences of the sample nucleic acid to be identified, provided that the second nucleic acid reagent cannot hybridize with the first nucleic acid reagent:
(c) washing said solid carrier to substantially remove said label which is not incorporated in said hybrid molecule; and (d) measuring said label on the washed solid carrier, whereby determining whether the sample contains the nucleic acid to be identified.
10. The method of claim 9, wherein the first nucleic acid reagent is a DNA or RNA fragment.
11. The method of claim 9 or 10, wherein the second nucleic acid reagent is a DNA or RNA fragment.
12. The method of claim 9, wherein the solid carrier is a nitrocellulose sheet.
13. The method of claim 9, wherein the label is a radioisotope.
14. A kit for the detection of nucleic acids with a one-step sandwich hybridization test, the kit comprising in packaged combination a container of at least one pair of nucleic acid reagents, said reagents having been purified:
(a) a first nucleic acid reagent of said pair comprising a single-stranded fragment of nucleic acid affixed to a solid carrier, said first nucleic acid reagent being capable of forming a double-stranded hydrid molecule by complementary base pairing with a given sequence of the nucleic acid to be identified and (b) a second nucleic acid reagent of said pair comprising a single-stranded fragment of nucleic labeled with a detectable label, said second nu-cleic acid reagent being capable of forming a double-stranded hybrid molecule by complementary base pairing with a given sequence of the nucleic acid to be identified, provided that the second nucleic acid reagent cannot hybridize with the first nucleic acid reagent.
15. The diagnostic kit of claim 14, wherein the first nucleic acid reagent is a DNA or RNA fragment.
16. The diagnostic kit of claim 14 or 15, wherein the second nucleic acid reagent is a DNA or RNA fragment.
17. The diagnostic kit of claim 14, wherein the solid carrier is a nitrocellulose sheet.
18. The diagnostic kit of claim 14, wherein the label is a radioisotope.
CA000413539A 1981-10-16 1982-10-15 Method and reagent combination for the diagnosis of microorganisms Expired CA1192120A (en)

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