JPH0632637B2 - Combination of microbial identification method and microbial identification reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for identifying microorganisms that mainly involves sandwich hybridization of nucleic acids onto a solid support, and to a combination of reagents used in the method.

In traditional microbial identification, the presence of a microorganism in a given sample is established by isolating the microorganism in question. After enrichment, the microorganism is identified based on its biochemical properties or using immunological methods. This type of identification requires that the microorganism in the sample be viable. Furthermore, identification by isolation is a laborious process, requiring 4-6 weeks in the case of viruses.

The present invention relates to a method of identification in which the presence of microorganisms in a sample is established by identifying the genetic material, i.e., nucleic acids, obtained using sensitive and specific nucleic acid hybridization techniques.
It is an old and well-known method for investigating the identity of nucleic acids. Complementary nucleic acid strands have the ability to form tight double-stranded structures according to the rules of base pairing, and the resulting hybrids can be separated from the remaining single-stranded nucleic acids.

Several methods based on nucleic acid identification have already been used for the differentiation of microorganisms.
coli) was identified from fecal samples by colony hybridization using the toxin-producing gene as a probe. Positive hybridization was verified by autoradiography (Moseley, SL et al., J. Infect. Dis. (1980) 1
42. 892-898). The colony hybridization method is based on the method first developed by Grunstein and Hogness (Proc. Natl. Acad. Sci. USA (1975) 72. 3961-
Hybridization has also been used as a method to differentiate between herpes simplex virus types 1 and 2 (Brautigam,
AR et al., J. Clin. Microbiol (1980) 12 , 226-234). However, this method was not a rapid identification method, and the virus type was determined after enrichment culture.
In this method, double-stranded hybrids are separated from nucleic acid components that remain single-stranded in solution by affinity chromatography.

Recently, it has been shown that DNA from cells infected with Epstein-Barr virus (sample) can be directly fixed onto a filter after appropriate pretreatment. The nucleic acid of interest is identified by hybridizing the filter with a radioactive probe, and positive hybridizations are detected by autoradiography (Brandsam, I. & Mi
ller, K. (1980) Proc. Natl. Acad. Sci. USA 77 . 6851
-6855).

A method similar to that described above is disclosed in German Patent No. 2,950,299.
German Patent No. 2,950,295, a method for producing a radioactively labeled DNA probe by recombinant DNA technology, is described in the specification of German Patent No. 2,950,295 and in The Lancet, October 10, 1982, pp. 765-767. The above-mentioned West German Patent No. 2,950,295 describes a method for producing a radioactively labeled DNA probe by DNA recombinant technology. This DNA probe is made from hepatitis B virus. The above-mentioned Lancet also describes the use of this probe in a method for detecting hepatitis B virus. In this method, DNA obtained from a sample is transferred to a nitrocellulose filter, and hepatitis DNA from the fixed sample is detected using this probe as a reagent.

The present invention aims to provide a novel method for distinguishing microorganisms using two reagents and the reagents. Because the method of the present invention uses two reagents, it is highly specific compared to conventional methods and allows the creation of different kits. This allows various microorganisms or groups of microorganisms to be detected from the same sample without impairing the specificity of the differential test for each target microorganism or group of microorganisms. In the method of the present invention,
Two different nucleic acid fragment reagents (one affixed to a solid support and the other labeled with an appropriate label) are used, and the sample DNA is in a liquid state.

The invention described herein is based on the sandwich hybridization technique (Dunno et al., 2001).
The technique is based on the method described by Hassell, AR & Hassell, JA (1977) Cell 12 , 23-36, and simplifies sample handling and detection of hybridization, making the technique particularly suitable for diagnostic applications.

In the method described in this invention, all desired microorganisms or groups of microorganisms can be identified from one and the same sample containing denatured single-stranded nucleic acid strands of the microorganisms or groups of microorganisms to be identified, without separating the samples. This method requires two nucleic acid reagents for each microorganism or group of microorganisms to be identified. The reagents are two separate nucleic acid fragments generated from the genome of the microorganism to be identified, which do not share any causal linkage in common and are preferably located close to each other in the genome. The reagents can be generated directly from the genome of the microorganism or by using established recombinant DNA techniques. One of the two nucleic acid fragments is denatured and then immobilized on a solid support, preferably a nitrocellulose filter.
The remaining single-stranded nucleic acid is labeled with a suitable label. When two different reagents, i.e., the nucleic acid reagents described above, for each immobilized microorganism or group of microorganisms are contacted with the immobilized single-stranded nucleic acid in the sample, the nucleic acid anneals to a complementary nucleic acid fragment on the solid support. The hybrids thus formed on the support become labeled after annealing with a labeled complementary nucleic acid fragment. The labeled nucleic acid fragments do not hybridize alone to the nucleic acid fragments fixed to the support, but only to the corresponding single-stranded nucleic acid from the sample. Thus, only those supports to which complementary nucleic acid from the sample has hybridized can become labeled. These supports are easily washed, and their labels can be measured by established methods.

The method according to the invention is applicable in principle to all DNA containing microorganisms such as viruses, bacteria, fungi and yeasts.
This method has the distinct advantage of allowing all bacteria and viruses of interest to be identified simultaneously and from the same sample, regardless of whether they contain DNA or RNA. A preferred combination of reagents allows a "kit" to be developed in which each microorganism to be identified has its own solid support with a labeled nucleic acid reagent. All filters included in the reagent combination can be added to the sample simultaneously with the labeled nucleic acid reagent. Once hybridization has occurred, the solid supports are washed and their labels are measured. The only supports that remain labeled are those that contain a causal linkage complementary to the microbial genome in the sample being investigated.

This method and reagent combination are used, for example, in pharmaceutical microbiology, veterinary microbiology, and food hygiene research, as well as in the microbial identification of plant diseases. Suitable sample materials include blood, feces, and all animal and plant tissue homogenates, such as nasal and urethral mucus, as well as patient secretions. The method is considered sufficiently sensitive to detect levels of microorganisms commonly present in clinical samples. Of course, preliminary enrichment by culturing the microorganisms present in the sample prior to identification testing is possible, and in some cases may be necessary. This method is also suitable for investigating samples in which the microorganisms can no longer be cultured but contain a significant amount of microbial contamination (e.g., after antibiotic treatment has been initiated), or when culturing the microorganisms is particularly time-consuming and difficult (e.g., anaerobic bacteria present in large numbers in purulent samples in the case of infections caused by anaerobic bacteria).

This method employs the following combination of reagents in the form of a "kit", although some of the reagents may of course be used separately.

Respiratory infections a) Bacteria: β-Streptococcus (A-group), Haemophilus influenzae, Streptococcus pneumoniae, Mycoplasma pneumoniae, Mycobacteria b) Viruses: Influenza A, Influenza B,
Parainfluenza 1-3, respiratory syncytial virus, adenovirus, coronavirus, rhinovirus diarrhea a) Bacteria: Salmonella, Shigella, Versinia enterocolitica, enterotoxigenic Escherichia coli, Clostridium difficile
difficile, Campylobacteria b) Viruses: Rotavirus, Parvovirus, Adenovirus, Enteric viral diseases a) Bacteria: Neisseria gonorrhoeae, Treponema pallidum, Chlamydia trachomatis b) Viruses: Herpex monocytogenes c) Yeasts: Candida albicans d) Protozoa: Tricomonas vaginalis Sepsis a) Bacteria: β-Streptococcus (group A), Streptococcus pneumoniae, Enteric bacteria as a single group Food hygiene a) Bacteria: Salmonella, Clostridium perfringens (group A)
Depending on the choice of agent, by selecting identifying agents from common genetic regions, the specificity of the differentiation can be limited to a particular genus of microorganisms (e.g., Salmonella) or an entire group of microorganisms (e.g., enterobacteria).

The nucleic acid reagents required for the sandwich hybridization technique described in this invention are produced by recombinant DNA techniques.
The reagent preparation and testing procedures for Example 1 are described below.

The reagent adenovirus type 2 (strains deposited at KTL, e.g., the National Institute for Public Health, Helsinki) was cultivated and purified, after which DNA was isolated (Petterson, U & Sambrook, J.
(1973) J. Mol. Biol. 73 , 125-130) (hereinafter simply Ad ₂ -D
This DNA was digested with BamHI restriction enzyme (BRL, Bethesda Research Laboratories) which split it into four reproducible fragments. Two of the four fragments were:
With the help of T4-ligase (BRL), the vector plasmid pB
The fragments were inserted into the BamHI site of R322 (BRL) (the fragments were not separated before ligation, but the inserts added to the plasmids were identified individually only after cloning).
gal ^- , pro ^- , leu ^- , hrs ^- , hrm ^- , recA, str ^r , F ^- ) (KTL
(obtained from the University of California, San Diego, CA) was modified with plasmid DNA constructed from a recombinant plasmid, i.e., a molecule that received a fragment of adenovirus DNA (Cohen, SN et al. (1972) Proc.
c. Natl. Acad. Sci. USA 69 , 2110-2114). Among the modified bacterial clones, those most likely to contain the recombinant plasmid were selected. Ampicillin and tetracycline resistance genes were transferred to the bacteria using the pBR322 plasmid (Boliver F. et al. (1977) Gene 2 , 95-11
3) However, bacteria containing recombinant plasmids
The BamHI restriction site is located within the tetracycline gene, and foreign DNA inserted into this region will disrupt the gene, making it sensitive to tetracycline. The insert of the plasmid was characterized by determining the size of the restriction fragment after BamHI digestion using agarose gel electrophoresis after plasmid enrichment.
The adjacent BamHI D- and C-fragments of Ad2 _- DNA (cf.
Gene map) was selected as a test subject (Soderlund, H.
(1976) Cell 7 , 585-593). The desired recombinant plasmids, Ad ₂ C-pBR322, KTL No. E 231 and Ad ₂ D-pBR322, KTL No.
o. EH230 was cultured and purified as described in the literature (Cle
w-ell, DB & Helinski, DR (1969) Proc. Natl. Aca.
d. Sci.USA 62 , 1159-1166).

The recombinant plasmid Ad ₂ D-pBR322 was used as a filter reagent. Removal of the plasmid causal linkage was unnecessary in this method, since the sample did not contain the pBR322-causal linkage. However, for radioactive labeling, the nucleic acid was separated from the pBR322-DNA by agarose gel electrophoresis after BamHI digestion. The C-fragment was isolated by phenol extraction or electroelution.
The antibody was isolated from LGT-agarose (Marine Colloids, Inc.) by the method described in (Wieslander, L. (1979) Anal. Biochem.
98 , 305-309) and concentrated by ethanol precipitation.

To avoid background contamination of the labeled nucleic acid reagent by direct hybridization of residual plasmid sequences with the filter, it is particularly advantageous to subclone the nucleic acid fragment selected for labeling into another vector. The single-stranded DNA phage M13mp7 (BRL) is used as the vector of choice, into which DNA fragments obtained by BamHI digestion can be readily transferred (Messing J. et al. (1981) Nucl
eic Acids Res. 9 , 309-323).

Attachment of DNA to filters The recombinant plasmid Ad ₂ D-pBR322 was denatured to a single-stranded form and randomly nicked at several sites by treatment with 0.2 N NaOH (5 min, 100°C).
The NA was cooled and neutralized immediately before transfer to the filter, and the transfer solution, 4x SSC medium/ice (SSC = 0.15M NaCl, 0.015M
The DNA was pipetted into four times the volume of the 4x SSC solution (since it was sodium citrate, this meant ice-cold 0.6M sodium chloride and 0.06M sodium citrate). Filters (Schleicher & Schull BA85 nitrocellulose) were thoroughly wetted (approximately 2 hours) in 4x SSC solution before applying the DNA. DNA was attached to the filters in dilute solution (0.5-1.0 μg/ml) by drawing the solution through the filter under a slight vacuum. Filters can absorb up to approximately 180 μg/ ^cm² of DNA (Kafatos, FC et al. (1979) Nucleic Acids Res. 7 ,
1541-1552). We used DNA concentrations between 0.5 μg DNA/2.5 cm filter diameter and 1.0 μg DNA/0.7 cm filter diameter. After DNA filtration, the filters were washed in 4×SSC, dried at room temperature, and finally baked in a vacuum oven for 2 hours at 80°C. The DNA on the filters then remains stable, and the filters can be stored at room temperature for long periods of time (Southern, EM (1
975) J. Mol. Biol. 98 , 503-517).

Labelling of radioactive nucleic acid fragments The radioactive label used was ^{the 125} I-isotope.
This isotope can be detected using gamma counters available in most large laboratories.
The half-life of this isotope is 60 days, giving a useful life of ¹²⁵ I-labeled agents of 4 months.

Nick-translation labeling: The principle of this method is to replace one of the nucleotides in a nucleic acid with a radioactive one, which makes the entire DNA molecule labeled. This is described by Rigby,
The method published by PWJ et al. (J. Mol. Biol. (197
7) 113 , 237-251). In this reaction,
The solution contains ^125I -labeled deoxynucleotide triphosphates, in this case ^125I -dCTP (Radiochemica
DNA becomes radioactively labeled when the substrate contains a specific activity of 10 ^cpm /µg of DNA (I Centre, Amersham; >1500 Ci/mmol).
The labeled DNA is purified from the remaining nucleotides in the reaction mixture by simple gel filtration, for example using BioGel P30 (Bio-Rad).

Alternative labeling methods Single-stranded nucleic acid reagents produced in M13mp7 phage are labeled by chemical iodination, in which reactive ^125I is covalently attached to the nucleic acid (Commenford, S.
L. (1971) Biochemistry 10 ,1993-2000, Orosz, JM &
Wetmur, J.G. (1974) Biochemistry 13 ,5467-5473).
Alternatively, nucleic acids can be made radioactive by end-labeling with radioactive nucleotides via terminal transposition (Roychoudhury, R & Wu, R. (1988)
0) Meth. Enzymol. 65 , 43-62).

The preparation of the above-mentioned reagents concerns microorganisms whose genetic material is in the form of DNA. In the case of RNA viruses, cloning of genomic fragments occurs in such a way that a DNA copy of the viral RNA (cDNA) is first made with the help of the opposite transcriptase, then the second DNA strand is copied by DNA polymerase, after which the DNA is cleaved as described above (Salser,
W. (1979) Genetic Engineering, Ed. AM Chakraba
rty, CRC Press, pp 53-81).

The most preferred cloning method is selected depending on the microorganism being used. Not only the vector but also the host may vary. Vectors include λ-phage, other plasmids,
It may also contain cosmids, cloning in Bacillus subtilis bacteria, etc. (Recombinant DNA,
Benchmarck Papers in Microbiology, Vol 15, Eds,
KJ Denniston & LWEnqvist, Dowden, Hutchinson &
Ross, Inc. (1981): Ish-Horowicz, D. & Burke, JF.
(1981) Nucleic Acids Res. 9 ,2989-2998).

Test execution Sample processing The nucleic acids of the microorganisms to be investigated must be removed from the microorganism itself and from the infected cells, and then denatured to a single-stranded form. The viral genome is removed by treating the sample material with 1% sodium dodecyl sulfate (SDS) and then denaturing the proteins that protect the genome with proteinase K.
By destroying it by treatment (1 mg/ml, 37°C, 60 minutes),
The bacterial sample must be further fractionated using lysozyme and ETDA treatment.

If the sample contains a large amount of viscous, high molecular weight cellular DNA, the sample must be sheared in several places to reduce its viscosity, for example, by sonication or by passing the sample several times through a fine needle.

Hybridization hybridization was performed in 4×SSC Denhardt's solution containing 1% SDS and 0.5 mg/ml DNA (salmon sperm or calf thymus).
ardt, DT (1966) Biochem Biophys. Res, Commun, 23 ,
641-646) in 50% formaldehyde (deionized and stored at -20°C) at 37°C and 16-20°C.
The hybridization is carried out for 1 hour, usually overnight. The filters selected for testing are incubated in a suitable container, to which the hybridization mixture is added and hybridization begins. The hybridization mixture contains: (a) pretreated samples (which contain 5
(b) a mixture containing concentrated formamide, SSC, and Denhardt's solution, which is pipetted into the denatured and cooled nucleic acid mixture (a). After mixing, the hybridization mixture is pipetted onto the filters in the hybridization vessel. After hybridization, the filters are carefully washed and individually counted in a γ-counter.

The invention will be clarified by the following several examples.

Example 1 Detection of Adenovirus by Sandwich Hybridization (See Table 1) Test details are provided in the text up to Table 1. The sandwich hybridization method can detect viral DNA from solutions, but viral genomes can be detected equally well from infected cells.

Hybridization background is measured in tubes containing only the filter and labeled nucleic acid reagent without sample. The background is generated by pBR322 sequences occurring in the labeled nucleic acid reagent. These sequences hybridize directly to the filter without the sample to mediate it. A filter containing calf thymus and no DNA is used in the test as a control. This indicates on the one hand the specificity of the hybridization and on the other hand the level of nonspecific background caused, for example, by insufficient washing.

In the following table, the reagent background was subtracted from the cpm values hybridized to the filters.

Filter: 1) _Ad2D -pBR322-plasmid, 2 μg 2) Calf thymus DNA 1 μg (Boehringer Mannheim) 3) Blank (no DNA) Labeled nucleic acid reagent: _Ad2 -BamHI C-fragment, purified, specific activity 90x1
^0.6 cpm/μg (200,000 cpm ¹²⁵ I/reaction) Hybridization: 50% formamide, 4x SSC, 0.5 mg/ml salmon sperm D
Denhardt's solution containing NA and 1% SDS, 37°C;
Washing: 0.1% SSC, room temperature, 40 min. Sample: Adenovirus type 2 DNA (BRL). Infection with adenovirus type 2 occurred in HeLa cells. The cells were then treated with 1% SDS and 1 mg
/ml proteinase-K enzyme (Sigma) at 37°C;
The fragments were digested for 30 minutes. Before denaturation, the samples were passed through a fine needle. The values presented in the table were corrected by subtracting the reagent background.
The background of the reagent was obtained by carrying out a similar hybridization without the sample.

Example 2: Detection of RNA viruses by sandwich hybridization (Table 2)
The model RNA virus used was Semliki Forest virus (London School of Hygiene and Tropical Medicine).
The virus was a prototype strain obtained from the University of California, San Diego (1989). Its genome is single-stranded RNA. Using the viral genome as a template, cDNA was generated as described by Garoff et al. (Proc. Natl. Acade. Sci. (1988)
0) USA 77 , 6376-6380) into the PstI site of the pBR322 plasmid. The recombinant plasmid thus obtained is pKTH312 KTL No. EH232. The insert of this plasmid, which originates from the viral genome, is approximately 1400 nucleotides long and spans the protein region of the construct, from approximately nucleotide 200 to nucleotide 1600 (numbering starts from the beginning of the construct gene) (Garoff, H. et al. 1980). For reagent production, the entire recombinant plasmid pKTH312 was transformed into E. coli.
Concatenation with the coRI restriction enzyme (BRL) (the Semliki Forest virus-derived conjugate does not contain a recognition site for the EcoRI enzyme), and the concatenated plasmid xhoII enzyme (BRL).
The latter restriction site was located in the Semliki Forest virus sequence. The larger EcoRI-XhoI fragment A (approximately 3900 base pairs)
adheres to the filter and the smaller fragment B (approximately
The fragment (1850 base pairs) was labeled with ¹²⁵ I using the "nicking" technique.

Both free Semliki Forest virus and virus-infected cells were used as samples in this test. In both cases, the virus-specific nucleic acids of the samples were all R
It consisted of NA.

Filter: 1) EcoRI-XhoI fragment A of the pKTH312 plasmid
(1.2 μg) 2) 1 μg calf thymus DNA 3) Blank (no DNA) Labeled nucleic acid reagent: EcoRI-XhoI-fragment B of plasmid pKTH312, specific activity 90×10 ⁶ cpm/μg DNA (200,000 cpm ¹²⁵ I/reaction) Hybridization: As described in Table 1.

Cleaning: As described in Table 1.

Sample: Semliki Forest virus (30 μg) was diluted with SD before testing.
The infected cells were treated as described in Table 1. Infection with Semliki Forest virus was
This was carried out in BHK-21 cells.

The values shown in the table were corrected for reagent background obtained by a similar hybridization without the sample.

Example 3 Viral samples in which viral messenger RNA is detected by sandwich hybridization (see Table 3) Sandwich hybridization reagents were prepared as described by Lebowitz & Weissman (Curr. Topics in Microbiol. Immunol. 2002).
nol. 87 , 43-172), PstI-enzyme (Boehringer Mannheim)
SV40 DNA was cut into two parts using
- viral DNA (BRL). The fragments were isolated and purified by agarose gel electrophoresis. Fragment A (4000 base pairs) was purified by nicking with ¹²⁵ I.
and fragment B (1220 base pairs) was attached to the filter.

The DNA fragments were selected so that each contained coding regions for both fast and slow messengers: fragment B
contains approximately 700 bases from the component protein gene VPI and more than 600 bases from the gene for the fast messenger. Because SV40 viral DNA is a covalently closed ring, it is impossible to detect it by the test before it becomes chain-like. Therefore, if infected cells are used as samples, it is possible to determine how well the method is suited to detecting RNA copies of the viral genome. As is clear from the results in Table 3, this test is very well suited to examining infected cells. The table also demonstrates that the same reagent can be used to examine both viral DNA and the mRNA produced from it.

Filter: 1) PstI-digested circular SV40 virus D
1) the shorter fragment PstIA of SV40 virus DNA (0.2 μg); 2) 1 μg of calf thymus DNA; 3) blank (no DNA). Labeled nucleic acid reagents: the longer PstIA fragment of SV40 virus DNA, specific activity 28×10 ⁶ cpm/μg DNA (200,000 cpm ¹²⁵ I/
Reaction) Hybridization: As described in Table 1. Hybridization time is 40 hours. Washing: As described in Table 1.

Sample: SV40 viral DNA (BRL) was linearized with EcoRI restriction enzyme (BRL). CV1 cells (Biomedical Center, Uppsala,
University) is the SV40 virus (Janice Y. Chou & Ro
(obtained from Dr. Robert G. Martini, NIH, Bethesda)
Cells were harvested 40 hours post-infection. Sample treatment was as described in Table 1.

The values shown in the table were corrected for reagent background obtained from similar hybridizations performed without the sample.

Example 4 Detection of Bacillus amyloliquefaciens by sandwich hybridization (see Table 4) The reagent was B. amyloliquefaciens E18 (Technical R
A fragment of the α-amylase gene from the Research Center of Finland, VTT was isolated for this test by restriction enzyme digestion and subsequent agarose gel electrophoresis from the recombinant plasmid pKTH10 (Palva, I. et al. (1981)
The fragment used in this test was isolated from the ClaI-EcoRI fragment region (460 base pairs) of the α - amylase gene (ClaI Boehringer Mann
The EcoRI-BamHI fragment was attached to a filter, and the ClaI-EcoRI fragment was radioactively labeled with ¹²⁵ I by nicking.

As can be seen from Table 4, B. amyloliquefaciens in the samples was identified by sandwich hybridization based on the single α-amylase gene, and E. coli gave a negative result in this test (it was not recognizable from the background).

Filter: 1) Ec of the α-amylase gene from plasmid pKTH10
1) Calf thymus DNA, 1 μg; 2) Blank (no DNA); Labeled nucleic acid reagent: ClaI-E fragment of the α-amylase gene from the plasmid pKTH10.
coRI fragment, specific activity 35×10 ⁶ cpm/μg (200000 cpm
¹²⁵ I/reaction) Hybridization: As described in Table 1 Washing: As described in Table 1 Sample: Bacterial samples were incubated with lysozyme (67μL) at 37°C for 30 minutes.
The E. coli samples were treated with 5 mM EDTA (2% final concentration). After this treatment, SDS was added to all samples (2% final concentration), which were then passed twice through a fine needle to reduce their viscosity before being denatured by boiling, as described in the text regarding sample handling.

The values presented in the table were corrected for reagent background obtained by similar hybridizations without the sample.

Example 5: Performance of the Reagent Combination Kit by Sandwich Hybridization (See Table 5) The samples investigated in this test were cells infected with three viruses (adenovirus, SV40 virus, and herpes simplex virus) and a sample containing Bacillus amyloliquefaciens bacteria. The following reagents were added simultaneously to each sample, five filters: SV40 virus, adenovirus, and Bacillus amyloliquefaciens alpha. The filters were then used to infect the SV40 virus, adenovirus, and Bacillus amyloliquefaciens alpha.
-amylase gene and one type D from calf thymus
One containing NA and one containing no DNA. In addition, 200,000 cpm of the following labeled nucleic acid reagent was added:

SV40 virus, adenovirus, and α-amylase gene DNA reagents. This experiment demonstrates that by adding a combination of reagents to a sample, it is possible to simultaneously investigate an appropriate array of microorganisms without dividing or diluting the sample. The sample may contain both viral and bacterial nucleic acids. The filter may be signed (marked)
The signature identifies the chain that the filter contains. The microorganisms were attached to the filter and hybridized. The signature may be a number or letter, such as 1, SV40 2, or Ad, or other designation such as * for SV40, △ for AD, or ○ for Bacillus.

Filter: 1) Same as Table 3 2) Same as Table 1 3) Same as Table 4 4) Calf thymus DNA, 1 μg 5) Blank (does not contain DNA) Labeled nucleic acid reagent: SV40 virus as in Table 3, adenovirus as in Table 1,
Same α-amylase gene hybridization as in Table 4: Same washes as in Table 1: Same samples as in Table 1: Cell samples infected with SV40 virus and adenovirus are listed in Tables 3 and 1, respectively.

¹⁰⁶ Vero cells were infected with herpes simplex virus type 1. The cells were harvested 20 hours postinfection to allow for cellular effects. Samples were treated as described for adenovirus-infected cells (see Table 1).

Bacillus amyloliquefaciens sample: The values in the table, same as Table 4, are corrected for the reagent background obtained by performing an equivalent hybridization without the sample.

Example 6 Detection of E. coli by sandwich hybridization (see Table 6) A reagent was made from the ompA gene (outer membrane protein A gene) of E. coli.

Hybrid plasmids pKTH40 and pKTH used as starting materials
45 is from Henning et al. (1979) Proc. Natl. Acad. Sci. USA 7
It was prepared from the pTU 100 plasmid described in [6 , 4360-4364].

Plasmid pKTH 45 used as a filter reagent
(e.g. National Public Health Institute, Helsinki
The plasmid pBR3 (deposited at the KTL as No. ...) consists of 740 base pairs, stretching from the 5' end of the ompA gene to the
22 inserted into the plasmid.

Plasmid pKTH40 contains 300 kDa from the 3' end of the ompA gene.
The pKTH40 plasmid was cleaved with the BamHI restriction enzyme to receive the E. coli DNA fragment (which contains the above 1700 base pairs). This fragment was cloned using the BamHI restriction enzyme from Messing et al. (1981), Nucl. Acids Res. 9 , 309.
-321, Heidecker et al. (1980), Gene 10 , 69-73, Gardner
(1981) Nucl. Acids Res. 9 , 2871-2888, using the single-stranded bacteriophage M13mp7.
The recombinant phage mKTH 1207 (deposited at KTL, No.
...) was labeled with ¹²⁵ I isotope as described above under "Other labeling methods" and used as a probe in sandwich hybridization.

DNA from the cloned E. coli cells was isolated,
As with purified DNA from E. coli, it can be detected by sandwich hybridization as shown in Table 6.

Filter: 1) pKTH45 plasmid 1.088 μg (2 × ¹⁰ molecules) 2) Calf thymus DNA 1.088 μg 3) Blank (contains no DNA) Labeled nucleic acid reagent: mKTH 1207, specific activity 8 × 10 ^cpm /μg DNA (200,000 cpm
/reaction) Hybridization: 4x SSC, 1x Denhardt's solution without BSA (bovine serum albumin), 0.25% SDS, 200 μg/ml Hering sperm DNA, 17.5 hours, +65°C Washing: same as in Table 1 Samples: E. coli K12 HB101-DNA was prepared according to the method of Marmur (1961) J.Mo1. Boil.
3 , 208-218, DNA was denatured with 7 mM NaOH (+100°C, 5 min).

The cells were incubated in lysozyme (500 μg/ml), EDTA (70 mM, +37
The DNA was then treated with 0.25% SDS (+65°C, 30 min), and the free DNA was denatured by boiling in 14 mM NaOH (+100°C, 5 min).

The values shown in the table were corrected for reagent background obtained from a similar hybridization without the sample.

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Claims (8)

[Claims] Claim 1: Derived from a specific microorganism or group of microorganisms,
A method for distinguishing microorganisms based on sandwich hybridization of nucleic acids on a solid support using at least a pair of nucleic acid reagents, one of which is a single-stranded nucleic acid fragment attached to a solid support and the other of which is another single-stranded nucleic acid fragment labeled with an appropriate label, wherein the pair of nucleic acid reagents are incapable of hybridizing with each other but are capable of hybridizing to different positions of a target nucleic acid contained in a microorganism or group of microorganisms to be identified;
A method for simultaneously identifying all microorganisms or groups of microorganisms to be identified in a single, undivided sample by first rendering the target nucleic acid contained in the microorganism or group of microorganisms single-stranded and then contacting each pair of nucleic acid reagents with all nucleic acids contained in the sample in a single step, thereby hybridizing the specified target nucleic acid in the sample with its complementary nucleic acid fragment attached to the solid support, and labeling the hybrids attached to the solid support by hybridization with the labeled nucleic acid fragment complementary to the specified target nucleic acid, and then measuring the label attached to the support. Claim 2: Derived from a specific microorganism or group of microorganisms,
A method for distinguishing microorganisms based on sandwich hybridization of nucleic acids on a solid support using at least a pair of nucleic acid reagents, one of which is a single-stranded nucleic acid fragment attached to a solid support and the other of which is another single-stranded nucleic acid fragment labeled with an appropriate label, wherein the pair of nucleic acid reagents are incapable of hybridizing with each other but are capable of hybridizing to different positions of a target nucleic acid contained in a microorganism or group of microorganisms to be identified;
A microorganism differentiation reagent for use only in a method for simultaneously identifying all microorganisms or groups of microorganisms by making target nucleic acids contained in a microorganism or group of microorganisms to be identified in a single, undivided sample single-stranded, and then contacting each pair of said nucleic acid reagents with all nucleic acids contained in the sample in a single step, thereby hybridizing the predetermined target nucleic acids in the sample with complementary nucleic acid fragments attached to said solid supports, and labeling the hybrids attached to the solid supports by hybridization with the labeled nucleic acid fragments complementary to the predetermined target nucleic acids, and then measuring the labels attached to the supports, said method comprising the steps of:
A combination of microbial differentiation reagents, each pair corresponding to a respective microorganism or group of microorganisms to be identified in a sample, characterized in that the pair of nucleic acid reagents consists of two nucleic acid fragments that are mutually non-hybridizable but complementary to different positions in the nucleic acid of the microorganism to be identified, the fragments being derived directly from the genome of the microorganism or using DNA recombinant technology, and the two nucleic acid fragments of the microorganism are single-stranded, one of which is attached to a solid support and the other is labeled with an appropriate label. 3. The combination of reagents according to claim 2 for the identification of adenovirus, wherein the nucleic acid reagent attached to the carrier is the adenovirus recombinant plasmid Ad ₂ DpBR3.
22, and the labeled nucleic acid reagent is an adenovirus Ad
₂ -BamHI C-fragment, a combination of microbial differentiation reagents. 4. A combination of reagents according to claim 2 for the identification of Semliki Forest virus, wherein the nucleic acid reagent attached to the carrier is EcoRI-XhoI fragment A of the pKTH312 plasmid and the labeled nucleic acid reagent is pKTH31
The EcoRI-XhoI fragment B of the 2 plasmid is a combination of microbial differentiation reagents. 5. The combination of reagents of claim 2 for identifying the SV40 virus, wherein the nucleic acid reagent attached to the solid support is the PstI B fragment of the SV40 virus and the labeled nucleic acid reagent is the PstI B fragment of the SV40 virus.
A combination of microbial differentiation reagents, which are tI A fragments. Claim 6: Bacillus amyloliquefaciens (Bacil
The reagent combination of claim 2 for the identification of Bacillus amyloliquefaciens, wherein the nucleic acid reagent attached to the solid support is Bacillus amyloliquefaciens.
the EcoRI-BamHI fragment of the α-amylase gene of Bacillus amyloliquefaciens (B) plasmid pKTH10, and the labeled nucleic acid reagent is
The microbial differentiation reagent combination is the ClaI-EcoRI fragment of the α-amylase gene of the plasmid pKTH10 of Acillus amyloliquefaciens. [Claim 7] A combination of reagents according to claim 2 for identifying Escherichia coli (E. coli), wherein the nucleic acid reagent attached to the solid support is the plasmid pKTH45 and the labeled nucleic acid reagent is the recombinant phage mKTH1207. 8. A method for producing a strain of a virus comprising an adenovirus, an SV40 virus, and a bacillus amyloliquefaciens strain.
different DNA- and R-selected from the group consisting of
10. A combination of microorganism differentiation reagents according to claim 2 for identifying NA viruses and bacteria in a single sample containing nucleic acids derived from these different viruses and bacteria, comprising the adenovirus recombinant plasmid Ad ₂ DpBR322, the PstI B fragment of SV40 virus, and the Bacillus amyloliquefaciens plasmid pKTH, each attached to a carrier.
A first set of reagents consisting of ten EcoRI-BamHI fragments of the α-amylase gene and adenovirus Ad ₂ -BamHI C-fragments each labeled with a label;
PstI A fragment of SV40 virus and Bacillus amyloliquefaciens
and a second set of reagents consisting of the ClaI-EcoRI fragment of the α-amylase gene of pKTH10.