JP2015039320A - Simultaneous detection method of multiple bacteria and/or simultaneous quantitative method of multiple bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of detecting and quantitating multiple bacteria simultaneously all at once.SOLUTION: The invention relates to a simultaneous detection method of at least five kinds of bacteria and/or a simultaneous quantitative method of at least five kinds of bacteria, wherein bacterium genes are detected and/or quantitated by mixing primers and probes respectively specific to the at least five kinds of bacterium genes which can be amplified by PCR (polymerase chain reaction) under same reaction condition, with DNAs extracted from a sample to perform PCR under the reaction condition. The invention also relates to a kit for simultaneously detecting and/or quantitating at least five kinds of bacteria, the kit containing a primer consisting of any nucleotide sequence among sequences of sequence numbers of 1-36.

Description

  The present invention relates to a method for simultaneous detection and / or quantification of a plurality of bacteria.

Prompt detection and quantification of pathogens is critical for proper treatment, prevention of infection spread, and assessment of infection risk. However, since there are multiple types of pathogens, it has been difficult to detect them simultaneously by the conventional detection / quantification technique.
The quantitative PCR method is a method for quantifying the abundance of a target gene by monitoring the amount of a PCR reaction product, and is roughly divided into a Sybr Green method and a TaqMan probe method. The quantitative PCR method using a TaqMan probe is suitable for quantifying a gene present in a trace amount because of its high detection sensitivity and specificity. Many methods have been reported for quantitatively detecting individual pathogens by using PCR primers and TaqMan probes specific for the virulence factor gene sequences (for example, Malorny et al. 2004 (Non-patent Document 1). )). Two to four pathogens can be simultaneously quantified by multiplex quantitative PCR using fluorescent dyes with different wavelengths (LaGier et al. 2004 (Non-patent Document 2)), but more There is still no method for quantitative detection of various pathogens simultaneously.

  On the other hand, as an attempt to detect and quantify multiple pathogens, an approach that analyzes a large amount of 16S rRNA gene is attracting attention. A PhyloChip based on microarray technology, a method based on a pyrosequencing method (Ye and Zhang 2011 (non-patent document 3)), and the like are known. However, in the analysis for 16S rRNA gene, it is impossible to detect a 16S rRNA gene that cannot be distinguished from non-pathogenic E. coli, such as pathogenic E. coli.

  In recent years, there have been many attempts to apply integrated circuit design techniques used in semiconductors to the biotechnology field. One of these technologies is a miniaturization laboratory that allows chemical and biochemical experiments to be performed on a single chip. These are collectively referred to as Lab-on-a-chip (LOC). Yes. In 2011, VereFoodborne, a multi-species pathogen detection kit using LOC, was announced by Veredus Laboratorie (Singapore). However, this kit is difficult to apply to risk assessment and management because it is not quantitative. In addition, a technique for detecting and quantifying multiple pathogens by quantitative PCR using the Sybr Green method using a Biotrove OpenArray system (Applied Biosystems, California, USA) has been reported (Stedtfeld et al. 2008 (Non-patent Document 4). )), However, has not yet become widespread due to insufficient detection sensitivity.

  In 2007, Bioig, a company that performed quantitative PCR using the TaqMan probe method on LOC, was sold by Fluidigm (headquartered in California, USA). So far, it has been mainly used as an alternative to microarray analysis, such as gene expression analysis in human cells and gene mutation detection (Spurgeon et al. 2008 (Non-Patent Document 5)). There are no examples of application to quantitative detection of species pathogenic microorganisms.

Malorny B, Paccassoni E, Fach P, Bunge C, Martin A, Helmuth R. 2004. Diagnostic real-time PCR for detection of Salmonella in food.Appl.Environ.Microbiol. 70: 7046-7052. LaGier MJ, Joseph LA, Passaretti TV, Musser KA, and Cirino NM. 2004.A real-time multiplexed PCR assay for rapid detection and differentiation of Campylobacter jejuni and Campylobacter coli. Mol. Cell.Probes 18: 275-282. Ye L, Zhang T. 2011. Pathogenic bacteria in sewage treatment plants as revealed by 454 pyrosequencing. Environ Sci. Technol. 45: 7173-7179. Stedtfeld RD, Baushke SW, Tourlousse DM, Miller SM, Stedtfeld TM, Gulari E, Tiedje JM, Hashsham SA. 2008. Development and experimental validation of a predictive threshold cycle equation for quantification of virulence and marker genes by high-throughput nanoliter-volume PCR on the OpenArray platform. Appl. Environ. Microbiol. 74: 3831-3838. Spurgeon SL, Jones RC, Ramakrishnan R. 2008. High throughput gene expression measurement with real time PCR in a microfluidic dynamic array.PLOS One 3: e1662.

  An object of the present invention is to provide a technique for simultaneously detecting and quantifying a plurality of bacteria.

  The present inventors designed a qPCR assay by the TaqMan probe method targeting 18 genes that can be reacted under the same conditions. In addition, a total of 20 assays were used in this study, combining two previously established TaqMan qPCR assays. As a result, 9 types of pathogenic bacteria typical of food poisoning and aquatic infections (pathogenic Escherichia coli, Salmonella, Campylobacter, Listeria, Legionella, Clostridium perfringens, Shigella, Vibrio parahaemolyticus, Vibrio cholerae), 1 general Escherichia coli, process control A TaqMan qPCR assay for the NH8B strain could be constructed.

  For quantification, a plasmid into which the target gene had been incorporated was linearized, and a serial dilution after concentration measurement by the PicoGreen colorimetric method was used as a calibration curve. Conventional qPCR (hereinafter C-qPCR) uses ABI Prism 7000 Sequence Detection System (Applied Biosystems), and microfluidic device qPCR (hereinafter MF-qPCR; Fig. 1) uses BioMark (Fluidigm). went.

  Prior to MF-qPCR, DNA pre-amplification (Specific Target Amplification; STA) reaction was performed. This is a 14-cycle multiplex PCR performed by mixing the primer pairs used in the above 20 assays. We carefully examined whether the STA reaction affects the quantitative performance of qPCR. In addition, we performed C-qPCR and MF-qPCR using genomic DNA extracted from 35 strains of 11 types of microorganisms, and verified the specificity of the assay.

Human feces were inoculated with 9 pathogens and process control strains alone or mixed to extract DNA. In addition, E. coli O157: H7 and a process control strain were inoculated into the lake water so that each concentration would be 10 ¹ to 10 ⁷ cells / L, followed by pressure filtration. The bacterial suspension was recovered from the membrane after filtration, and DNA was extracted. C-qPCR and MF-qPCR were performed on DNA obtained from feces and environmental water.

In this study, we were able to develop 20 specific and sensitive TaqMan qPCR assays. The assay had high amplification efficiency (90-110%) and a wide amplification range (2-2 × 10 ⁶ copies / μL) (FIG. 2). Since the calibration curve for the STA reaction took a small Cq value (y-axis in Fig. 2), it was found that the detection sensitivity increased. The slope of the calibration curve was the same regardless of the presence or absence of the STA reaction, so it was judged that there was no significant difference in amplification efficiency. Furthermore, there was no significant difference in the amplification range regardless of the presence or absence of STA reaction. Based on the above, it was determined that the STA reaction did not affect qPCR quantitative performance.

  As a result of specificity verification using a pure strain, target genes (iap, hlyA) were not amplified from Listeria monocytogenes serovar 4c JCM7679, but each target gene was detected from other strains (Fig. 3). . Therefore, the TaqMan qPCR assay developed in this study was judged to be specific.

When pathogens were inoculated into feces and environmental water samples, only the inoculated strain could be detected specifically (FIGS. 4 and 5). Specific pathogens could also be detected specifically from samples such as feces, which contain a large number of contaminating bacteria. The detection sensitivity at that time was 500 to 5 × 10 ⁴ cells / g stool and 100 cells / L water, respectively. When the bacterial cells were inoculated in serial dilution, the quantitative value decreased according to the amount of the bacterial cells (FIG. 6). Therefore, quantification by MF-qPCR may be applicable to environmental samples. The present invention has been completed based on these findings.

The gist of the present invention is as follows.
1. Primers and probes specific for each of at least five bacterial genes that can be amplified by PCR under the same reaction conditions are mixed with DNA extracted from the sample, and PCR is performed under the reaction conditions to detect and / or detect bacterial genes. Alternatively, a method for simultaneous detection and / or quantification of at least five kinds of bacteria, characterized in that quantification is performed.
2. PCR is performed using a primer comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 34, and non-pathogenic E. coli, pathogenic E. coli, Salmonella, Campylobacter, Listeria, Legionella, Welsh bacteria, Shigella, Vibrio parahaemolyticus, and Vibrio cholerae 2. The method according to 1, wherein at least 5 kinds of bacteria selected from the group consisting of are simultaneously detected and / or quantified.
3. 3. The method according to 2, wherein PCR is performed using at least one primer pair selected from the group consisting of the following primer pairs (1) to (17).
(1) A primer pair consisting of the nucleotide sequence of SEQ ID NO: 1 and a primer consisting of the nucleotide sequence of SEQ ID NO: 2 (target gene: ftsZ)
(2) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 3 and primer consisting of nucleotide sequence of SEQ ID NO: 4 (target gene: uidA)
(3) Pair of primer consisting of the nucleotide sequence of SEQ ID NO: 5 and primer consisting of the nucleotide sequence of SEQ ID NO: 6 (target gene: eaeA)
(4) A primer pair comprising the nucleotide sequence of SEQ ID NO: 7 and a primer comprising the nucleotide sequence of SEQ ID NO: 8 (target gene: stx ₁ )
(5) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 9 and a primer consisting of the nucleotide sequence of SEQ ID NO: 10 (target gene: stx ₂ )
(6) Pair of primer consisting of the nucleotide sequence of SEQ ID NO: 11 and primer consisting of the nucleotide sequence of SEQ ID NO: 12 (target gene: ipaH 7.8)
(7) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 13 and primer consisting of nucleotide sequence of SEQ ID NO: 14 (target gene: ipaH all)
(8) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 15 and a primer consisting of the nucleotide sequence of SEQ ID NO: 16 (target gene: virA)
(9) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 17 and primer consisting of nucleotide sequence of SEQ ID NO: 18 (target gene: invA)
(10) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 19 and a primer consisting of the nucleotide sequence of SEQ ID NO: 206 (target gene: ttrC)
(11) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 21 and a primer consisting of the nucleotide sequence of SEQ ID NO: 22 (target gene: cadF)
(12) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 23 and a primer consisting of the nucleotide sequence of SEQ ID NO: 24 (target gene: ciaB)
(13) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 25 and primer consisting of nucleotide sequence of SEQ ID NO: 26 (target gene: cpe)
(14) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 27 and primer consisting of nucleotide sequence of SEQ ID NO: 28 (target gene: plc)
(15) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 29 and a primer consisting of the nucleotide sequence of SEQ ID NO: 30 (target gene: mip)
(16) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 31 and primer consisting of nucleotide sequence of SEQ ID NO: 32 (target gene: iap)
(17) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 33 and a primer consisting of the nucleotide sequence of SEQ ID NO: 34 (target gene: hlyA)
4). Determine the recovery rate of the added internal standard bacteria by adding bacteria as the internal standard of known concentration to the target sample, performing DNA extraction and PCR, and detecting and quantifying the internal standard bacteria The method according to any one.
5. 5. The method according to 4, wherein Pseudogulbenkiania sp. NH8B strain is used as an internal standard bacterium, and PCR is performed targeting NH8B_3641.
6). 6. The method according to 5, wherein PCR is performed using NH8B_3641 as a target, using a primer pair consisting of the nucleotide sequence of SEQ ID NO: 35 and a primer sequence consisting of the nucleotide sequence of SEQ ID NO: 36.
7). Before performing PCR targeting bacterial genes that can be amplified under the same conditions, DNA amplification is performed by mixing all kinds of primer pairs used in the PCR and performing multiplex PCR. The method of crab.
8). A standard curve is prepared in advance by linearizing a plasmid into which a target gene has been incorporated, measuring the concentration, and then serially diluting it, and using this calibration curve, the amount or concentration of bacteria in the target sample is obtained 1 The method in any one of -7.
9. A kit for simultaneous detection and / or quantification of at least 5 types of bacteria, comprising a primer comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 36.

  According to the present invention, a plurality of bacteria can be simultaneously detected and / or quantified. The method of the present invention is expected to be applicable as a rapid diagnostic tool for pathogen identification and as an accurate risk assessment technique. Moreover, it is thought that it can be used as a safety evaluation method for drinking water, foods, etc. by using it for quantification of fecal indicator bacteria (E. coli).

Principle of quantitative PCR (MF-qPCR) method using microfluidic equipment. In actual MF-qPCR, up to 96 samples and 96 targets of qPCR (9,216 reactions / run in total) can be performed in one run. Calibration curves from 20 TaqMan qPCR assays. The calibration curve (●) prepared after STA reaction and the calibration curve (◯) prepared without STA reaction are shown for each assay. In addition, the equation of the calibration curve and the r ² value are shown. Specificity verification of 20 TaqMan qPCR assays. DNA was extracted from a pure strain of the pathogen and subjected to TaqMan qPCR. The same results were obtained with C-qPCR and MF-qPCR. Application to stool samples. Human feces were inoculated with pathogens of known concentration and subjected to MF-qPCR after DNA extraction. Quantitative values by MF-qPCR are shown in gray scale (the lighter the color, the larger the quantitative value). Sample names Mix E6 to Mix E2 represent 10 pathogens (enterohemorrhagic Escherichia coli (EHEC) O157, Salmonella, Campylobacter, Clostridium perfringens, Legionella, Listeria, Vibrio cholerae, Vibrio parahaemolyticus, and process control NH8B). All were mixed and inoculated at a concentration of 10 ⁶ to 10 ² . Application to environmental water samples. Intestinal hemorrhagic Escherichia coli (EHEC) O157 and process control NH8B strain were mixed in lake water and inoculated at a concentration of 10 ⁷ to 10 ¹ . Quantitative values by MF-qPCR are shown in gray scale (the lighter the color, the larger the quantitative value). Relationship between the amount of inoculum and the quantitative value by MFq-PCR. A regression line was drawn with the concentration of pathogen inoculated in the environmental water sample on the x-axis and the quantitative value by MF-qPCR on the y-axis. The four regression lines are based on the quantitative results targeting eaeA (◇), stx1 (□), stx2 (△), and NH8B_3641 (●). Alignment of nucleotide sequences used to design qPCR primers. Sequences were selected to cover the target gene diversity. Primer and probe annealing sites are shown above the aligned nucleotide sequences. Alignment of nucleotide sequences used to design qPCR primers. Alignment of nucleotide sequences used to design qPCR primers. Alignment of nucleotide sequences used to design qPCR primers. Alignment of nucleotide sequences used to design qPCR primers. Alignment of nucleotide sequences used to design qPCR primers.

  Hereinafter, the present invention will be described in detail.

  In the present invention, a primer and probe specific for each of at least five bacterial genes that can be amplified by PCR under the same reaction conditions are mixed with DNA extracted from a sample, and PCR is performed under the reaction conditions. There is provided a method for simultaneous detection and / or quantification of at least five kinds of bacteria, characterized in that

  Genes targeted by PCR are genes that are present in the bacteria to be detected and / or quantified, and are the same as the PCR reaction conditions for amplifying other bacterial genes to be detected and / or quantified simultaneously. Any material that can be amplified by PCR under reaction conditions may be used. The target gene is preferably a gene that exists specifically in the target bacterium (for example, invA that exists specifically in Salmonella), but even if it is a universally existing gene, it is sufficient if a specific primer can be designed for the target bacterium. . For example, ftsZ is commonly present in Escherichia coli and Salmonella, but due to its high sequence diversity, primers specific to Escherichia coli can be designed. The length of the PCR amplification product may be 60 to 150 nucleotides long, the primer length may be 18 to 27 nucleotides long, and the probe length may be 8 to 30 nucleotides long However, it is not limited to these. PCR reaction conditions are 5 to 30 seconds at 95 ° C (thermal denaturation of DNA strands) and 30 to 60 seconds at 60 ° C (primer annealing and DNA extension reaction), 30 to 40 cycles. It is not limited.

  Examples of the sample include feces, environmental water, tap water, drinking water, tap water, sewage, and the like, but are not limited thereto.

In the method of the present invention, PCR is performed using a primer consisting of any one of the nucleotide sequences of SEQ ID NOS: 1 to 34, non-pathogenic E. coli, pathogenic E. coli, Salmonella, Campylobacter, Listeria, Legionella, Clostridium perfringens, Shigella, At least five kinds of bacteria selected from the group consisting of Vibrio parahaemolyticus and Vibrio cholerae can be simultaneously detected and / or quantified. For example, PCR may be performed using at least one primer pair selected from the group consisting of the following primer pairs (1) to (17).
(1) A primer pair consisting of the nucleotide sequence of SEQ ID NO: 1 and a primer consisting of the nucleotide sequence of SEQ ID NO: 2 (target gene: ftsZ)
(2) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 3 and primer consisting of nucleotide sequence of SEQ ID NO: 4 (target gene: uidA)
(3) Pair of primer consisting of the nucleotide sequence of SEQ ID NO: 5 and primer consisting of the nucleotide sequence of SEQ ID NO: 6 (target gene: eaeA)
(4) A primer pair comprising the nucleotide sequence of SEQ ID NO: 7 and a primer comprising the nucleotide sequence of SEQ ID NO: 8 (target gene: stx ₁ )
(5) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 9 and a primer consisting of the nucleotide sequence of SEQ ID NO: 10 (target gene: stx ₂ )
(6) Pair of primer consisting of the nucleotide sequence of SEQ ID NO: 11 and primer consisting of the nucleotide sequence of SEQ ID NO: 12 (target gene: ipaH 7.8)
(7) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 13 and primer consisting of nucleotide sequence of SEQ ID NO: 14 (target gene: ipaH all)
(8) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 15 and a primer consisting of the nucleotide sequence of SEQ ID NO: 16 (target gene: virA)
(9) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 17 and primer consisting of nucleotide sequence of SEQ ID NO: 18 (target gene: invA)
(10) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 19 and a primer consisting of the nucleotide sequence of SEQ ID NO: 206 (target gene: ttrC)
(11) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 21 and a primer consisting of the nucleotide sequence of SEQ ID NO: 22 (target gene: cadF)
(12) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 23 and a primer consisting of the nucleotide sequence of SEQ ID NO: 24 (target gene: ciaB)
(13) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 25 and primer consisting of nucleotide sequence of SEQ ID NO: 26 (target gene: cpe)
(14) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 27 and primer consisting of nucleotide sequence of SEQ ID NO: 28 (target gene: plc)
(15) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 29 and a primer consisting of the nucleotide sequence of SEQ ID NO: 30 (target gene: mip)
(16) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 31 and primer consisting of nucleotide sequence of SEQ ID NO: 32 (target gene: iap)
(17) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 33 and a primer consisting of the nucleotide sequence of SEQ ID NO: 34 (target gene: hlyA)
In the method of the present invention, by adding a bacterium which is an internal standard of known concentration to the target sample, DNA extraction and PCR are performed, and the internal standard bacterium is detected and quantified, thereby increasing the recovery rate of the added internal standard bacterium. Judgment can be made. In Examples described later, Pseudogulbenkiania sp. NH8B strain was used as an internal standard bacterium, and PCR was performed targeting NH8B_3641. For PCR, a pair of a primer (IAC_23F) consisting of the nucleotide sequence of SEQ ID NO: 35 and a primer (IAC_92R) consisting of the nucleotide sequence of SEQ ID NO: 36 was used. In the experiments shown in FIGS. 5 and 6, environmental water samples were inoculated simultaneously with a known concentration of Pseudogulbenkiania sp. NH8B and a known concentration of Escherichia coli O157: H7 Sakai, and quantified by simultaneous detection after DNA extraction. The Pseudogulbenkiania sp. NH8B strain was quantified by qPCR targeting NH8B_3641, and the E. coli O157: H7 Sakai strain was quantified by qPCR targeting eaeA, stx1, and stx2 genes. As a result, the recovery rate was almost the same for Pseudogulbenkiania sp. NH8B strain and Escherichia coli O157: H7 Sakai strain. From this, it can be said that the Pseudogulbenkiania sp. NH8B strain is suitable as an internal standard (in the examples, process control) for quantifying Escherichia coli O157: H7. Furthermore, since E. coli O157: H7 and other enteropathogenic bacteria have similar cell structures (ie, DNA extraction efficiency is considered to be almost the same), Pseudogulbenkiania sp. NH8B strains are commonly used for enteropathogenic bacteria (such as Salmonella and Campylobacter). It can be used as an internal standard for quantification. NH8B_3641 is mutated and has an extremely short sequence compared to similar genes. It is considered that there is no gene with the exact same sequence as NH8B_3641 except for Pseudogulbenkiania sp. NH8B strain, and when the sequence was actually searched in the database, it did not hit other bacteria. Therefore, it is considered that only the inoculated Pseudogulbenkiania sp. NH8B strain can be specifically detected and quantified by targeting NH8B_3641. Desirable conditions for the internal standard are as follows.
(1) Not present in the target sample (environmental water, food, etc.).
(2) Does not affect the detection and quantification of target microorganisms (eg, pathogenic bacteria, fecal indicator bacteria (E. coli), etc.).
(3) Not pathogenic.
(4) Simple culture and easy inoculation of samples.

  The Pseudogulbenkiania sp. NH8B stock satisfies all of the above.

  In the method of the present invention, before performing PCR targeting bacterial genes that can be amplified under the same conditions, by performing PCR by mixing all kinds of primer pairs used in the PCR, Good. In MF-qPCR, qPCR is performed in a 10 nl chamber. In consideration of dilution by qPCR master mix etc., a DNA sample of at least 222 copies / μl is required to detect and quantify the target gene by MF-qPCR. Since the concentration of pathogenic microorganisms in the environmental sample is often low, a case where this concentration is not satisfied is assumed. When the pre-amplification process called STA reaction is performed, the target gene can be specifically amplified, so this problem can be solved. The STA reaction is a 10-14 cycle multiplex PCR. If the number of STA reaction cycles is increased too much, the quantitative performance may be affected, but the 14-cycle STA reaction does not affect the subsequent qPCR quantitative performance (see Example 1). The target gene in the sample is amplified about 1,000 to 15,000 times by the STA reaction by 14 cycles.

  There are two types of quantitative PCR, the Sybr Green method and the TaqMan probe method. The Sybr Green method uses a reagent that emits fluorescence by binding to double-stranded DNA (intercalator), so there is no need to prepare a probe for each target gene, the cost is low, and the construction of a reaction system is easy However, detection sensitivity and specificity are not high. On the other hand, the method using the TaqMan probe uses different fluorescently labeled probes for each target gene, so the cost is high, but the detection sensitivity and specificity are high. In the present invention, PCR is preferably quantified by the TaqMan probe method. The TaqMan probe is not particularly limited as long as it specifically hybridizes to the template DNA, and is commercially available. In the examples described later, commercially available products (UPL probe: Roche) shown in Table 2 were used. Moreover, the location where a UPL probe hybridizes is shown in FIGS.

  In the method of the present invention, a standard curve is prepared in advance by linearizing a plasmid into which a target gene has been incorporated, measuring the concentration and then serially diluting, and using this calibration curve, The amount or concentration should be determined. In the examples described below, for quantification, the plasmid into which the target gene has been incorporated (one target gene incorporated into one plasmid) is linearized, and after concentration measurement by the PicoGreen colorimetric method, all plasmid solutions are mixed. To prepare a calibration curve solution.

  By carrying out the method of the present invention on a microarray or LOC, a large amount of sample can be analyzed in a short time.

  By the method of the present invention, pathogenic bacteria, fecal contamination indicator bacteria, and the like can be simultaneously detected and / or quantified.

  The present invention also provides a kit for simultaneous detection and / or quantification of at least 5 types of bacteria, comprising a primer comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 36.

The kit of the present invention further includes a cell serving as an internal standard, a medium and an antibiotic for culturing the cell serving as the internal standard, DNA extracted from the cell serving as the internal standard, a DNA extraction reagent, and a fluorescently labeled probe , Materials for preparing calibration curves (eg vectors for cloning PCR products (eg plasmids) and hosts, plasmid isolation reagents, enzymes for linearizing plasmids, gel extraction reagents, DNA concentration measurement Reagent, etc.), buffer, calibration curve, instruction manual, software for converting the amount of bacteria from the measured value, and the like.

Hereinafter, the present invention will be described in more detail with reference to examples.
[Example 1]
1-1. Example 1 shows an example in which a plasmid incorporating a target gene shown in Table 2 is mixed and detected and quantified by a normal PCR apparatus. Reagent adjustment was performed in a HEPA / UV PCR workstation (UVP) to prevent contamination (external DNA contamination).
1-2. For each target gene shown in Table 2, PCR was performed using a forward primer and a reverse primer. The PCR reaction solution was prepared as shown in Table 3, and the PCR reaction was carried out with the following program: 95 ° C. (10 minutes), 30 cycles (95 ° C. (30 seconds), 60 ° C. (30 seconds), 72 ° C. (30 Seconds)). The strain shown in bold in Table 1 was used as the template for the PCR reaction.
1-3. The PCR product was incorporated into pCR 2.1-TOPO vector (Invitrogen) and introduced into E. coli DH5α by electroporation. This operation followed the product manual of pCR 2.1-TOPO vector. The introduced plasmid was amplified in E. coli DH5α and extracted using GeneJET Plasmid Mini Kit (Fermentas). The extracted plasmid was cleaved with Bam HI (Takara Bio), linearized, and purified using FastGene Gel / PCR Extraction Kit.
1-4. The concentration of the linear plasmid after purification was quantified using PicoGreen dsDNA quantification reagent (Molecular Probes) and diluted to 10 ⁸ copies / μl. Since plasmids are prepared for each target gene, 20 types of plasmid solutions (concentration: 10 ⁸ copies / μl) are prepared.
1-5. 2 μl of 20 kinds of plasmid solutions were taken and mixed with 60 μl of PCR grade water (Ambion). By this operation, a mixed solution having a concentration of 2 × 10 ⁶ copies / μl of each plasmid is prepared. This is called the 2E6 standard. 2E6 standard is serially diluted and the concentration of each plasmid is 2 × 10 ⁵ copies / μl, 2 × 10 ⁴ copies / μl, 2 × 10 ³ copies / μl, 2 × 10 ² copies / μl, 20 copies / μl And a 2 copy / μl mixture was made. They are called 2E5 standard, 2E4 standard, 2E3 standard, 2E2 standard, 2E1 standard and 2E0 standard, respectively.
1-6. About each object gene shown in Table 2, it mixed so that the density | concentration of a forward primer and a reverse primer might be 20 micromol, respectively (refer Table 4). This is called a primer pair mix. By the above operation, 20 kinds of primer pair mixes are created.
1-7. In order to carry out the STA reaction, 20 kinds of primer pair mixes were mixed in equal amounts and adjusted so that the concentration of each primer was 0.2 μM (see Table 5). This is called the STA primer mixture.
1-8. The STA reaction is a 14-cycle multiplex PCR using a STA primer mixture. The STA reaction solution was adjusted to a volume of 10 μl as shown in Table 6, and the reaction was performed using ABI 7000 Sequence Detection System (Applied Biosystems). The STA reaction was carried out with the following program: 95 ° C. (10 minutes), 14 cycles (95 ° C. (15 seconds), 60 ° C. (4 minutes)).
1-9. A normal qPCR reaction (C-qPCR) was prepared with a volume of 20 μl as shown in Table 7, and performed using ABI 7000 Sequence Detection System (Applied Biosystems). As template DNA, 2E0-2E6 standard before STA reaction and 2E0-2E6 standard after STA reaction were diluted 60 times with TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0).
1-10. The C-qPCR reaction was performed with the following program: 95 ° C. (10 minutes), 40 cycles (95 ° C. (10 seconds), 60 ° C. (1 minute)). In each cycle, fluorescence intensity readings (FAM and ROX) were taken at 60 ° C. Since FAM is fluorescence generated by the degradation of the TaqMan probe, the fluorescence intensity increases as the PCR product is amplified. ROX is a reference dye included in the qPCR master mix and was used to correct FAM fluorescence intensity.
1-11. The number of cycles (Threshold cycle; Ct) when the fluorescence intensity threshold was reached was determined using ABI Prism 7000 SDS Software (Applied Biosystems). A calibration curve was created by plotting the Ct value on the x-axis and the number of DNA copies contained in the template DNA (the number of DNA copies before the STA reaction when the STA reaction was performed) on the y-axis and drawing a regression line. FIG. 3 shows two calibration curves obtained from 60-fold dilutions of the 2E0-2E6 standard before STA reaction and the 2E0-2E6 standard after STA reaction for each gene. The amplification efficiency (E) of qPCR was determined from the slope of the calibration curve (S) and the following formula and expressed as a percentage (%).
E = 10 ^{(-1 / S)} -1
1-12. Each qPCR assay had high amplification efficiency (90-110%) and a wide amplification range (2-2 × 10 ⁶ copies / μL) (FIG. 2). The slope of the calibration curve was the same regardless of the presence or absence of the STA reaction, and there was no significant difference in the amplification range regardless of the presence or absence of the STA reaction. Therefore, it was determined that the STA reaction did not affect the quantitative performance of qPCR.
1-13. Since the calibration curve for the STA reaction took a small Ct value (y-axis in FIG. 2), it was found that the detection sensitivity was increased. Since the difference in Ct value was 4-8, considering the dilution of the post-STA reaction (60-fold dilution), calculated amounts of template DNA are increased to 960～15,360 times (approximately 2 ^{10-2 14-fold)} become. STA reaction because multiplex PCR of 14 cycles, theoretically gene copy number should increase to 2 ¹⁴ times. From the above, although the amplification factor varies depending on the target gene, it can be determined that the STA reaction is suitable for amplifying a low-concentration DNA copy number.

[Example 2]
2-1. Example 2 shows an example in which the strains shown in Table 1 were simultaneously detected by the C-qPCR method and the MF-qPCR method. Reagent adjustment was performed in a HEPA / UV PCR workstation (UVP) to prevent contamination (external DNA contamination).
2-2. DNA was extracted from the strains shown in Table 1 using DNeasy Blood & Tissue Kit (Qiagen).
2-3. The 2E1-2E6 standard prepared in Example 1 was used as a calibration curve sample for quantification.
2-4. C-qPCR was carried out in the same manner as in Example 1 (1-9 to 1-11).
2-5. Hereafter, MF-qPCR operation is shown. Using the DNA samples shown in 2-2 and 2-3, the STA reaction was performed in the same manner as in Example 1 (1-6 to 1-8). The STA reaction product was diluted 6-fold with TE buffer.
2-6. A sample premix (Table 8) was prepared using the STA reaction product diluted 6-fold.
2-7. A 10X assay (Table 9) was prepared for each target gene shown in Table 2. As the probe, the UPL probe (Roche) shown in Table 2 or the probe shown in SEQ ID NO: 41 or SEQ ID NO: 42 (Table 3) modified with FAM and MGB / NFQ was used.
2-8. The individual package of the Dynamic Array 48.48 chip (Fluidigm) was opened, the control line fluid (Fluidigm) was poured into a predetermined part of the chip, and then inserted into the IFC Controller MX (Fluidigm). The Dynamic Array 48.48 chip was initialized by the Prime (113x) program.
2-9. 5 μl each of the sample premix prepared in 2-6 and the 10X assay prepared in 2-7 were injected into predetermined wells on the Dynamic Array 48.48 chip after initialization. The Dynamic Array 48.48 chip was inserted into the IFC Controller MX, and the reaction solution was injected into the microchannel and chamber by the Load Mix (113x) program and mixed.
2-10. After completion of Load Mix (113x), Dynamic Array 48.48 chip was inserted into BioMark HD Reader (Fluidigm), and qPCR reaction was started. The reaction program is as follows: 50 ° C (2 minutes), 95 ° C (10 minutes), 40 cycles (95 ° C (15 seconds), 70 ° C (5 seconds), 60 ° C (1 minute)). In each cycle, fluorescence intensity readings (FAM and ROX) were taken at 60 ° C. Since FAM is fluorescence generated by the degradation of the TaqMan probe, the fluorescence intensity increases as the PCR product is amplified. ROX is a reference dye included in the qPCR master mix and was used to correct FAM fluorescence intensity.
2-11. The number of cycles (Threshold cycle; Ct) when the fluorescence intensity threshold was reached was determined using Real-Time PCR Analysis software version 3.0.2 (Fluidigm). For the calibration curve samples, Ct values were plotted on the x-axis, DNA copy number before STA reaction was plotted on the y-axis, and a calibration curve was drawn by drawing a regression line. Using the calibration curve, the DNA copy number of the sample of unknown concentration was determined.
2-12. Nine pathogenic bacteria (pathogenic Escherichia coli, Salmonella, Campylobacter, Listeria, Legionella, Clostridium perfringens, Shigella, Vibrio parahaemolyticus, Vibrio cholerae), general Escherichia coli 1 Species, process control NH8B strain could be detected specifically (FIG. 3). The same results were obtained from the C-qPCR method and the MF-qPCR method.
2-13. Enteropathogenic E. coli (EPEC) with eaeA, Shiga-toxin producing E. coli (STEC) with stx ₁ or stx ₂ , eaeA and stx Enterohemorrhagic E. coli (EHEC) and the like possessing ₁ or stx ₂ exist, and these could be identified by this method.
2-14. Shigella is considered to be a type of E. coli in phylogenetics, so it is no doubt that genes (ftsZ and uidA) targeting E. coli are detected.
2-15. For Listeria 4c serotype JCM7679, the target genes (iap, hlyA) were not amplified, but each target gene was detected from other Listeria strains. Since most Listeria isolated from human patients are serotypes 1 / 2a, 1 / 2b, and 4b and isolated cases of 4c serotype are rare, Listeria 4c serotype JCM7679 is not detected by this method This is not expected to have a significant impact on use for diagnostic and public health purposes.

Example 3
3-1. In Example 3, human feces of healthy subjects were inoculated with Escherichia coli O157 strain, Shigella, Salmonella, Campylobacter, Clostridium perfringens, Legionella, Listeria, Vibrio parahaemolyticus, and process control NH8B strain, and by MF-qPCR method. An example of simultaneous detection is shown. Reagent adjustment was performed in a HEPA / UV PCR workstation (UVP) to prevent contamination (external DNA contamination).
3-2. Feces were collected from 6 healthy individuals, Escherichia coli O157 Sakai strain, Shigella RIMD 0509763 strain, Salmonella JCM 1652 strain, Campylobacter jejuni JCM 2013 strain, Clostridium perfringens JCM 1290 strain, Legionella JCM 7571 strain, Listeria JCM 7671 strain, Vibrio cholerae RIMD 2203246, Vibrio parahaemolyticus RIMD 2210633, and process control Psuedogulbenkiania sp. NH8B were inoculated alone or in combination. The inoculum concentration was adjusted to 10 ⁷ cells / 200 mg stool in the case of single inoculation, and 10 ² to 10 ⁶ cells / 200 mg stool in the case of mixed inoculation.
3-3. DNA was extracted from the stool sample prepared in 3-2 using QIAamp DNA Stool minikit (QIAGEN).
3-4. The 2E1-2E6 standard prepared in Example 1 was used as a calibration curve sample for quantification.
3-5. Using the DNA samples shown in 3-3 and 3-4, the STA reaction was performed in the same manner as in Example 1 (1-6 to 1-8). The STA reaction product was diluted 6-fold with TE buffer.
3-6. Sample premixes and 10X assays were made as in Example 2 (2-6 to 2-7).
3-7. The individual package of the Dynamic Array 96.96 chip (Fluidigm) was opened, the control line fluid (Fluidigm) was poured into a predetermined part of the chip, and then inserted into the IFC Controller HX (Fluidigm). The Dynamic Array 96.96 chip was initialized by the Prime (136x) program.
3-8. 5 μl each of the sample premix prepared in 3-6 and the 10X assay were injected into predetermined wells on the Dynamic Array 96.96 chip after initialization. The Dynamic Array 96.96 chip was inserted into the IFC Controller HX, and the reaction solution was injected into the microchannel and chamber by the Load Mix (136x) program and mixed.
3-9. After completion of Load Mix (136x), a Dynamic Array 96.96 chip was inserted into BioMark HD Reader (Fluidigm), and qPCR reaction was started. The reaction program is the same as in Example 2 (2-10).
3-10. In the same manner as in Example 2 (2-11), the obtained qPCR results were analyzed to determine the DNA copy number of the sample with unknown concentration.
3-11. The quantitative results are shown in FIG. Quantitative values by MF-qPCR are shown in gray scale (the lighter the color, the larger the quantitative value). Sample names Mix E6 to Mix E2 are inoculated with all 10 types of bacterial strains mixed and diluted to a concentration of 10 ⁶ to 10 ² .
3-12. It was possible to specifically detect pathogenic bacteria inoculated into human feces. However, because E. coli is a resident bacterium in the human intestine, genes (ftsZ and uidA) targeting E. coli have also been detected from human stool samples not inoculated with E. coli (FIG. 4).
3-13. Since the number of gene copies detected from human fecal samples inoculated with serial dilutions of bacteria decreased according to the inoculation concentration, MF-qPCR is considered to be capable of quantifying not only the target bacteria. . From the above, it was shown that the target bacteria can be specifically detected and quantified from environmental samples containing a large number of contaminating bacteria such as human feces.

Example 4
4-1. Example 4 shows an example in which E. coli O157 strain and process control NH8B strain were mixed and inoculated into lake water, and simultaneously detected and quantified by MF-qPCR method. Reagent adjustment was performed in a HEPA / UV PCR workstation (UVP) to prevent contamination (external DNA contamination).
4-2. 40 liters of Onoike water from Hokkaido University was collected. The water quality of the collection day (November 9, 2012) is as follows: 10 ° C, pH 5.7, conductivity 11.5 S / m, suspended material 9.0 mg / l, total nitrogen 0.273 mg / l, total phosphorus 0.364 mg / l.
4-3. Escherichia coli O157 Sakai strain and Pseudogulbenkiania sp. NH8B strain were inoculated to 5 liters of environmental water so that each concentration would be 10 ¹ to 10 ⁷ cells / l.
4-4. The cells were collected on a polyethersulfone membrane (Millipore) having a pore size of 0.20 μm by pressure filtration of environmental water. The collected membrane was divided into four equal parts with a cutter knife, placed in a 50 ml centrifuge tube, and vigorously shaken in 25 ml of PBS containing 0.1% gelatin (pH 7.2) to separate the cells from the membrane. Thereafter, the cells were centrifuged at 10,000 × g for 15 minutes to form pellets.
4-5. DNA was extracted from the cell pellet using PowerSoil DNA Isolation Kit (MoBio Laboratories).
4-6. The 2E1-2E6 standard prepared in Example 1 was used as a calibration curve sample for quantification.
4-7. Using the DNA samples shown in 4-5 and 4-6, the STA reaction was performed in the same manner as in Example 1 (1-6 to 1-8). The STA reaction product was diluted 6-fold with TE buffer.
4-8. The target bacteria were simultaneously detected and quantified by MF-qPCR in the same manner as in Example 3 (3-6 to 3-10).
4-9. The quantitative results are shown in FIG. Quantitative values by MF-qPCR are shown in gray scale (the lighter the color, the larger the quantitative value). Sample names Mix E6 to Mix E2 are inoculated with all 10 types of bacterial strains mixed and diluted to a concentration of 10 ⁶ to 10 ² . As in Example 3, only the inoculated strain could be specifically detected.
4-10. Since many ducks fly to Ohno Pond, the water in this pond may be contaminated with feces derived from ducks. Therefore, E. coli genes (ftsZ and uidA) are detected in E. coli low-concentration inoculated samples and non-inoculated environmental water samples from E. coli, which is a resident bacteria in the duck gut. It is thought that.
4-11. Since the number of gene copies detected from the environmental water sample inoculated with serial dilutions of bacteria in the same manner as in Example 3 decreased according to the inoculation concentration, MF-qPCR not only detected the target bacteria but also quantified it. It is considered possible. FIG. 6 shows this relationship more clearly. Quantitative values of E. coli O157 Sakai strain (eaeA, stx ₁ , stx ₂ ) and Pseudogulbenkiania sp. NH8B strain (NH8B_3641) showed a positive correlation between inoculum concentration and slope 1 (r ² > 0.97) Therefore, it can be judged that the quantitative value by MF-qPCR reflects the number of bacteria present in the environmental water.
4-12. In addition, the cell recovery rate obtained by comparing the quantified value (cells / l) obtained by MF-qPCR and the amount of cells inoculated (cells / l) was obtained for E. coli O157 Sakai and Pseudogulbenkiania sp. NH8B. The values were similar to 27.4 ± 5.0% and 22.6 ± 3.1%, respectively. From this, it can be said that Pseudogulbenkiania sp. NH8B strain is suitable as an internal standard (process control) for quantifying Escherichia coli O157. That is, a recovery rate of the NH8B strain is obtained by inoculating an environmental sample with a known concentration of Pseudogulbenkiania sp. NH8B and performing quantification by DNA extraction and MF-qPCR. Since the recovery rate of E. coli O157 is the same as that of the NH8B strain, the concentration of E. coli O157 in the environmental sample can be determined from the quantitative value of MF-qPCR and the recovery rate of NH8B. By considering the recovery rate, it is possible to correct for DNA loss during DNA extraction and reaction inhibition during qPCR, so that more accurate concentration calculation is possible.
4-13. Since Escherichia coli O157: H7 and other enteropathogenic bacteria have similar cell structures (ie, DNA extraction efficiency is considered to be almost the same), Pseudogulbenkiania sp. NH8B is a quantifier for enteropathogenic bacteria in general (such as Salmonella and Campylobacter). Can be used as an internal standard.
The strains used in this example are shown in Table 1.

  Table 2 shows qPCR primers and probes used in this example. As a primer targeting the ctxA gene of Vibrio cholerae, forward primer (5′-TTTGTTAGGCACGATGATGGAT-3 ′) (SEQ ID NO: 37) and reverse primer (5′-ACCAGACAATATAGTTTGACCCACTAAG-3 ′) (SEQ ID NO: 38) are used. (84 base pair amplicon within the ctxA gene). As a probe, the sequence in the ctxA gene, 5 ′ end of 5′-TGTTTCCACCTCAATTAGTTTGAGAAGTGCCC-3 ′ (SEQ ID NO: 41) is labeled with FAM, and 3 ′ end is labeled with Non-fluorescence Quencher (NFQ) and Minor Grove Binder (MGB) The one labeled with was used. In addition, as primers targeting the tdh gene of Vibrio parahaemolyticus, forward primer: 5′-AAACATCTGCTTTTGAGCTTCCA-3 ′ (SEQ ID NO: 39) and reverse primer: 5′-CTCGAACAACAAACAATATCTCATCAG-3 ′ (SEQ ID NO: 40) were used ( 75 base pair amplicon). As the probe, 5 ′ end of 5′-TGT CCC TTT TCC TGC CCC CGG-3 ′ (SEQ ID NO: 42) is labeled with FAM, 3 ′ end is Non-fluorescence Quencher (NFQ) and Minor Grove Binder (MGB) The one labeled with was used.

  The method of the present invention is expected to be applicable as a rapid diagnostic tool for pathogen identification and as an accurate risk assessment technique. Moreover, it is thought that it can be used as a safety evaluation method for drinking water, foods, etc. by using it for quantification of fecal indicator bacteria (E. coli).

  The sequence numbers and sequences are summarized in the following table.

Claims (9)

Primers and probes specific for each of at least five bacterial genes that can be amplified by PCR under the same reaction conditions are mixed with DNA extracted from the sample, and PCR is performed under the reaction conditions to detect and / or detect bacterial genes. Alternatively, a method for simultaneous detection and / or quantification of at least five kinds of bacteria, characterized in that quantification is performed. PCR is performed using a primer comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 34, and non-pathogenic E. coli, pathogenic E. coli, Salmonella, Campylobacter, Listeria, Legionella, Welsh bacteria, Shigella, Vibrio parahaemolyticus, and Vibrio cholerae The method according to claim 1, wherein at least 5 kinds of bacteria selected from the group consisting of are simultaneously detected and / or quantified. The method according to claim 2, wherein PCR is performed using at least one primer pair selected from the group consisting of the following primer pairs (1) to (17).
(1) A primer pair consisting of the nucleotide sequence of SEQ ID NO: 1 and a primer consisting of the nucleotide sequence of SEQ ID NO: 2 (target gene: ftsZ)
(2) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 3 and primer consisting of nucleotide sequence of SEQ ID NO: 4 (target gene: uidA)
(3) Pair of primer consisting of the nucleotide sequence of SEQ ID NO: 5 and primer consisting of the nucleotide sequence of SEQ ID NO: 6 (target gene: eaeA)
(4) A primer pair comprising the nucleotide sequence of SEQ ID NO: 7 and a primer comprising the nucleotide sequence of SEQ ID NO: 8 (target gene: stx ₁ )
(5) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 9 and a primer consisting of the nucleotide sequence of SEQ ID NO: 10 (target gene: stx ₂ )
(6) Pair of primer consisting of the nucleotide sequence of SEQ ID NO: 11 and primer consisting of the nucleotide sequence of SEQ ID NO: 12 (target gene: ipaH 7.8)
(7) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 13 and primer consisting of nucleotide sequence of SEQ ID NO: 14 (target gene: ipaH all)
(8) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 15 and a primer consisting of the nucleotide sequence of SEQ ID NO: 16 (target gene: virA)
(9) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 17 and primer consisting of nucleotide sequence of SEQ ID NO: 18 (target gene: invA)
(10) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 19 and a primer consisting of the nucleotide sequence of SEQ ID NO: 206 (target gene: ttrC)
(11) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 21 and a primer consisting of the nucleotide sequence of SEQ ID NO: 22 (target gene: cadF)
(12) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 23 and a primer consisting of the nucleotide sequence of SEQ ID NO: 24 (target gene: ciaB)
(13) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 25 and primer consisting of nucleotide sequence of SEQ ID NO: 26 (target gene: cpe)
(14) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 27 and primer consisting of nucleotide sequence of SEQ ID NO: 28 (target gene: plc)
(15) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 29 and a primer consisting of the nucleotide sequence of SEQ ID NO: 30 (target gene: mip)
(16) Pair of primer consisting of nucleotide sequence of SEQ ID NO: 31 and primer consisting of nucleotide sequence of SEQ ID NO: 32 (target gene: iap)
(17) A pair of a primer consisting of the nucleotide sequence of SEQ ID NO: 33 and a primer consisting of the nucleotide sequence of SEQ ID NO: 34 (target gene: hlyA) Claim 1 to determine the recovery rate of the added internal standard bacteria by adding bacteria as an internal standard of known concentration to the target sample, performing DNA extraction and PCR, and detecting and quantifying the internal standard bacteria 4. The method according to any one of 3. The method according to claim 4, wherein Pseudogulbenkiania sp. NH8B strain is used as an internal standard bacterium, and PCR is performed targeting NH8B_3641. The method according to claim 5, wherein PCR is performed using NH8B_3641 as a target by using a primer pair consisting of the nucleotide sequence of SEQ ID NO: 35 and a primer pair consisting of the nucleotide sequence of SEQ ID NO: 36. Before performing PCR which targets the bacterial gene which can be amplified on the same conditions, DNA is preamplified by mixing all the primer pairs used for said PCR, and performing PCR. The method of crab. A standard curve is prepared in advance by linearizing a plasmid incorporating the target gene, measuring the concentration and then serially diluting, and using this calibration curve, the amount or concentration of bacteria in the target sample is determined. Item 8. The method according to any one of Items 1 to 7. A kit for simultaneous detection and / or quantification of at least 5 types of bacteria, comprising a primer comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 36.