WO2011004725A1

WO2011004725A1 - Method for detection of pathogenic bacteria, and kit for use in the method

Info

Publication number: WO2011004725A1
Application number: PCT/JP2010/060940
Authority: WO
Inventors: 克英三宅; 裕之綱島; 新太片山
Original assignee: 国立大学法人名古屋大学
Priority date: 2009-07-08
Filing date: 2010-06-28
Publication date: 2011-01-13

Abstract

Disclosed is a method for detecting a pathogenic bacterium that is suspected to be present in a sample in a simple, rapid and specific manner. A nucleic acid amplification reaction that targets for a capsular polysaccharide gene of the pathogenic bacterium. The presence or absence of the pathogenic bacterium in the sample is determined on the basis of the results of the amplification.

Description

Pathogen detection method and kit used therefor

The present invention relates to a pathogen detection technology. Specifically, the present invention relates to a pathogen detection method and a kit used therefor. In particular, the present invention relates to a method for simultaneously detecting two or more types of pathogenic bacteria and a kit used therefor. This application claims priority based on Japanese Patent Application No. 2009-161351 filed on Jul. 8, 2009, the entire contents of which are incorporated by reference.

Even in the present day when hygiene is said to be good, people are regularly exposed to the threat of pathogenic bacteria. In order to prevent health damage caused by pathogenic bacteria, it is important to quickly detect pathogenic bacteria in foods and the environment to eliminate the risk of infection and spread of contamination. Examples of pathogen detection methods developed so far include a method of culturing on an agar plate and confirming colonies, and an ELISA method using an antibody reaction (Patent Document 1). As a result of recent progress in genome analysis of many bacteria, a method for confirming 16S rRNA gene by PCR (Patent Document 2) or a method for amplifying a specific gene and confirming it with a DNA chip (Patent Document 3) ) Etc. are being proposed.

JP 2000-78999 A JP 2000-83699 A Special table 2008-515423

A wide variety of pathogenic bacteria may exist in soil samples and sewage samples. In such a case, in order to ensure safety, it is desired to rapidly detect various pathogenic bacteria at once. Moreover, since it is naturally expected that microorganisms other than the pathogenic bacteria are mixed, it is necessary to increase the specificity for the pathogenic bacteria. Conventional detection methods are difficult to meet these demands. For example, in the case of detection method using 16S rRNA (Patent Document 2), since 16S rRNA gene is present in all bacteria, no matter how much the sequence is used to increase the specificity, there is a possibility that nonspecific amplification will occur. I can't deny it. In addition, the detection method (patent document 3) using a probe of a gene (toxin gene or the like) specific for each pathogen can ensure specificity, but is not appropriate as a universal pathogen detection method. Then, this invention makes it a subject to detect the pathogenic microbe which can exist in a sample simply, rapidly, and specifically.

In order to solve the above problems, it is necessary to develop a method that uses only traits common to many pathogenic bacteria as well as specific bacteria. The present inventors paid attention to a biosynthetic gene group of capsular polysaccharides specific to pathogenic bacteria. Capsular polysaccharides are present in almost all pathogens and are closely related to pathogenicity. The capsular polysaccharide usually has a shape protruding from the lipid of the cell membrane and functions mainly for defense from the host. Its sugar chain structure is extremely diverse and varies depending on the type of fungus. On the other hand, there are many common structures, and the sugar that first binds to lipids in the root part, that is, the cell membrane, is usually glucose. In addition, the commonality of enzymes and genes involved in the synthesis of capsular polysaccharide is very high. As shown in the examples below, the commonality and specificity of the capsular gene for pathogens such as Streptococcus, Pseudomonas, and Staphylococcus are examined in detail. In addition to being found to be high, the inventors have succeeded in identifying a gene that is particularly preferable as a detection target (target), that is, a gene having extremely high commonality. In addition, the gene was shown to be extremely specific. On the other hand, as a result of performing the PCR method targeting the gene identified in this way, non-specific amplification was not observed, and the high specificity and practicality of the gene were demonstrated. Moreover, as a result of attempting to detect pathogens using two primer sets designed for specific pathogens, it was possible to detect two types of pathogens at the same time as expected. The present invention described below is mainly based on the above results.
[1] A method for detecting a pathogenic bacterium in a specimen, comprising performing a nucleic acid amplification reaction targeting the capsular polysaccharide gene of the pathogenic bacterium, and determining the presence or absence of the pathogenic bacterium based on the amplification result.
[2] The method according to [1], wherein the nucleic acid amplification reaction is a PCR method.
[3] Two or more types of pathogens selected from Streptococcus, Staphylococcus, Clostridium, Pseudomonas, pathogenic Escherichia coli, and Salmonella The method according to [2], wherein the PCR method is a multiplex PCR method.
[4] The capsular polysaccharide gene is a cpsE gene for Streptococcus, a capE gene for Staphylococcus, a kpsF gene for Clostridium, a wzz gene for Pseudomonas, and pathogenic The method according to [3], wherein E. coli is a wcaA gene, and Salmonella is a wbaP gene or an rfc gene.
[5] A method for simultaneously detecting a plurality of types of pathogenic bacteria, including the following steps (1) to (4):
(1) preparing a DNA sample;
(2) a step of preparing a plurality of primer sets, wherein each primer set is a primer set targeting capsular polysaccharide genes of different target pathogens;
(3) performing a nucleic acid amplification reaction using the DNA sample as a template and simultaneously using the plurality of sets of primers;
(4) A step of detecting an amplification product.
[6] The method according to [5], wherein the nucleic acid amplification reaction is a multiplex PCR method.
[7] The target pathogen is selected from two or more selected from Streptococcus, Staphylococcus, Clostridium, Pseudomonas, pathogenic Escherichia coli, and Salmonella The method according to [5] or [6], comprising a pathogen.
[8] A primer set targeting the cpsE gene of Streptococcus, a primer set targeting the capE gene of Staphylococcus, a primer set targeting the kpsF gene of Clostridium, A primer set targeting the wzz gene of Pseudomonas, a primer set targeting the wcaA gene of pathogenic Escherichia coli, a primer set targeting the wbaP gene of Salmonella, and the rfc gene of Salmonella A kit for detecting pathogens, comprising two or more primer sets selected from the group consisting of primer sets targeted at.

Structure of capsular polysaccharide (schematic diagram) of pathogenic bacteria and structure of capsular polysaccharide gene. The result of DNA amplification from 200 ng (Kuridashi) of a soil DNA sample using a primer designed for Streptococcus agalactie cpsE gene (in the case of Streptococcus agalactie, called cpsIaE gene; hereinafter referred to as cpsIaE gene). The leftmost lane is a molecular weight marker. The target amplification product is the band indicated by the arrow. Results of DNA amplification from 200 ジ ng (Kuridashi) soil DNA sample using primers designed to target the Wzz gene of Pseudomonas aeruginosa. The rightmost lane is a molecular weight marker. The target amplification product is the band indicated by the arrow. Results of multiplex PCR using a primer set targeting the cpsIaE gene of Streptococcus agaractie and a primer set targeting the cpsE gene of Streptococcus anginosas. The case where only the former primer set was used (left lane), the case where only the latter primer set was used (middle lane), and the case where both primer sets were used (right lane) were compared. Streptococcus agalactie genomic DNA and Streptococcus anjinosas genomic DNA were used as template DNA. The band indicated by the arrow (upper: Streptococcus anjinosas, lower: Streptococcus agalactie) is the target amplification product. On the left is the result of DNA amplification from genomic DNA using primers designed to target the capE gene (381 bp) of Staphylococcus aureus. The rightmost lane is a molecular weight marker. The target amplification product is the band indicated by the arrow. On the right is the result of DNA amplification from genomic DNA using a primer set designed to target the rfc gene (311 bp) of Salmonella enterica. The leftmost lane is a molecular weight marker. The target amplification product is the band indicated by the arrow. Results of multiplex PCR using a primer set targeting the Staphylococcus aureus capE gene and a primer set targeting Salmonella enterica rfc gene. The template DNA used was Staphylococcus aureus genomic DNA and Salmonella enterica genomic DNA. A band corresponding to each target gene is observed.

(Pathogen detection method)
The first aspect of the present invention relates to a method for detecting pathogenic bacteria. The detection method of the present invention is characterized in that a nucleic acid amplification reaction targeting a capsular polysaccharide gene of a pathogenic bacterium is performed. That is, in the detection method of the present invention, a nucleic acid amplification reaction targeting the capsular polysaccharide gene of a pathogenic bacterium is performed, and the presence or absence of the pathogenic bacterium is determined based on the amplification result. When a specific amplification product is recognized, the target pathogen is detected. Examples of nucleic acid amplification reactions include PCR (Polymerase chain reaction) method or its modification, LAMP (Loop-Mediated Isothermal Amplification) method (Tsugunori Notomi et al. Nucleic Acids Research, Vol.28, No.12, e63,
2000; Kentaro Nagamine, Keiko Watanabe et al. Clinical Chemistry, Vol. 47, No. 9,
1742-1743, 2001), ICAN (Isothermal and Chimeric
primer-initiated Amplification of Nucleic Acids (Patent No. 3433929, Patent No. 3883476), NASBA (Nucleic Acid Sequence-Based Amplification) method, LCR (Ligase Chain Reaction) method, 3SR (Self-sustained Sequence Replication) method, Examples include SDA (Standard Displacement Amplification), TMA (Transcription Mediated Amplification), and RCA (Rolling Circle Amplification). Among them, it is preferable to carry out an amplification reaction by the PCR method or a modified method thereof.

In the detection method of the present invention, a capsular polysaccharide gene that has been found to be highly common among pathogens of the same genus is targeted for detection. In this specification, the “target of detection” is also called “target” or “detection probe” in accordance with the conventional practice. By targeting the capsular polysaccharide gene, it becomes possible to specifically detect multiple pathogens of the same gene.

As a result of the study by the present inventors, among the capsular genes, a highly common one is a chain length determining gene present on the 5 ′ side of the operon, glucose that synthesizes the first sugar of the sugar chain unit, galactose, Alternatively, it was a N-acetylglucosamine transferase gene, a flippase gene that transfers a sugar chain unit to the outside of the cell membrane. In particular, the commonality of the chain length determining gene and the glucose, galactose, or N-acetylglucosamine transferase gene among the pathogenic bacteria is high. Therefore, the chain length determining gene or the first glycosyltransferase gene is preferably targeted.

The pathogenic bacteria to be detected (hereinafter, “pathogenic bacteria to be detected” is also referred to as “target pathogenic bacteria”) is not particularly limited. Examples of target pathogens include Streptococcus, Staphylococcus, Clostridium, Pseudomonas, pathogenic E. coli (enteropathogenic E. coli (EPEC), intestinal tissue invasive E. coli (EIEC) ), Enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), intestinal agglutinating E. coli (EAggEC)), and Salmonella. When detecting using a nucleic acid amplification reaction capable of simultaneously amplifying a plurality of targets, such as multiplex PCR, two or more, preferably three or more, more preferably four of the pathogenic bacteria exemplified here. The above is the target pathogen.

As shown in Examples described later, the present inventors have succeeded in identifying genes with extremely high commonality and specificity for each genus for a plurality of types of pathogenic bacteria. In a preferred embodiment of the present invention, the specified gene is targeted and the detection specificity and accuracy are improved. Below, the gene which becomes a target in the said aspect is shown for every kind of pathogenic microbe (Table 1).

As an example of the sequence of the target gene of the genus Streptococcus (cpsE gene), the sequence of S. anginosus is SEQ ID NO: 1, the sequence of S. gordonii is SEQ ID NO: 2, and the sequence of S. oralis The sequence is shown in SEQ ID NO: 3, the sequence of S. pneumoniae is shown in SEQ ID NO: 4, and the sequence of S. agalactiae is shown in SEQ ID NO: 5, respectively. Similarly, as an example of the target inheritance (capE gene) of the genus Staphylococcus, the sequence of S. aureus is shown in SEQ ID NO: 6. Further, as an example of a Clostridial target gene (kpsF gene), the sequence of Tetani (C. テ tetani) is shown in SEQ ID NO: 7. As an example of a target gene of the genus Pseudomonas (wzz gene), the sequence of aeruginosa (P. 番号 aeruginosa) is shown in SEQ ID NO: 8. An example of the sequence of the target gene (wcaA gene) of pathogenic E. coli is shown in SEQ ID NO: 9. Examples of sequences of Salmonella target genes (wbaP gene, rfc gene) are shown in SEQ ID NO: 10 (wbaP gene) and SEQ ID NO: 23 (rfc gene).

As a result of the study by the present inventors, if an appropriate highly common gene is selected as a target, one to several primer sets (about 5 at most depending on the genus) are prepared for one genus. As a result, it was found that the main pathogenic bacteria of the genus can be comprehensively detected. Therefore, for example, when trying to detect pathogens of one genus (for example, Streptococcus genus) at once, a nucleic acid amplification reaction using one to several primer sets (for example, PCR with a single primer set, multiple primer sets) In the case of a set, multiplex PCR) will be performed. On the other hand, in the case of detection for a plurality of genera, a nucleic acid amplification reaction (for example, multiplex PCR) may be carried out using a primer set (one to several sets for each genera) prepared for each genera.

Here, the example of the primer set which can be used by this invention is shown below.
(1) Primer set for Streptococcus (designed based on the sequence of the cpsIaE gene of Streptococcus agaractie)
Direct: 5'-CAATCAAATGACAGGGCTAAT-3 '(SEQ ID NO: 11)
Reverse: 5'-TAAAACTAAGGCGTCGCTT-3 '(SEQ ID NO: 12)
(2) Primer set for Streptococcus genus (designed based on the sequence of Streptococcus anjinosas cpsE gene)
Direct: 5'-TGTACGTAGATGCCGAGG-3 '(SEQ ID NO: 13)
Reverse: 5′-TTAAAGCTCAATCGCCGC-3 ′ (SEQ ID NO: 14)
(3) Primer set for Pseudomonas (designed based on the sequence of Pseudomonas aeruginosa wzz gene)
Direct: 5'-CGTGAAGATCGTAT-3 '(SEQ ID NO: 15)
Reverse: 5'-GGAATAAAAGGATCATC-3 '(SEQ ID NO: 16)
(4) Primer set for Staphylococcus (designed based on the sequence of Staphylococcus aureus capE gene)
Direct: 5'-ATTAATTACTGGGGGCACAG-3 '(SEQ ID NO: 17)
Reverse: 5′-TAGCATTAATAGGATACGCT-3 ′ (SEQ ID NO: 18)
(5) Primer set for the genus Clostridium (designed based on the sequence of the kpsF gene of Clostridium tetani)
Direct: 5'-CATGGAGATTTAGGAATGAT-3 '(SEQ ID NO: 19)
Reverse: 5′-ATCTCCTAAAAC-3 ′ (SEQ ID NO: 20)
(6) Salmonella (designed based on the rfc gene sequence of Salmonella enterica)
Direct: 5'-CATACCTGATAATAACAGTC-3 '(SEQ ID NO: 21)
Reverse: 5'-GGTTAATTCGTATATTCTTC-3 '(SEQ ID NO: 22)

Each oligonucleotide constituting the primer set may be prepared by a conventional method. For example, it can be chemically synthesized using a general-purpose DNA synthesizer.

The detection method of the present invention typically uses a PCR method. This improves versatility and quickness. Preferably, a multiplex PCR method is employed. The multiplex PCR method is a PCR method using a plurality of primer sets at the same time, and enables detection of a plurality of targets at once (for example, US Pat. No. 5,582,989, Journal of Microbiological Methods 68 (2007) 52 − (See 59). Therefore, if the multiplex PCR method is used, the types of pathogenic bacteria that can be detected at a time are increased, and the rapidity and convenience are further improved. In the detection method of the present invention, typically, the following steps (1) to (4) are performed to detect a plurality of types of pathogenic bacteria simultaneously.
(1) Step of preparing a DNA sample (2) Step of preparing a plurality of primer sets, each primer set being a primer set targeting capsular polysaccharide genes of different target pathogens (3) A step of performing a nucleic acid amplification reaction using the DNA sample as a template and simultaneously with the plurality of sets of primers (4) A step of detecting an amplification product

In step (1), prepare a DNA sample. The detection method of the present invention can be used to detect pathogenic bacteria in various test materials (specimens). Examples of test materials include soil, sewage, food, wiped samples, urine, stool, urine storage, sputum, and vomit. After removing impurities or diluting as necessary, a DNA sample is prepared from the test material, and the detection method of the present invention is applied. A DNA sample may be prepared by a conventional method. Many kits for preparing DNA samples are also commercially available, and DNA samples can be easily obtained by using such kits.

In step (2), in order to be able to simultaneously amplify the target genes (capsular polysaccharide genes) of multiple types of pathogenic bacteria in the subsequent step (3), each primer set has a different target pathogenic capsular polysaccharide. Prepare multiple primer sets targeting genes. The number of primer sets is, for example, 2 to 5 sets. As the number of primer sets increases, the number of pathogenic bacteria that can be detected simultaneously increases, but there is a problem that the possibility of non-specific amplification increases and the design of primers becomes difficult. Therefore, the number of primer sets is preferably 5 to 30 sets. The number of primer sets prepared for one genus of pathogenic bacteria may not be one. That is, two or more primer sets (for example, 2, 3, 4, or 5) may be prepared for one genus.

In order to facilitate detection of amplification products (PCR products), a primer set labeled with a labeling substance may be used. Examples of labeling substances are fluorescent substances, chemiluminescent substances, biotin, and radioisotopes.

In step (3), the nucleic acid amplification reaction is performed using the DNA sample prepared in step (1) as a template and using the plurality of primer sets prepared in step (2) simultaneously. Typically, multiplex PCR is performed. Each step of the nucleic acid amplification reaction may be performed by a conventional method. If PCR is employed, for example, a commercially available PCR device (for example, thermal cycler personal manufactured by Takara Bio Inc.) can be used. Numerous PCR kits that include enzymes, reagents, etc. are also commercially available. By using such a kit, each step of PCR can be carried out easily. PCR conditions may be set in consideration of primer Tm and the like. Examples of PCR conditions are as follows. That is, heat denaturation is 90 ° C to 98 ° C, annealing is 30 ° C to 65 ° C, and extension reaction is 65 ° C to 75 ° C. The number of reaction cycle repetitions is, for example, 20-40. For example, Ex Taq (registered trademark, Takara Bio Inc.), gene taq (Nippon Gene), KOD plus (Toyobo Co., Ltd.), etc. can be used as the polymerase.

In step (4), the amplification product is detected, but the detection method is not particularly limited. For example, a method using electrophoresis, a method using chromatography, a method using a DNA array, and the like can be employed. A specific example of the method using electrophoresis is as follows. The reaction solution after PCR is applied to a gel such as agarose and subjected to electrophoresis, followed by staining with ethidium bromide. The presence or absence of the target DNA fragment (amplified product) is determined using the position (movement distance) of the band that appears by staining as an index. In addition, the amount of the target DNA fragment may be determined based on the density of the band. For electrophoresis, for example, 2% TAE (Tris acetate, EDTA) agarose or 5% TBE (Tris borate, EDTA) polyacrylamide gel can be used.

(Pathogen detection kit)
Another aspect of the present invention provides a kit for detecting pathogenic bacteria. The detection kit of the present invention is a primer set targeting the Streptococcus cpsE gene, a primer set targeting the Staphylococcus capE gene, and Clostridium spp. Primer set targeting kpsF gene, primer set targeting Pseudomonas wzz gene, primer set targeting wcaA gene of pathogenic E. coli, primer set targeting wbaP gene of Salmonella, and rfc of Salmonella It includes two or more primer sets selected from the group consisting of primer sets targeting genes.

Multiple primer sets may be used for one genus of pathogenic bacteria. An example of this embodiment (an example of a kit capable of detecting Streptococcus spp. And Pseudomonas spp.) Is shown. As a primer set targeting the Streptococcus cpsE gene, the cpsE gene of a specific species (one or more) And a primer set targeting a cpsE gene of one or more species different from the species, and a primer set targeting a Pseudomonas wzz gene as a specific species ( A kit comprising a primer set targeting one or more wzz genes and a primer set targeting a wzz gene of a species (one or more) different from the species. In this example, two primer sets are used for one genus of pathogenic bacteria, but the present invention is not limited to this.

The primer constituting the primer set is not particularly limited as long as it can specifically amplify the target gene or a part thereof. The length of the primer is not limited as long as it functions as a primer (for example, a PCR primer) in the nucleic acid amplification reaction employed, and is, for example, 15 to 30 bp, preferably 20 to 30 bp, more preferably 20 to 25 bp. There may be 1 to several, preferably 1 to 5, more preferably about 1 to 3 mismatches between the primer and the region of the target gene to which it hybridizes.

Specific examples of the primer set include the above-mentioned primer set for the genus Streptococcus (SEQ ID Nos. 11 and 12: designed based on the sequence of the cpsIaE gene of Streptococcus agalactie), and the primer set for the genus Streptococcus (SEQ ID Nos. 13 and 14: Streptococcus anjinosas based on the sequence of cpsE gene), Pseudomonas genus primer set (SEQ ID NOs: 15 and 16), Staphylococcus genus primer set (SEQ ID NOs: 17 and 18), Clostridium genus primer A set (SEQ ID NOs: 19 and 20) and a primer set (SEQ ID NOs: 21 and 22) for Salmonella.

Reagents necessary for each step of nucleic acid amplification reaction (typically PCR) (DNA polymerase, buffer solution, etc.), reagents required for detection (reagents for gel preparation, stain solution, etc.), containers, instruments, etc. It may be included in the detection kit of the present invention. Usually, an instruction manual is attached to the detection kit of the present invention.

1. Search for target genes Capsular polysaccharides are present in almost all pathogens and are closely related to pathogenicity (Fig. 1). The capsular polysaccharide usually has a shape protruding from the lipid of the cell membrane and functions mainly for defense from the host. Its sugar chain structure is extremely diverse and varies depending on the type of fungus. On the other hand, there are many common structures, and the sugar that first binds to the lipids of the cell membrane, ie, the cell membrane, is usually glucose or galactose. In addition, the commonality of enzymes and genes involved in the synthesis of capsular polysaccharide is very high.

Aiming to establish a simple method for detecting pathogens in the environment such as soil samples, we examined the commonality and specificity of capsular genes for several pathogens based on the genetic information registered in public databases. . As a result, it was found that the pathogenic bacteria belonging to the genus Streptococcus have high commonality and specificity of the cpsE gene encoding glucose transferase (FIG. 1). In addition, the results of a detailed study of the structure of the Pseudomonas aeruginosa polysaccharide (called O antigen) gene suggests that in Pseudomonas, the wzz gene (the CpsC homologue of Streptococcus) is highly common and specific. It was. On the other hand, a detailed examination of the structure of the capsular polysaccharide gene of Staphylococcus aureus revealed that it was similar to the gene structure of Staphylococcus spp. And it was suggested that the specificity is high. In addition, when the kpsF gene, which is a polysialic acid synthesis system gene of Clostridium tetani, was examined, all the hits in the homology search were pathogenic bacteria, and no Clostridium bacteria without pathogenicity were hit. Thus, in the case of Clostridium pathogens, it was suggested that the kpsF gene is promising as a highly common and specific gene. Furthermore, it was found that the commonality of the wcaA gene was high for pathogenic E. coli, and the commonality of the wbaP gene and the rfc gene (corresponding to cpsE gene and capE gene) was high for Salmonella.

As mentioned above, cpsE gene for Streptococcus pathogens, wzz gene for Pseudomonas pathogens, capE gene for Staphylococcus pathogens, kpsF gene for Clostridium pathogens, pathogenicity The wcaA gene was found for E. coli and the wbaP and rfc genes were found for Salmonella.

2. Design of detection primer set A detection primer set was designed for each target gene as follows.
(Primer set targeting the cpsIaE gene of Streptococcus agalactie)
Direct: 5'-CAATCAAATGACAGGGCTAAT-3 '(SEQ ID NO: 11)
Reverse: 5'-TAAAACTAAGGCGTCGCTT-3 '(SEQ ID NO: 12)
(Primer set targeting the cpsE gene of Streptococcus anginosas)
Direct: 5'-TGTACGTAGATGCCGAGG-3 '(SEQ ID NO: 13)
Reverse: 5′-TTAAAGCTCAATCGCCGC-3 ′ (SEQ ID NO: 14)
(Primer set targeting the wzz gene of Pseudomonas aeruginosa)
Direct: 5'-CGTGAAGATCGTAT-3 '(SEQ ID NO: 15)
Reverse: 5'-GGAATAAAAGGATCATC-3 '(SEQ ID NO: 16)
(Primer set targeting the capE gene of Staphylococcus aureus)
Direct: 5'-ATTAATTACTGGGGGCACAG-3 '(SEQ ID NO: 17)
Reverse: 5′-TAGCATTAATAGGATACGCT-3 ′ (SEQ ID NO: 18)
(Primer set targeting the kpsF gene of Clostridium tetani)
Direct: 5'-CATGGAGATTTAGGAATGAT-3 '(SEQ ID NO: 19)
Reverse: 5′-ATCTCCTAAAAC-3 ′ (SEQ ID NO: 20)
(Primer set targeting the rfc gene of Salmonella enterica)
Direct: 5'-CATACCTGATAATAACAGTC-3 '(SEQ ID NO: 21)
Reverse: 5'-GGTTAATTCGTATATTCTTC-3 '(SEQ ID NO: 22)

3. Confirmation of Specific Detection (1) Specificity of primer set targeting the cpsIaE gene of Streptococcus agaractie The specificity of the above primer sets (SEQ ID NO: 11 and SEQ ID NO: 12) was examined by the following procedure. DNA was prepared from soil (Kuridashi) using a DNA extraction kit (ISOL, Nippon Gene Co., Ltd.). The following test groups: negative control (no DNA sample), soil DNA sample (200 ng) only, soil DNA sample (200 ng) and Streptococcus agalactiae genomic DNA (1 fg: 2x10 ^-25 mol), soil DNA sample (200 ng) ) And Streptococcus agalactie genomic DNA (1 pg: 2x10 ^-22 mol), soil DNA sample (200 pg) and Streptococcus agalactia genomic DNA (1 ng: 2x10 ^-19 mol), and the above primer set (SEQ ID NO: 11) And SEQ ID NO: 12). KOD plus DNA polymerase (Toyobo Co., Ltd.) was used and the PCR conditions were as follows.
<PCR conditions>
2 minutes at 96 ° C, 10 seconds at 98 ° C; 30 seconds at 50 ° C; 20 seconds at 68 ° C (35 cycles)

Each sample after PCR was subjected to electrophoresis (2% TAE (Tris acetic acid, EDTA) agarose) to confirm the presence or absence of amplification. As shown in FIG. 2, the target gene of Streptococcus agalactie was not amplified from the soil sample, and the specificity of the used primer was confirmed.

(2) Specificity of the primer set targeting the P. aeruginosa wzz gene The specificity of the above primer sets (SEQ ID NO: 15 and SEQ ID NO: 16) was examined as follows. DNA was prepared from soil (Kuridashi) using a DNA extraction kit (ISOL, Nippon Gene Co., Ltd.). The following test groups: negative control (no DNA sample), soil DNA sample (200 ng) only, soil DNA sample (200 ng) and Pseudomonas aeruginosa genomic DNA (1 fg), soil DNA sample (200 ng) and Pseudomonas aeruginosa Set genomic DNA (1 fg), soil DNA sample (200 pg) and Pseudomonas aeruginosa genomic DNA (1 ng), soil DNA sample (200 pg) and Pseudomonas aeruginosa genomic DNA (1 μg), and set the above primer set (sequence) PCR was performed using No. 15 and SEQ ID No. 16). KOD plus DNA polymerase (Toyobo Co., Ltd.) was used and the PCR conditions were as follows.
<PCR conditions>
2 minutes at 96 ° C, 10 seconds at 98 ° C; 30 seconds at 40 ° C; 20 seconds at 68 ° C (35 cycles)

Each sample after PCR was subjected to electrophoresis (2% TAE (Trisacetate, EDTA) agarose) to confirm the presence or absence of amplification. As shown in FIG. 3, the target gene of Pseudomonas aeruginosa was not amplified from the soil sample, confirming the high specificity of the primers used.

(3) Specific amplification by multiplex PCR Two types of primer sets (a primer set targeting the cpsIaE gene of Streptococcus agalactie (SEQ ID NO: 11 and SEQ ID NO: 12)) and a primer set targeting the cpsE gene of Streptococcus anjinosas (sequence) No. 13 and SEQ ID NO: 14)), and multiplex PCR was performed using as a template a mixture of Streptococcus agalactie genomic DNA (10 ng) and Streptococcus anginosas genomic DNA (10 ng). The PCR and electrophoresis conditions were the same as in (1) above.

As shown in FIG. 4, when two primer sets were used together (right lane), bands corresponding to each primer set were recognized. That is, it was shown that the target gene can be amplified and detected simultaneously by multiplex PCR.

(4) Specificity of primer set targeting Staphylococcus aureus capE gene The specificity of the above primer sets (SEQ ID NO: 17 and SEQ ID NO: 18) was examined by the following procedure. Set the following test groups, ie negative control (no DNA sample), Staphylococcus aureus genomic DNA (0.1 ng, 1 ng, 10 ng), and PCR using the above primer sets (SEQ ID NO: 17 and SEQ ID NO: 18) Went. KOD plus DNA polymerase (Toyobo Co., Ltd.) was used and the PCR conditions were as follows.
<PCR conditions>
94 ° C for 2 minutes, 98 ° C for 10 seconds; 50 ° C for 30 seconds; 68 ° C for 20 seconds (35 cycles)

Each sample after PCR was subjected to electrophoresis (2% TAE (Tris acetic acid, EDTA) agarose) to confirm the presence or absence of amplification. As shown in the left of FIG. 5, specific amplification of the target gene was observed. It was also shown that it can be detected with high sensitivity from a small amount of sample. As described above, the high specificity of the primer was confirmed.

(5) Specificity of the primer set targeting the rfc gene of Salmonella enterica The specificity of the above primer sets (SEQ ID NO: 21 and SEQ ID NO: 22) was examined by the following procedure. Set the following test groups, ie, negative control (no DNA sample), genomic DNA of Salmonella enterica (0.1 ng, 1 ng, 10 ng) and perform PCR using the above primer sets (SEQ ID NO: 21 and SEQ ID NO: 22). It was. KOD plus DNA polymerase (Toyobo Co., Ltd.) was used and the PCR conditions were as follows.
<PCR conditions>
94 ° C for 2 minutes, 98 ° C for 10 seconds; 40 ° C for 30 seconds; 68 ° C for 20 seconds (35 cycles)

Each sample after PCR was subjected to electrophoresis (2% TAE (Tris acetic acid, EDTA) agarose) to confirm the presence or absence of amplification. As shown in the right of FIG. 5, the target gene could be detected with high specificity and sensitivity. That is, the usefulness of the primer was shown.

(6) Specific amplification by multiplex PCR Two types of primer sets (primer sets targeting the capE gene of Staphylococcus aureus (SEQ ID NO: 17 and SEQ ID NO: 18) and primer sets targeting the rfc gene of Salmonella enterica (SEQ ID NO: 21 and SEQ ID NO: 22)), multiplex PCR was performed using a mixture of Staphylococcus aureus genomic DNA and Salmonella enterica genomic DNA as a template. The PCR and electrophoresis conditions were the same as in the case of (5) above.

As shown in FIG. 6, bands corresponding to each primer set were recognized. That is, it was shown that two target genes can be amplified and detected simultaneously by multiplex PCR.

In the present invention, pathogenic bacteria in a specimen are detected by targeting a capsular polysaccharide gene that is highly common and closely related to pathogenicity. Typically, a PCR method that is versatile and excellent in rapidity is used. According to the present invention having these features, pathogenic bacteria can be detected from the environment simply, quickly and specifically. Compared to conventional methods targeting 16S rRNA genes that are not related to pathogenicity, the present invention exhibits high specificity for pathogenic bacteria. The detection method of the present invention is suitable for simultaneously detecting a plurality of pathogenic bacteria that may be present in samples such as soil, sewage, and food.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Sequence numbers 11-22: Description of artificial sequence: Primer

Claims

A method for detecting a pathogenic bacterium in a specimen, comprising performing a nucleic acid amplification reaction targeting the capsular polysaccharide gene of the pathogenic bacterium and determining the presence or absence of the pathogenic bacterium based on the amplification result.
The method according to claim 1, wherein the nucleic acid amplification reaction is a PCR method.
The pathogen includes two or more pathogens selected from Streptococcus, Staphylococcus, Clostridium, Pseudomonas, pathogenic Escherichia coli, and Salmonella, The method according to claim 2, wherein the PCR method is a multiplex PCR method.
The capsular polysaccharide gene is the cpsE gene for Streptococcus, the capE gene for Staphylococcus, the kpsF gene for Clostridium, the wzz gene for Pseudomonas, and the pathogenic E. coli The method according to claim 3, which is a wcaA gene, and is Salmonella wbaP gene or rfc gene.
A method for simultaneously detecting multiple types of pathogens, including the following steps (1) to (4):
(1) preparing a DNA sample;
(2) a step of preparing a plurality of primer sets, wherein each primer set is a primer set targeting capsular polysaccharide genes of different target pathogens;
(3) performing a nucleic acid amplification reaction using the DNA sample as a template and simultaneously using the plurality of sets of primers;
(4) A step of detecting an amplification product.
The method according to claim 5, wherein the nucleic acid amplification reaction is a multiplex PCR method.
The target pathogen includes at least two types of pathogens selected from Streptococcus, Staphylococcus, Clostridium, Pseudomonas, pathogenic Escherichia coli, and Salmonella The method according to claim 5 or 6.
Primer set targeting Streptococcus cpsE gene, primer set targeting Staphylococcus capE gene, primer set targeting Clostridium kpsF gene, Pseudomonas genus ( Pseudomonas) primer set targeting wzz gene, pathogenic Escherichia coli wcaA gene targeting primer set, Salmonella wbaP gene targeting primer set, and Salmonella rfc gene targeting A kit for detecting pathogens, comprising two or more primer sets selected from the group consisting of prepared primer sets.