JP2007159533A - Primer for detecting clostridium perfringens and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly and specifically detecting Clostridium perfringens or Clostridium perfringens holding a enterotoxin gene in an environmental examination, a food examination, a clinical examination, or the like. <P>SOLUTION: A primer set for detecting Clostridium perfringens or Clostridium perfringens holding an enterotoxin gene by LAMP method is characterized by comprising at least four kinds of LAMP primers capable of being annealed in a specific region of the cpa gene of the Clostridium perfringens. A primer set comprises at least four kinds of LAMP primers capable of being annealed in a specific region of the cpe gene of the Clostridium perfringens. Also, the method using these LAMP primer sets is provided for detecting the Clostridium perfringens or Clostridium perfringens holding an enterotoxin gene. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a primer set and method for rapidly and accurately detecting Clostridium perfringens .

  More specifically, the present invention relates to the detection of Clostridium perfringens with or without the enterotoxin gene, comprising detecting amplification of the cpa gene or cpe gene of Clostridium perfringens by LAMP (loop-mediated isothermal amplification) method. And a primer set.

  Since Clostridium perfringens is a major cause of food poisoning, it is considered to be a very important bacterial species in terms of pathogenicity. This bacterium is widely distributed in various environments such as mammals, birds, meat, milk and dairy products, feed, soil and water. As a food contamination source, it is widely distributed in the environment (Non-Patent Document 1).

  The main cause of food poisoning caused by C. perfringens is enterotoxin, a protein toxin with a molecular weight of 35 kDa that is expressed during spore formation. It is thought that enterotoxin is produced at the time of spore formation and is released along with the release of mature spore from the cells (Non-patent Document 2).

  Clostridium perfringens is a gram-positive obligate anaerobic bacterium that forms spores ubiquitously. Conventionally, when judging C. perfringens by biochemical properties, it has been determined by performing motility, lecithinase, lecithinase control test, nitrate reduction, gelatin liquefaction, lactose fermentation, saccharolytic test, etc. (Non-patent Document 3).

  For detection of Clostridium perfringens, various biochemical tests and the like are performed through enrichment and isolation culture. Since these methods require time to obtain results, gene-based test methods have attracted attention. For example, a PCR (Polymerase Chain Reaction) method for amplifying and confirming a gene region specific to Clostridium perfringens is widely used (Non-patent Document 4).

  However, the bacterial detection method using the PCR method requires a special instrument for performing the reaction, and agarose gel electrophoresis, which is one of the final determination methods, requires a lot of time and labor.

  In addition, Clostridium perfringens produces at least 14 types of toxins. Of these, enterotoxins are toxins that cause food poisoning, and therefore, enterotoxin production tests are required for the detected Welsh strains. As a result of investigations on various Clostridium perfringens isolates, it has been reported that possession of cpe gene or enterotoxin production ability is observed only in a relatively small number of strains of 6% or less of the investigated strains (non- Patent Document 5 and Non-Patent Document 6).

  Enterotoxin is a protein having a molecular weight of 35,317 consisting of 319 amino acid residues, and is a heat-labile toxin that is inactivated by heating at 60 ° C. for 10 minutes or at pH 4.0 or less (Non-patent Document 3). The entire nucleotide sequence of the enterotoxin gene (cpe gene) derived from Clostridium perfringens type A has already been reported (Non-patent Documents 7 and 8).

  For the enterotoxin test of Clostridium perfringens, the enterotoxin protein produced by the isolated strain is detected by immunological techniques, and the presence or absence of the enterotoxin structural gene encoded on the DNA of Clostridium perfringens is determined by genetic techniques. The detection method is mainly used.

  As an immunological detection method, the reverse passive latex agglutination method (RPLA method) is known, and enterotoxin (antigen) produced in the medium is immunized with latex particles sensitized with an anti-enterotoxin antibody. Although it is a detection method, it takes at least two days to obtain a result (Non-patent Document 3).

  On the other hand, as a genetic detection method, a PCR method for amplifying and confirming a gene region specific to the enterotoxin of the present bacterium is widely used (Patent Document 1 and Non-Patent Document 5). However, as described above, the PCR method has a drawback that it requires a lot of time and labor.

  Recently, a method using the LAMP reaction as one of new gene amplification methods was developed by Eiken Chemical Co., Ltd. (Tochigi, Japan). This method is an isothermal nucleic acid amplification method, has high specificity and amplification efficiency, and can be performed in about 1 hour from reaction to detection (Patent Document 2 and Non-Patent Document 9).

Japanese Patent Laid-Open No. 11-239484 JP 2002-330796 A Food-contaminating pathogenic microorganisms, 2003, 312-326 Solar eclipse, 1997, Vol.14, No1, 35-42 Antibacterial fungus, 2001, Vol.29, No10, 675-685 Let. Appl. Microbiol., 1997,25,339-344 J. Clin. Microbiol., 1994,32,2533-2539 Am. J. Vet. Res., 1996,57,496-501 Infect Immun., 1993,61,3429-3439 J. Gen. Microbiol., 1989,135,903-909 T. Notomi et al., Nucleic Acids research, 2000, 28 (12), e63

An object of the present invention is to provide a primer set that can specifically detect Clostridium perfringens , particularly Clostridium perfringens , which has or does not have an enterotoxin gene, by the LAMP method.

  Another object of the present invention is to provide a method for specifically detecting the Clostridium perfringens using the primer set.

  As a result of studies to solve the above-mentioned problems, the present inventor produced an oligonucleotide primer that selectively hybridizes to the cpa gene of Clostridium perfringens, and amplifies this oligonucleotide as a primer by the LAMP method. The present invention was completed by discovering that Clostridium perfringens was detected specifically, conveniently, quickly and with high sensitivity from a specimen.

  At the same time, the present inventor prepared an oligonucleotide primer that selectively hybridizes to the C. perfringens cpe gene, and amplifies it by the LAMP method using this oligonucleotide as a primer. The present invention has been completed by finding that detection is simple, simple, rapid and highly sensitive.

That is, the present invention can be summarized as follows.
In the first aspect, the present invention provides a LAMP primer set for detecting C. perfringens, each of which has the base sequences FIP, BIP, F3 and SEQ ID NOs: A set comprising 4 primers consisting of B3 is provided.

  In the embodiment, the primer set further includes two kinds of primers consisting of base sequences Loop-F and Loop-B shown in SEQ ID NOs: 5 and 6, respectively, in addition to the four kinds of primers of SEQ ID NOS: 1-4. Can be included.

  In the second embodiment, the present invention provides a LAMP primer set for detecting a C. perfringens having an enterotoxin gene, which can be annealed to a specific region of a cpe gene of C. perfringens. A set comprising four kinds of primers consisting of base sequences FIP, BIP, F3 and B3 is provided.

  In the embodiment, the primer set further includes two kinds of primers consisting of base sequences Loop-F and Loop-B shown in SEQ ID NOs: 11 and 12, respectively, in addition to the four kinds of primers of SEQ ID NOs: 7 to 10. Can be included.

  In the third aspect, the present invention is a method for detecting Clostridium perfringens that has or does not have an enterotoxin gene, the method comprising detecting amplification of the cpa gene or cpe gene of Clostridium perfringens by the LAMP method.

  In that embodiment, the LAMP method comprises (i) a LAMP primer set comprising the four primers of SEQ ID NOs: 1 to 4 above, or (ii) the four primers of SEQ ID NOs: 1-4, This is carried out using a LAMP primer set further comprising two kinds of primers 5 and 6.

  In another embodiment thereof, the LAMP method comprises (i) a LAMP primer set comprising the four primers of SEQ ID NOs: 7 to 10 above, or (ii) four primers of SEQ ID NOs: 7 to 10, This is performed using a LAMP primer set further comprising two primers of SEQ ID NOs: 11 and 12.

  Any of the primer sets for amplifying the C. perfringens cpa gene of the present invention can specifically detect C. perfringens by the LAMP method (Table 1 described later). As a result of testing on various bacterial strains, all the Welsh strains were positively detected by the primer set of the present invention. On the other hand, negative results were obtained for all other Clostridium species.

  In addition, any of the primer sets for amplifying the C. perfringens cpe gene of the present invention can specifically detect C. perfringens having the enterotoxin gene by the LAMP method (Table 2 described later). As a result of testing various bacterial strains, only the strains possessing the C. perfringens enterotoxin gene were detected positively by the primer set of the present invention.

  According to the present invention, Clostridium perfringens that has or does not have an enterotoxin gene can be detected specifically, simply, rapidly, and with high sensitivity.

  Clostridium perfringens produces a wide variety of toxins, and among them, the main toxins α, β, ε, and ι toxins are classified into five toxin types A to E (bacterial and fungal protection, 2001). , 29 (10), 675-685). Among these, alpha toxin is a toxin produced in common in A to E types. The cpa gene product of Clostridium perfringens is an alpha toxin, which shows hemolysis, skin necrosis, neutrophil activation, platelet aggregation, etc. (Bacterial toxin handbook, 2002, 131-138).

  Moreover, the Clostridium perfringens cpe gene product is enterotoxin, and exhibits biological activities such as pore formation in the cell membrane, membrane permeability change due to pore formation, cell shape change, and cell death.

  The base sequence of C. perfringens cpa gene and cpe gene was compared with other bacteria, the sequence specific to C. perfringens was identified, and the LAMP primer of the present invention was designed. The base sequences of the bacterial cpa gene and cpe gene can be obtained from databases (NCBI) such as GenBank, for example, and have registration numbers of L43546 and Y16009 (or X81849, M31795, etc.), respectively.

In particular, the cpe gene detection primer was determined as follows.
The Clostridium perfringens enterotoxin molecule has one cysteine residue at position 186 of its 319 amino acids, and can be divided into functional domains on the N-terminal side and C-terminal side across this cysteine residue. . In the N-terminal domain, the 45th to 116th amino acid region is a domain necessary to cause cell shape change due to membrane pore formation. On the other hand, the region from amino acid 290 to 319 in the C-terminal domain is essential for binding to the enterotoxin receptor. Based on the base sequence of the cpe gene, the present inventor designed a primer targeting the region encoding the C. perfringens enterotoxin gene (381-1341st base sequence; registration number Y16009). In designing the primer, the region where the Tm value of each region was about 58 to 65 ° C. was selected targeting the above base sequence, and the region between the F2 and B2 regions was designed to be about 120 bp. Primers for cpe gene detection were set by narrowing down regions that satisfy these several conditions, which are important points in designing LAMP primers. As a result of various studies, it was found that an optimal LAMP primer can be constructed in the 880 to 1150th base sequence in GenBank accession number Y16009.

  According to the present invention, the region amplified by the cAMP gene amplification LAMP primer and the cpe gene amplification LAMP primer is a portion that does not show homology as compared with the base sequences of other bacterial species available in GenBank, etc. Was confirmed.

  As described above, the base sequences determined as regions that can be annealed to specific regions of the C. perfringens cpa gene and the cpe gene are preferably (i) the bases represented by SEQ ID NOs: 1-4 and 5-6, respectively. Sequence (ii) is the base sequence shown in SEQ ID NOs: 7-10 and 11-12.

  When the LAMP method is performed using a primer set of these sequences, Clostridium perfringens can be detected separately (ie, specifically) from other Clostridium species or bacteria of other genera. Moreover, the presence or absence of the enterotoxin gene in C. perfringens can also be detected.

  Therefore, the LAMP primer set for the detection of Clostridium perfringens of the present invention utilizes a region in which a base sequence different from that of other bacteria on the cero gene of Clostridium perfringens exists, and has at least four types of primers (FIP , BIP, F3 and B3). In addition, two types (Loop-F, Loop-B) of primers are added to accelerate the nucleic acid amplification reaction, and a total of at least six types of primers may be used as one set.

  The LAMP primer set for detecting the Clostridium perfringens enterotoxin gene of the present invention uses a region on the cpe gene of Clostridium perfringens where a base sequence different from that of other bacteria is present, and at least four types of primers (FIP , BIP, F3 and B3). Further, two types (Loop-F, Loop-B) primers for accelerating the nucleic acid amplification reaction may be added, and a total of at least six types of primers may be used as one set.

  According to the present invention, the LAMP method is used as a method for detecting Clostridium perfringens having or without the enterotoxin gene.

  In the LAMP method, four primers (generally called FIP, BIP, F3 and B3) are set for six regions of the target gene, and a nucleic acid can be amplified isothermally using a strand displacement reaction. (JP 2002-330796 A; T. Notomi et al., Nucleic Acids research, 2000, 28 (12), e63). This method will be described below.

  First, for the target gene, the three regions F3c, F2c, and F1c from the 3 ′ end are defined, and the regions B1, B2, and B3 are defined toward the 5 ′ end of the target gene. Four types of primers are designed: FIP, F3, BIP and B3. Here, regions complementary to the F3c, F2c, and F1c regions are F3, F2, and F1, respectively, and regions complementary to the B1, B2, and B3 regions are B1c, B2c, and B3c, respectively.

FIP is a primer designed to have an F2 region complementary to the F2c region of the target gene on the 3 ′ end side and the same sequence as the F1c region of the target gene on the 5 ′ end side. If necessary, a restriction enzyme site can be introduced between F1c and F2 of the FIP primer.
F3 is a primer designed to have an F3 region complementary to the F3c region of the target gene.

BIP is a primer designed to have a B2 region complementary to the B2c region of the target gene on the 3 ′ end side and the same sequence as the B1c region of the target gene on the 5 ′ end side. If necessary, a restriction enzyme site can be introduced between B1c and B2 of the BIP primer.
B3 is a primer designed to have a B3 region complementary to the B3c region of the target gene.

  When the restriction enzyme site is contained in the FIP and BIP primers, the amplified product can be observed as one band after electrophoresis by treating the amplification product with the restriction enzyme after the reaction. In this case, if the target DNA has a restriction enzyme site, it is not necessary to artificially introduce the restriction enzyme site into the primer.

  In addition, when using Loop-F and Loop-B primers, these primers bind to loop portions that are not used in the nucleic acid amplification process. Is accelerated (JP 2002-345499 A).

  The LAMP reaction can be performed in a one-step process until detection by keeping the sample gene, primer, strand-displacing DNA synthase, substrate and the like together at a constant temperature (about 60 to 65 ° C.).

  In this reaction, strand displacement type DNA synthase is used. This enzyme is inexpensive, unlike thermostable DNA polymerase in PCR, and can synthesize DNA while unfolding the double strand of template DNA. it can. For this reason, in the LAMP method, it is not necessary to thermally denature double-stranded DNA into a single strand in advance, unlike the PCR method.

As the LAMP reaction reagent, it is convenient to use a Loopamp DNA amplification reagent kit (excluding the primer set) commercially available from Eiken Chemical Co., Ltd. (Tochigi, Japan). Specifically, examples of the reaction solution are as follows. That is, a buffer solution for double concentration: 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH ₄ ) ₂ SO ₄ , 16 mM MgSO ₄ , 0.2% Tween 20, 1.6 M Betain; each final concentration of 2.8 mM dATP, dCTP, dGTP, dTTP; DNA polymerase: Bst DNA Polymerase 8 units / μl; each primer of the present invention (final concentration): FIP and BIP 40 μM each, F3 and B 35 μM each, Loop-F and Loop-B 20 μM each .

  As the LAMP reaction solution, for example, 3.5 μl of sterilized distilled water, 12.5 μl of buffer solution for double concentration reaction, 1 μl of each primer of FIP, BIP, F3, B3, Loop-F and Loop-B are added, and DNA polymerase Add 1 μl and 2 μl of sample solution (template DNA) to prepare a total volume of 25 μl of reaction solution.

The reaction is performed through the following steps.
(i) By the action of the strand displacement type DNA polymerase, a DNA strand complementary to the template DNA is synthesized starting from the 3 ′ end of the F2 region of FIP.
(ii) The F3 primer anneals to the outside of the FIP, and DNA synthesis extends while peeling the DNA strand from the previously synthesized FIP by the action of the strand displacement DNA polymerase starting from the 3 ′ end.
(iii) The DNA strand synthesized from the F3 primer and the template DNA are double-stranded.
(iv) The DNA strand previously synthesized from FIP is peeled off by the DNA strand from the F3 primer to become single-stranded DNA. This DNA strand has complementary regions F1c and F1 on the 5 ′ end side. Then, self-annealing occurs and a loop is formed.
(v) BIP anneals to the DNA strand that formed a loop in the process of (iv) above, and complementary DNA synthesis is performed starting from the 3 ′ end of this BIP. Furthermore, the B3 primer anneals to the outside of BIP, and DNA synthesis extends while peeling the DNA strand from the previously synthesized BIP by the action of the strand displacement DNA polymerase starting from its 3 ′ end.
(vi) Double-stranded DNA is formed in the process of (v) above.
(vii) Since the DNA strand synthesized from the BIP peeled off in the process of (v) has a complementary sequence at both ends, it self-anneals and forms a loop to form a dumbbell-like structure.
(viii) Starting from the DNA strand having the dumbbell structure, a desired DNA amplification cycle is performed through FIP and BIP annealing.

Each primer set of the present invention causes DNA strand synthesis at the same time as annealing at about 60 to 65 ° C. (eg, 65 ° C.). It is possible to amplify the nucleic acid by 10 ⁹ to 10 ¹⁰ times by carrying out the reaction for about 1 hour by annealing reaction and DNA strand synthesis.

  When a specific DNA region of Clostridium perfringens is amplified, the reaction solution becomes cloudy due to the influence of magnesium pyrophosphate formed as a by-product, so the presence or absence of amplification can be visually determined based on this turbidity, or a turbidity measuring device Turbidity can also be measured optically using. If necessary, the presence or absence of DNA fragments can also be confirmed and detected using agarose gel electrophoresis or the like.

  Samples for detection of Clostridium perfringens and enterotoxin gene-bearing Clostridium perfringens by nucleic acid amplification include food samples such as dairy products, meat products, vegetables and seafood, and environmental samples such as soil and water, and clinical samples. For example, feces may be used.

  When these specimens are used as samples for the LAMP method, operations such as concentration and separation of bacteria present in the specimen, separation of nucleic acids from bacterial cells, and concentration of nucleic acids can also be performed as pretreatment. As a method for concentrating and separating bacteria, filtration, centrifugation, and the like are known and can be appropriately selected. For release of nucleic acids from bacterial cells present in food samples, environmental samples, etc., there is a method of treating the bacterial cells with, for example, Lysozyme or Proteinase K and releasing the nucleic acids from the bacterial cells by heating at 100 ° C. If there is a need for further purification, especially for food samples, the nucleic acid is purified by phenol / chloroform treatment, ethanol precipitation, centrifugation, etc., and finally re-dissolved in TE buffer and used as template DNA for testing. (Let. Appl. Bacteriol, 1998, 26, 382-386).

  For example, an enrichment culture of Clostridium perfringens, which is considered to be present in foods, is performed in an appropriate medium, DNA is isolated from colonies formed on the agar medium, and a LAMP reaction is performed on the DNA using the above-mentioned primers. Amplify specific gene region of fungus.

  As described above, the reaction solution becomes cloudy due to the influence of magnesium pyrophosphate formed as a by-product by the amplification reaction. Therefore, the turbidity of the reaction solution is visually checked or optical nucleic acid amplification using a turbidity measuring device is performed. Can be easily confirmed. If nucleic acid amplification of the cpa gene or a specific region of the cpe gene is observed, it means that the target gene is present, and the cpa gene amplification results in Welsh bacteria, and the cpe gene amplification results in enterotoxin gene-bearing Welsh bacteria. , Indicating that each was detected, ie, positive (+). On the contrary, when nucleic acid amplification is not observed, it means that the target gene is absent, and as a result, welsh bacterium or enterotoxin gene-carrying welsh bacterium is negative (-).

  As described above, according to the present invention, a primer comprising the base sequences shown in SEQ ID NOs: 1 to 4 that amplify a specific gene region derived from the cpa gene, and SEQ ID NOs: 5 to 6 that can be added as appropriate. By using the set as a LAMP primer set, Clostridium perfringens can be specifically detected. Here, “specifically” means that the detection reaction is negative for other Clostridium species and bacteria of other genera.

  In addition, by using a primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 7 to 10 for amplifying a specific gene region derived from the cpe gene and SEQ ID NOs: 11 to 12 that can be added as appropriate, as a LAMP primer set And the C. perfringens enterotoxin gene can be specifically detected. Here, “specifically” means that the detection reaction is negative for bacteria of the genus Clostridium perfringens or other genera that do not carry the Clostridium perfringens enterotoxin gene.

  The method for detecting Clostridium perfringens by the LAMP method of the present invention can be used as a test method that can quickly determine the presence or absence of Clostridium perfringens present in test objects such as foods, environmental samples, and clinical samples. It can be used for detection of contamination of Clostridium perfringens in meat products, vegetables and seafood. Further, the present invention can be used as a test method that can quickly determine whether or not the detected C. perfringens possesses an enterotoxin gene.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to these Examples.

1. LAMP reaction
The concentration of each reagent used in the LAMP reaction is as follows. A LoopAMP DNA amplification reagent kit manufactured by Eiken Chemical Co., Ltd. was used as the LAMP reaction reagent. Double concentration buffer for reaction: 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH ₄ ) ₂ SO ₄ , 16 mM MgSO ₄ , 0.2% Tween 20, 1.6 M Betain. DATP, dCTP, dGTP, dTTP each at a final concentration of 2.8 mM. DNA polymerase: Bst DNA Polymerase 8units / μl. Each primer (final concentration) of the present invention: FIP, BIP 40 μM each, F3, B3 5 μM each, Loop-F, Loop-B 20 μM each. (Refer to the instruction manual of EMPKEN DNA amplification reagent kit LMP201)
LAMP reaction solution is 3.5 μl of sterile distilled water, 12.5 μl of double buffer solution, 1 μl of each primer of FIP, BIP, F3, B3, Loop-F and Loop-B, 1 μl of DNA polymerase, the above 2 μl of the sample solution (template DNA) obtained was added to prepare a reaction solution with a total volume of 25 μl.

  For the LAMP reaction, a turbidity measuring device LA-200F manufactured by Teramex was used, an isothermal reaction at 65 ° C. was performed for 60 minutes, and then an enzyme deactivation treatment was performed at 80 ° C. for 2 minutes. The turbidity measuring device can observe turbidity due to magnesium pyrophosphate, a by-product of the LAMP reaction, over time, and those that increase in turbidity are positive for C. perfringens or C. perfringens enterotoxin genes, and turbidity increases. Those not recognized were considered negative.

2. Primer design The CAMP gene of Clostridium perfringens was compared with the base sequences of other Clostridium spp. To design LAMP primers specific to Clostridium perfringens. In addition, we targeted the cpe gene for the detection of the C. perfringens enterotoxin gene and designed a LAMP primer specific for the C. perfringens enterotoxin gene.

3. Results Primer sets with SEQ ID NOs: 1-4 (FIP, BIP, F3 and B3, respectively) and addable SEQ ID NOs: 5-6 (Loop-F, Loop-B, respectively) are designed to amplify specific regions of the cpa gene. Designed. Actually, as a result of LAMP reaction using 7 strains of Clostridium perfringens and 16 strains of other genus Clostridium and 18 strains of other genera shown in Table 1, only the Clostridium perfringens was a by-product of the amplification reaction. A certain cloudiness of magnesium pyrophosphate was observed. In addition, cross-reactivity was not observed in other bacteria. The results are shown in Table 1.

  Primer sets with SEQ ID NOs: 7-10 (FIP, BIP, F3 and B3, respectively) and addable SEQ ID NOs: 11-12 (Loop-F, Loop-B, respectively) are designed to amplify specific regions of the cpe gene It is. As a result of actually performing the LAMP reaction on 7 strains of Clostridium perfringens shown in Table 2, white turbidity of magnesium pyrophosphate, which is a by-product of the amplification reaction, was observed only in the strain (ATCC 12915) having the Enterobacterial enterotoxin gene. In addition, cross-reactivity was not observed in other bacteria. The results are shown in Table 2.

  As a comparative example, detection by the PCR method (J. Clin. Microbiol., 1994, 32, 2533-2539), which is one of the detection methods for the Clostridium perfringens enterotoxin gene, resulted in the same results as the LAMP method. This indicates that the test according to the present invention gives correct results.

  In addition, as a result of detecting enterotoxin protein using the PET-RPLA "Seiken" (Denka Seiken Co., Ltd.) PET-RPLA detection kit for detecting C. perfringens by reverse passive latex agglutination, which is one of immunological methods, Only enterotoxin protein production was observed (Table 2).

  As shown in Table 1 and Table 2, by carrying out the LAMP method using the primer set for cpa gene amplification in the present invention, it is possible to specifically detect Clostridium perfringens and within 1 hour. The amplification reaction could be confirmed. Furthermore, by carrying out the LAMP method using the primer set for cpe gene amplification, it is possible to specifically detect the C. perfringens enterotoxin gene and to confirm the amplification reaction within 1 hour. It was.

  Thus, it was judged that the cpa gene amplification primer set was effective as a primer for specifically detecting C. perfringens. Moreover, it was judged that the primer set for cpe gene amplification was effective as a primer for specifically detecting C. perfringens having the enterotoxin gene.

  The detection method of Clostridium perfringens or enterotoxin gene-bearing Clostridium perfringens by the LAMP method of the present invention can quickly and specifically determine the presence or absence of Clostridium perfringens present in test objects such as foods, environmental samples, and clinical samples. Since it can be used as an inspection method, it is useful for detecting contamination of C. perfringens in dairy products, meat products, vegetables, seafood, and the like. In addition, since it is possible to quickly and specifically determine whether the detected C. perfringens possesses an enterotoxin gene, it is possible to quickly determine genetically the presence or absence of pathogenicity, It is useful for detecting the enterotoxin gene of Clostridium perfringens.

Claims (7)

  A set of LAMP primers for detecting C. perfringens, comprising 4 types of primers consisting of base sequences FIP, BIP, F3 and B3 shown in SEQ ID NOs: 1 to 4, respectively, which can be annealed to a specific region of C. perfringens cpa gene .   The LAMP primer set according to claim 1, further comprising two kinds of primers consisting of base sequences Loop-F and Loop-B shown in SEQ ID NOs: 5 and 6, respectively.   LAMP primer set for detecting C. perfringens carrying the enterotoxin gene, which can be annealed to a specific region of C. perfringens cpe gene from the base sequences FIP, BIP, F3 and B3 shown in SEQ ID NOs: 7 to 10, respectively. A set comprising the following four kinds of primers.   The LAMP primer set according to claim 3, further comprising two kinds of primers consisting of base sequences Loop-F and Loop-B shown in SEQ ID NOs: 11 and 12, respectively.   A method for detecting Clostridium perfringens that has or does not have an enterotoxin gene, the method comprising detecting amplification of the cpa gene or cpe gene of Clostridium perfringens by the LAMP method.   The method according to claim 5, wherein the LAMP method is performed using the LAMP primer set according to claim 1 or 2.   The method according to claim 5, wherein the LAMP method is performed using the LAMP primer set according to claim 3 or 4.