KR100457355B1 - Pcr primers for amplifying the gene of pathogenic microorganism, and method and test kit for detecting pathogenic microorganism by using the same - Google Patents

Abstract

The present invention performs a PCR primer pair capable of amplifying a specific gene of a pathogenic microorganism, and performs a polymerase chain reaction (PCR) using the same. A pathogenic microorganism capable of quickly and accurately detecting up to nine pathogenic microorganisms causing food poisoning A detection method, and a detection kit.

Description

Primer for gene amplification of pathogenic microorganism, method for detecting pathogenic microorganism using same, and detection kit {PCR PRIMERS FOR AMPLIFYING THE GENE OF PATHOGENIC MICROORGANISM, AND METHOD AND TEST KIT FOR DETECTING PATHOGENIC MICROORGANISM BY USING THE SAME}

According to the present invention, PCR primer pairs capable of amplifying specific genes of nine pathogenic microorganisms, and using the same, perform a multiplex-polymerase chain reaction (M-PCR) to quickly and accurately identify pathogenic microorganisms causing food poisoning. The present invention relates to a pathogenic microorganism detection method and a detection kit.

Despite the development of science and technology, it is still difficult to completely prevent food poisoning. Food poisoning is food poisoning caused by natural poisoning (blowfish poison, mushroom poison, etc.) based on the cause; Food poisoning due to food decay; And food poisoning by incorporation or reproduction of bacteria in food. Among these food poisonings, food poisoning caused by natural poisoning can be easily prevented by avoiding the ingestion of the foods that cause the food poisoning. Food corruption is easily detected by changes in the appearance, smell, etc. of food poisonings. Relatively easy. However, in the case of food poisoning caused by bacteria, that is, bacterial food poisoning, food poisoning is most difficult to prevent because it is generally not observed by the naked eye and is difficult to detect by professional methods. can do.

Recently, as food facilities, such as restaurants, order delivery, food centers, and fast food restaurants, are being used in large quantities, and many consumers use them, there is a risk that many people will get food poisoning in a short time when bacterial food poisoning occurs in these facilities. And because bacterial food poisoning is severe, such as severe vomiting, diarrhea, abdominal pain, and high fever, the occurrence of group food poisoning is a significant problem for both food suppliers and consumers. In addition, these food poisoning causative bacteria produce toxic substances such as endotoxins in the body of infected people, so the treatment should be performed after infection, and in many cases, the treatment is difficult. Especially, very serious such as hemorrhagic enteritis such as pathogenic E. coli O157: H7 Symptoms may be severe and lead to death in severe cases. Therefore, it can be said that there is an urgent need for early detection and prevention of the causative agent of pathogenic food poisoning.

As a method of detecting and confirming the causative agent of bacterial food poisoning, the collected sample is cultured using a selective medium, the colonies formed on the medium are visually observed, or other detection and confirmation means are used. In the test method using a polymerase chain reaction (PCR) has been developed and used.

Polymerase Chain Reaction (PCR) is a simple and convenient way to exponentially amplify DNA sequences of specific regions with very small amounts of DNA. Two different primers in the opposite direction and Taq DNA polymerase, which are functionally stable at high temperatures, are used in three different temperature cycles: denaturation step, annealing step, and extension step. Repeatedly amplify specific DNA. The detection method of pathogenic microorganisms using PCR is amplified by PCR reaction using primers that recognize specific genes of microorganisms to be detected and confirmed by electrophoresis, etc. Pathogenic microorganisms can also be detected.

As a prior art related to a pathogenic microorganism detection method using PCR, Patent Publication No. 1999-65107 discloses four specific genes (2 verotoxins) possessed by Escherichia coli (O157: H7) using a polymerase chain reaction method. A method for simultaneously detecting multiple genes (multiplex-PCR) capable of detecting barrier-associated pathogens and enzyme specific genes) is described. In addition, Japanese Patent Laid-Open No. 1999-88425 discloses oligonucleotides that selectively hybridize with the Verotoxin gene of EHEC or VTEC or the O antigen synthesis region gene of Escherichia coli O157, and perform PCR using this oligonucleotide as a primer. It describes a method for selectively detecting the bacteria producing E. coli and E. coli capable of producing vero toxin.

An object of the present invention is to provide a primer capable of amplifying a specific gene of a pathogenic microorganism causing food poisoning in humans.

Still another object of the present invention is to detect pathogenic microorganisms by polymerase chain reaction using primers that can amplify specific genes of pathogenic microorganisms causing food poisoning in humans. It is to provide a fast and accurate method for detecting pathogenic microorganisms that can be shortened to one day.

It is another object of the present invention to provide a detection kit for pathogenic microorganisms that can detect pathogenic microorganisms quickly and accurately in a short time by polymerase chain reaction using a primer that can amplify specific genes of pathogenic microorganisms causing food poisoning in humans. have.

Figures 1a, 1b, and 1c shows the results of electrophoresis after performing multiplex polymerase chain reaction (Multiplex-PCR) using the genomic DNA extracted after the mixed culture of the pathogenic microorganism as a template.

Figures 2a to 2i shows the results of electrophoresis after polymerase chain reaction using the genomic DNA extracted after culturing each of the nine pathogenic microorganisms as a template.

In order to achieve the above object, the present invention

Primer for amplification of Campylobacter jejuni having the nucleotide sequences shown in SEQ ID NOs: 1 and 2, E. coli O157: H7 ( E.coli O157: H7 ) having the nucleotide sequences shown in SEQ ID NOs: 3 and 4 Primer pair for gene amplification of Bacillus cereus having a base sequence shown in SEQ ID NOs: 5 and 6, a pair of primers for amplification of gene of Bacillus cereus , Listeria monocytogenes having a base sequence shown in SEQ ID NOs: 7 and 8 Primer pair for amplification of genes of the genome of Yersinia enterocolitica having a base sequence shown in SEQ ID NOs: 9 and 10, Salmonella sp having a base sequence shown in SEQ ID NOs: 11 and 12 . a pair of primers for amplification of the gene), SEQ ID NO: 13 and having the nucleotide sequence shown in 14 Staphylococcus gene increased the mouse fluorescein (Staphylococcus aureus) For a pair of primers, SEQ ID NO: 15 and a pair of primers for gene amplification of the Sh Gela (Shigella spp.) Having the nucleotide sequence shown in 16, and SEQ ID NO: 17 and Vibrio parahaemolyticus having a base sequence shown in the 18 gene amplification (Vibrio parahaemolyticus) Provided is a primer set for specific gene amplification of a pathogenic microorganism comprising one or more primer pairs selected from the group consisting of primer pairs.

In addition, the present invention is a method for detecting pathogenic microorganisms using a polymerase chain reaction (PCR), characterized in that detecting the pathogenic microorganisms that cause food poisoning by polymerase chain reaction (Polymerase Chain Reaction) using the primer set It relates to a method for detecting pathogenic microorganisms. Preferably, the detection of nine pathogenic microorganisms causing food poisoning by a multiplex-polymerase chain reaction using a primer set comprising primer pairs for gene amplification of two or more different pathogenic microorganisms. It is intended to provide a method for detecting two or more pathogenic microorganisms.

In addition, the present invention is a kit for detecting pathogenic microorganisms, including PCR primers, reaction buffer, Taq DNA polymerase to detect pathogenic microorganisms using a PCR polymerase chain reaction (PCR), the PCR primers in SEQ ID NO: 1 and 2 A primer pair for amplification of the gene of Campylobacter jejuni having the nucleotide sequence shown, and a pair of primers for the gene amplification of E. coli O157: H7 ( E. coli O157: H7 ) having the nucleotide sequence shown in SEQ ID NOs: 3 and 4. Primer pairs for gene amplification of Bacillus cereus having the nucleotide sequences shown in SEQ ID NOs: 5 and 6, primer pairs for gene amplification of Listeria monocytogenes having the nucleotide sequences shown in SEQ ID NOs: 7 and 8 , SEQ ID NO: 9, and example 10 having the nucleotide sequence shown in reusi California Enterococcus coli urticae a pair of primers for amplification of the gene (Yersinia enterocolitica), SEQ ID No. 11 and salmonella having the nucleotide sequence shown in 12, a pair of primers for gene amplification of the (Salmonella sp.), SEQ ID NO: 13 and having the nucleotide sequence shown in 14 Staphylococcus fluorescein-house primer pairs for gene amplification (Staphylococcus aureus), Primer pairs for gene amplification of Shigella spp. Having the nucleotide sequences shown in SEQ ID NOs: 15 and 16, and primer pairs for gene amplification of Vibrio parahaemolyticus having the nucleotide sequences shown in SEQ ID NOs: 17 and 18. The present invention relates to a pathogenic microorganism detection kit comprising a primer set for amplifying a specific gene of a pathogenic microorganism comprising at least one primer pair selected from the group consisting of:

According to the present invention, a PCR primer pair capable of amplifying a specific gene of a pathogenic microorganism, using the same, performs a Multiplex-Polymerase Chain Reaction (M-PCR) to quickly and accurately detect a pathogenic microorganism that causes food poisoning. The present invention relates to a pathogenic microorganism detection method and a detection kit. By using the pathogenic microorganism and the detection kit according to the present invention, pathogenic microorganisms can be detected quickly and accurately, which can shorten the diagnosis time from 4 days to 7 days. .

Hereinafter, the present invention will be described in more detail.

In the present invention, Campylobacter jejuni , Escherichia coli O157: H7 ( E. coli O157: H7 ), Bacillus cereus, Bacillus cereus , Listeria monocytogenes , Yersinia enterocholicica ( Yersinia) the specific gene to detect specific genes of pathogenic microorganisms of enterocolitica ), Salmonella sp. , Staphylococcus aureus , Staglococcus aureus , Shigella spp. , and Vibrio parahaemolyticus A primer pair for amplification of is provided. Preferably, the present invention may provide a primer set including one or more primer pairs selected from the group consisting of the primer pairs of the nine pairs of pathogenic microorganisms.

Nine PCR primer pairs are designed and synthesized so that specific genes of the nine pathogenic microorganisms can be detected simultaneously with different sizes.

The first PCR primer pair P1 / P2 was designed to specifically detect Campylobacter jejuni , and the size of the amplification product was 127 bp. The base sequence of this PCR primer is as follows.

PCR primer P1: 5'-attttcgctgattttgattttagg-3 '(SEQ ID NO: 1)

PCR primer P2: 5'-tattgatatctttggtggaggcga-3 '(SEQ ID NO: 2)

The second PCR primer pair P3 / P4 was designed to specifically detect E. coli O157: H7 ( E. coli O157: H7 ), and the size of the amplification product was 208 bp. The nucleotide sequence is as follows.

PCR primer P3: 5'-gacagcagttataccactctgcaa-3 '(SEQ ID NO: 3)

PCR primer P4: 5'-gacgaaattctctctgtatctgcc-3 '(SEQ ID NO: 4)

The third PCR primer pair P5 / P6 was designed to specifically detect Bacillus cereus , and the size of the amplification product was 305 bp. The base sequence is as follows.

PCR primer P5: 5'-gactacattcacgattacgcagaa-3 '(SEQ ID NO: 5)

PCR primer P6: 5'-ctatgctgacgagctacatccata-3 '(SEQ ID NO: 6)

The fourth PCR primer pair P7 / P8 was designed to specifically detect Listeria monocytogenes , and the size of the amplification product was 454 bp. The base sequence is as follows.

PCR primer P7: 5'-ctggcacaaaattacttacaacga-3 '(SEQ ID NO: 7)

PCR primer P8: 5'-aactactggagctgcttgtttttc-3 '(SEQ ID NO: 8)

The fifth PCR primer pair P9 / P10 was designed to specifically detect Yersinia enterocolitica , and the size of the amplification product was 551 bp.

PCR primer P9: 5'-cctgtttatcaattgcgtctgtta-3 '(SEQ ID NO: 9)

PCR primer P10: 5'-ataaatggggttcacttcactcag-3 '(SEQ ID NO: 10)

The sixth PCR primer pair P11 / P12 was designed to specifically detect Salmonella sp. , And the size of the amplification product was 678 bp.

PCR primer P11: 5'-gaatcctcagtttttcaacgtttc-3 '(SEQ ID NO: 11)

PCR primer P12: 5'-tagccgtaacaaccaatacaaatg-3 '(SEQ ID NO: 12)

The seventh PCR primer pair P13 / P14 was designed to specifically detect Staphylococcus aureus , and the size of the amplification product was 264 bp.

PCR primer P13: 5'-aatttaacagctaaagagtttggt-3 '(SEQ ID NO: 13)

PCR primer P14: 5'-ttcattaaagaaaaagtgtacgag-3 '(SEQ ID NO: 14)

The eighth PCR primer pair P15 / P16 was designed to specifically detect Shigella spp. And the size of the amplification product was 959 bp.

PCR primer P15: 5'-gacttgttaagtataaaggaaggc-3 '(SEQ ID NO: 15)

PCR primer P16: 5'-aagatttttattatctgaattggg-3 '(SEQ ID NO: 16)

The ninth PCR primer pair P17 / P18 was designed to specifically detect Vibrio parahaemolyticus , and the size of the amplification product was 375 bp.

PCR primer P17: 5'-ctcatttgtactgttgaacgccta-3 '(SEQ ID NO: 17)

PCR primer P18: 5'-aatagaaggcaaccagttgttgat-3 '(SEQ ID NO: 18)

The present invention also relates to a method for detecting a pathogenic microorganism by a multiplex polymerase chain reaction using a primer pair capable of amplifying a specific gene of the pathogenic microorganism.

In the present invention, Campylobacter jejuni , Escherichia coli O157: H7 ( E.coli ) by polymerase chain reaction using a primer set comprising at least one primer pair selected from the group consisting of the nine pairs of primer pairs. O157: H7 ), Bacillus cereus , Listeria monocytogenes , Yersinia enterocolitica , Salmonella sp. , Staphylococcus aureus , Staphylococcus aureus , Shigella spp. , And Vibrio parahaemolyticus can detect one or more pathogenic microorganisms. Preferably, two or more pathogenic microorganisms can be detected by multiplex polymerase chain reaction using a primer set including two or more of the primer pairs, and up to nine microorganisms can be detected simultaneously.

According to an example of the present invention, the detection method first collects and cultures food suspected of being contaminated with pathogenic microorganisms, heat-extracts DNA from the cultured microorganisms, and then uses the multiplex primer mixture as the template DNA. Perform polymerase chain reaction (M-PCR). After the M-PCR reaction, the amplified gene product is electrophoresed on agarose gel to determine which pathogenic microorganisms are contaminated in the food depending on the presence or absence of specific gene amplification.

Figures 1a, 1b, and 1c shows the results of M-PCR performed with the genomic DNA extracted after the mixed culture of the pathogenic microorganism as a template. In FIG. 1A, each lane is as follows.

Lane M: size marker,

Lane 1 is Campylobacter jejuni ,

Lane 2 is E. coli 0157: H7 ( E. coli 0157: H7 ),

Lane 3 is Staphylococcus aureus ,

Lane 4 is Bacillus cereus ,

Lane 5 is Vibrio parahaemolyticus ,

Lane 6 is Listeria monocytogenes ,

Lane 7 is Yersinia enterocolitica ,

Lane 8 is Salmonella sp. , And

Lane 9 is Shigella spp .

In FIG. 1, lane 1 is a result of performing a polymerase chain reaction with multiplex PCR primers by culturing the above 9 pathogenic microorganisms and culturing the extracted genomic DNA as a template.

Lanes 2 to 10 in FIGS. 1B and 1C are as follows.

Lane M: size marker,

Lane 2 is Campylobacter jejuni ,

Lane 3 is E. coli 0157: H7 ( E. coli 0157: H7 ),

Lane 4 is Staphylococcus aureus ,

Lane 5 is Bacillus cereus ,

Lane 6 is Vibrio parahaemolyticus ,

Lane 7 is Listeria monocytogenes ,

Lane 8 is Yersinia enterocolitica ,

Lane 9 is Salmonella sp. , And

Lane 10 is Shigella spp .

Lane 1-1 of FIG. 1C shows a culture after mixing Campylobacter jejuni , Staphylococcus aureus , Listeria monocytogenes , and Salmonella sp. The result of multiple polymerase chain reaction using genomic DNA extracted here as a template is shown. Lanes 1-2 of FIG. 1C show E. coli O157: H7 , Bacillus cereus , Vibrio parahaemolyticus , Yersinia enterocolitica , And Shigella spp. Were mixed and cultured, followed by multiple polymerase chain reaction using genomic DNA extracted as a template.

The detection method of the present invention can not only detect each pathogenic microorganism by using the one PCR primer, but also use a polymerase chain reaction (M-PCR) using a primer set including two or more PCR primers. ), The nine pathogenic microorganisms can be detected quickly and accurately at one time.

In addition, the present invention provides a pathogenic microorganism detection kit comprising a primer set containing one pair of primer pairs selected from the nine pairs of PCR primers. The detection kit includes the nine pairs of primers (SEQ ID NOs: 1 to 18) and general components of the microbial detection kit using PCR (reaction buffer, Taq DNA polymerase, etc.). In a preferred embodiment of the present invention, the detection kit is configured as follows.

(1) the PCR primer pair P1 / P2 of SEQ ID NOs: 1-18; P3 / P4; P5 / P6; P7 / P8; Primer set containing one or more primer pairs selected from P9 / P10, P11 / P12, P13 / P14, P15 / P16, and P17 / P18

(2) Reaction Buffer

(3) Taq DNA polymerase

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the examples.

Example 1

Design and Synthesis of Primer for Amplifying Specific Genes of 9 Pathogenic Microorganisms

Campylobacter jejuni , Escherichia coli O157: H7 ( E.coli O157: H7 ), Bacillus cereus , Listeria monocytogenes , Yersinia enterocolitica , Soluble to simultaneously detect nine pathogenic microorganisms: Salmonella sp. , Staphylococcus aureus , Shigella spp. , And Vibrio parahaemolyticus . Similarly, PCR primers for detecting pathogenic microorganisms were designed and synthesized as shown in Table 1 below.

primer Microbe detection Sequence (SEQ ID NO) order P1 Campylobacter jejuni 5'-attttcgctgattttgattttagg-3 ' One P2 Campylobacter jejuni 5'-tattgatatctttggtggaggcga-3 ' 2 P3 E.coli O157: H7 5'-gacagcagttataccactctgcaa-3 ' 3 P4 E.coli O157: H7 5'-gacgaaattctctctgtatctgcc-3 ' 4 P5 Bacillus cereus 5'-gactacattcacgattacgcagaa-3 ' 5 P6 Bacillus cereus 5'-ctatgctgacgagctacatccata-3 ' 6 P7 Listeria monocytogenes 5'-ctggcacaaaattacttacaacga-3 ' 7 P8 Listeria monocytogenes 5'-aactactggagctgcttgtttttc-3 ' 8 P9 Yersinia enterocolitica 5'-cctgtttatcaattgcgtctgtta-3 ' 9 P10 Yersinia enterocolitica 5'-ataaatggggttcacttcactcag-3 ' 10 P11 Salmonell sp. 5'-gaatcctcagtttttcaacgtttc-3 ' 11 P12 Salmonell sp. 5'-tagccgtaacaaccaatacaaatg-3 ' 12 P13 Staphylococcus aureus 5'-aatttaacagctaaagagtttggt-3 ' 13 P14 Staphylococcus aureus 5'-ttcattaaagaaaaagtgtacgag-3 ' 14 P15 Shigella sp. 5'-gacttgttaagtataaaggaaggc-3 ' 15 P16 Shigella sp. 5'-aagatttttattatctgaattggg-3 ' 16 P17 Vibrio parahaemolyticus 5'-ctcatttgtactgttgaacgccta-3 ' 17 P18 Vibrio parahaemolyticus 5'-aatagaaggcaaccagttgttgat-3 ' 18

Example 2: Construction of a Pathogenic Microorganism Detection Kit

A multiplex primer mixture was prepared containing 9 pairs of PCR primers (SEQ ID NOS: 1 to 18) synthesized in Example 1 to construct a detection kit as follows.

(Kit component)

5 × Reaction buffer

Multiplex Primer Mixture (SEQ ID NOS: 1-18)

Taq DNA Polymerase (1U / μl)

Example 3: Detection of Pathogenic Microorganisms Using Multiple Polymerase Chain Reaction

DNA extracted from the culture solution of the food contaminated with the pathogenic microorganism as a template, PCR reaction with the multiplex primer mixture prepared in Example 2 to amplify the specific gene of the microorganism. The amplified gene product was electrophoresed on agarose gel, and then the pathogenic microorganisms were determined according to the presence or absence of amplification of specific genes (see FIGS. 2A to 2I). The specific experimental method is as follows.

1) Preparation of cell heat extraction sample

After 25g of food suspected of being contaminated with pathogenic microorganisms was quantified, it was added to 225 ml of the seed culture and shaken at 37 ° C for 18 to 24 hours. 1 ml of the spawn culture was transferred to a 1.5 ml centrifuge tube, centrifuged at 12,000 rpm for 5 minutes, the supernatant was removed, and the collected cells were suspended in 200 µl of sterile distilled water and heated at 100 ° C. for 5 minutes. After the heat treatment, the mixture was centrifuged at 12,000 rpm for 10 minutes, and then the supernatant was collected as a heat extract sample.

2) PCR reaction solution (50 μl total reaction solution)

10 μl heat extract sample

5 × Reaction buffer 10 μl

5 μl multiplex primer mixture

1 μl of Taq DNA polymerase (1U / μl)

Residual sterile distilled water (50 μl total)

3) PCR condition

Multiple polymerase chain reaction is performed after initial denaturation at 94 ° C. for 2 minutes, followed by denaturation (30 seconds at 94 ° C.), annealing (30 seconds at 60 ° C.), and extension (72 ° C.). 30 seconds) was performed a total of 40 times.

4) Determination of food pathogenic microbial contamination

After completion of the reaction, the PCR product was developed by electrophoresis on a 2% agarose gel to confirm the contamination of the pathogenic microorganisms with or without the formation of strain-specific gene amplification products. Test results of the food contaminated with each pathogenic microorganism are shown in Figures 2a to 2i.

Lane 1: negative control

Lane 2 is Campylobacter jejuni ,

Lane 3 is E. coli 0157: H7 ( E. coli 0157: H7 ),

Lane 4 is Staphylococcus aureus ,

Lane 5 is Bacillus cereus ,

Lane 6 is Vibrio parahaemolyticus ,

Lane 7 is Listeria monocytogenes ,

Lane 8 is Yersinia enterocolitica ,

Lane 9 is Salmonella sp. , And

Lane 10 is Shigella spp .

Figure 2a is a test food was contaminated with Campylobacter jejuni (127 bp gene amplification product was confirmed, contaminated with E. coli O157: H7 ( E. coli O157: H7 ) was confirmed that the food amplification product In this case, a gene amplification product of 208 bp was identified. FIG. 2c shows a case where Staphylococcus aureus is detected in food, and FIG. 2d shows a case where Bacillus cereus is detected in food, and 264 bp and 305 bp gene amplification products were identified, respectively. . In addition, Figure 2e is when Vibrio parahaemolyticus is detected in the food, Figure 2f is a case of detecting the listeria monocytogenes ( Listeria monocytogenes ) in the food was confirmed that the gene amplification products of 375 bp, 454 bp, respectively. Figure 2g is Yersinia enterocolitica ( Yersinia enterocolitica ) is detected, Figure 2h is Salmonella ( Salmonella sp. ) Was detected in the food when the gene amplification products of 551 bp, 678bp, respectively. FIG. 2i shows a case where Shigella spp. Was detected in food and a gene amplification product of 959 bp was identified.

According to the present invention, it is possible to quickly and accurately determine whether the major pathogenic microorganisms causing food poisoning can be effectively used for the prevention of bacterial food poisoning, and in particular, the conventional food poisoning bacteria diagnosis time, which usually takes 4 to 7 days By shortening the diagnosis in a day, it has the advantage of significantly reducing time and expense.

<110> KOGENEBIOTECH CO., LTD. SAMSUNG EVERLAND INC. <120> PCR PRIMERS FOR AMPLIFYING THE GENE OF PATHOGENIC MICROORGANISM, AND METHOD AND TEST KIT FOR DETECTING PATHOGENIC MICROORGANISM BY USING THE SAME <130> DPP20010603 <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Campylobacter jejuni <400> 1 attttcgctg attttgattt tagg 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Camplylobacter jejuni <400> 2 tattgatatc tttggtggag gcga 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for E.coli O157: H7 <400> 3 gacagcagtt ataccactct gcaa 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for E. coli O157: H7 <400> 4 gacgaaattc tctctgtatc tgcc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer Bacillus cereus <400> 5 gactacattc acgattacgc agaa 24 <210> 6 <211> 24 <212> DNA <213 > Artificial Sequence <220> <223> PCR Primer for Bacillus cereus <400> 6 ctatgctgac gagctacatc cata 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer Listeria monocytogenes < 400> 7 ctggcacaaa attacttaca acga 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Listeria monocytogenes <400> 8 aactactgga gctgcttgtt tttc 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer Yersinia enterocolitca <400> 9 cctgtttatc aattgcgtct gtta 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Yersinia enterocolitica <400> 10 ataaatgggg ttcacttcac tcag 24 <210> 11 <211> 24 <212> DNA < 213> Artificial Sequence <220> PCR Primer for Salmonella sp. <400> 11 gaatcctcag tttttcaacg tttc 24 <210> 12 <211> 24 <212> DNA Artificial sequence <220> PCR Primer for Samonella sp. <400> 12 tagccgtaac aaccaataca aatg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Staphylococcus aureus <400> 13 aatttaacag ctaaagagtt tggt 24 <210> 14 <211 > 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Staphylococcus aureus <400> 14 ttcattaaag aaaaagtgta cgag 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR primer for Shigella sp. <400> 15 gacttgttaa gtataaagga aggc 24 <210> 16 <211> 24 <212> DNA Artificial sequence <220> PCR Primer for Shigella sp. <400> 16 aagattttta ttatctgaat tggg 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Vibrio parahaemolyticus <400> 17 ctcatttgta ctgttgaacg ccta 24 <210> 18 <211 > 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Vibrio parahaemolyticus <400> 18 aatagaaggc aaccagttgt tgat 24

Claims (8)

Primer pairs for gene amplification of Staphylococcus aureus having the nucleotide sequences shown in SEQ ID NOs: 13 and 14, for gene amplification of Shigella spp. Having the nucleotide sequences shown in SEQ ID NOs: 15 and 16 Specific gene amplification of a pathogenic microorganism comprising a primer pair and at least one primer pair selected from the group consisting of primer pairs for gene amplification of Vibrio parahaemolyticus having the nucleotide sequences shown in SEQ ID NOs: 17 and 18 Primer set for. delete delete delete The primer set according to claim 1, wherein the primer set comprises a pair of primers for amplifying a gene of Campylobacter jejuni having the nucleotide sequences shown in SEQ ID NOs: 1 and 2, and E. coli O157 having the nucleotide sequences shown in SEQ ID NOs: 3 and 4. : A7 primer pair for gene amplification of E. coli O157: H7, a pair of primers for gene amplification of Bacillus cereus having base sequences shown in SEQ ID NOs: 5 and 6, bases shown in SEQ ID NOs: 7 and 8 A pair of primers for gene amplification of Listeria monocytogenes with sequence, A pair of primers for gene amplification of Yersinia enterocolitica, SEQ ID NOs: 11 and 12 A disease further comprising one or more primer pairs selected from the group consisting of amplification primer pairs of Salmonella sp. Primer set for amplification of specific genes of native microorganisms. In the method for detecting pathogenic microorganisms using polymerase chain reaction (PCR), A method for detecting pathogenic microorganisms, comprising detecting pathogenic microorganisms by polymerase chain reaction using a primer set according to claim 1. The method according to claim 6, wherein the detection method comprises two or more kinds of causes of food poisoning by a multiplex-polymerase chain reaction using a primer set including primer pairs for gene amplification of two or more different pathogenic microorganisms. A method for detecting pathogenic microorganisms, wherein the pathogenic microorganisms are detected at the same time. In the detection kit of pathogenic microorganisms, including PCR primers, reaction buffer, Taq DNA polymerase to detect pathogenic microorganisms using polymerase chain reaction (PCR), The PCR primer is a pathogenic microorganism detection kit is a primer set according to claim 1 or 5.