KR100919261B1 - A primer for detecting food borne pathogenic micro-organism and a method for detecting food borne pathogenic micro-organism using the same - Google Patents

Abstract

The present invention relates to a primer for detecting food hazard microorganisms and a method for detecting food hazard microorganisms using the same. In particular, the present invention is E. coli O157: H7, Staphylococcus aureus, Bacillus cereus, Vibrio parahemolyticus, Listeria monocytogenes, Thermocinidium entericitis, Clostridium perfringens, Salmonella genus The present invention provides a PCR primer capable of rapidly and accurately amplifying a specific gene of each strain, Shigella genus strain, and Campylobacter jejuni, a food poisoning detection method using the primer, and a detection kit.

Description

A PRIMER FOR DETECTING FOOD BORNE PATHOGENIC MICRO-ORGANISM AND A METHOD FOR DETECTING FOOD BORNE PATHOGENIC MICRO-ORGANISM USING THE SAME}

The present invention relates to a primer for detecting food hazard microorganisms and a method for detecting food hazard microorganisms using the same, and more particularly, using SYBR Green I as well as a general polymerase chain reaction including a conventional PCR. The present invention relates to a method for rapidly and accurately detecting food harmful microorganisms, ie, pathogenic microorganisms causing food poisoning, using PCR primers that can be used for real time PCR.

Food safety has increased due to changes in dietary patterns such as the expansion of group meals such as school meals and increased dining opportunities and environmental changes such as global warming and room temperature. Measures for securing are urgently needed.

Ensuring food safety is directly related to ensuring the effectiveness of microbiological tests to ensure food safety. The most important point of the microbiological test is to identify and identify contamination of food poisoning-related microorganisms and rot microorganisms.

There are two significances in evaluating food in the context of microbiology. First, the purpose of food quality control is to identify the degree of microbial contamination of foods and the useful microorganisms used in the foods. Second, to analyze which foods are contaminated with which pathogenic microorganisms. The former test is used to ensure food freshness and to determine the extent of decay and decay. The latter is important for food hygiene, and tests conducted to determine the cause of food in food poisoning events also fall into this category. In other words, the microbiological test to ensure food stability can be said to be mainly related to the latter.

In relation to the microbiological test, the development of a management system that can detect and prevent bacterial foodborne diseases that can occur collectively in a short time, especially bacterial causes of food poisoning, and prevent the spread of group outbreaks is an important issue. .

More than 250 diseases are known to be a problem in humans due to food mediation, and livestock-related diseases account for a large portion, and about 25 major pathogens are known. Important pathogens transmitted through livestock products have been reported to cause food poisoning in humans such as Salmonella spp., Escherichia coli O157, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, and Clostridium perfringens. .

The food poisoning is a disease accompanied by symptoms such as fever, vomiting, vomiting, diarrhea and abdominal pain, most of which is bacterial food poisoning caused by bacteria. The bacterial food poisoning is classified into an infectious type and a toxin type according to the form of the onset. Type of food poisoning is caused by infection with bacteria during the intake of contaminated food proliferate in the intestinal tract of food poisoning, Salmonella sp (Salmonella spp ), Escherichia coli O157 ( Escherichia . coli O157 : H7 , E. coli 0157 : H7 ) or Vibrio parahemolyticus ( Vibrio parahaemolyticus ) is the main causative organism. Toxin-type food poisoning is food poisoning caused by ingesting toxins present in food by producing toxins as bacteria grow in food. Therefore, in this case, food poisoning may occur when the toxin remains, even if the causative organism has already been killed in the food, and Staphylococcus aureus ) or Clostridium perfusion perfringens ) is the main causative agent.

According to statistics from the Centers for Disease Control and Prevention (CDC), 5 million cases of foodborne illness occur annually, with about 4,000 deaths, most of which are caused by Salmonella strains. It occurs in 10,000 to 4 million people, of which about 2,000 are reported to die, about 0.1%. In addition, food poisoning caused by Escherichia coli O157: H7 caused about 10,000 deaths per year and about 300 deaths. In the case of Listeria monocytogenes, about 1,500 cases occur and about 400 deaths are known. The contamination of livestock foods by bacteria has been shown to be serious.

Looking at the status of food poisoning in Korea, meat and processed meat products account for more than 50% of the causative food, the causative pathogens are Salmonella about 50%, staphylococci about 20%. In order to correctly identify the causative organisms, biochemical tests should be carried out by culturing contaminated specimens or foods, which should take at least three to several weeks. Although such media and biochemical tests are widely used as accredited tests, there is a high probability of food poisoning already spreading before the causative agent is identified and epidemiological studies are completed.

For example, currently used Salmonella detection method, pre-cultured in Buffered Peptone Water (BPW) and cultured in a selective medium, the first verification and then confirmed by biochemical and serological analysis Usually takes about 5 to 6 days.

Therefore, by additionally screening for genetic screening, it is possible to enable early diagnosis of pathogens causing food poisoning. Polymerase chain reaction, first introduced as a genetic test, can quickly and accurately detect pathogen genes by amplifying specific toxin genes or specific genes in common. This method is faster than media and biochemical tests and is characterized by improved specificity. As described above, that is, the specific gene of the pathogen can be tested in a way that can accurately and quickly identify the causative pathogen, and the polymerase chain reaction method is widely used in clinical diagnosis.

However, in the case of the polymerase chain reaction method used previously, the culture process is performed in order to obtain analytical cell mass, and thus there is a problem that it takes more than one day to analyze, and electrophoresis for confirmation and determination after gene amplification process. The hassle of going through the dyeing process is constant. Therefore, in recent years, as the research on the method of detecting more with high sensitivity in the unique environment of food has been continuously conducted, various polymerase chain reaction methods have been developed, and among them, real-time polymerase chain reaction method has received the most attention. have.

As described above, using a variety of techniques, such as the real-time polymerase chain reaction method, the detection of food poisoning causative bacteria, which omitted the culture process of food poisoning causative bacteria, and the development of a detection method that can detect the degree of food contamination therefrom This is urgently needed.

In order to solve the problems of the prior art, an object of the present invention is to provide a primer for detecting microorganisms for food.

In addition, an object of the present invention is to provide a food hazard microorganism detection method using the primer for detecting food hazard microorganisms.

In addition, an object of the present invention is to provide a food hazard microorganism detection kit comprising the primer for the detection of food hazard microorganisms, reaction buffer and polymerase.

In order to achieve the above object, the present invention provides a primer for detecting microorganisms for food.

The food-hazard microorganism means a causative agent of a disease occurring in humans through food, preferably a pathogenic microorganism, more preferably a pathogenic microorganism causing food poisoning.

The pathogenic microorganism is preferably E. coli O157 ( E. coli O157 : H7 ), Staphylococcus aureus), Bacillus cereus (Bacillus cereus ), Vibrio parahemolyticus parahaemolyticus), L. monocytogenes to Ness (Listeria monocytogenes), ten o'clock Catania Entebbe recalls Lee Utica (Yersinia entericolitica), Clostridium flops presence (Clostridium perfringens), Salmonella sp (Salmonella spp . ), Shigella genus strain spp . ) And Camphylobacter jejunii may be one or more selected from the group consisting of.

The primer for detecting the harmful food microorganism of the present invention means a primer capable of detecting a food harmful microorganism by detecting a specific gene of the food harmful microorganism, and preferably by detecting the specific gene of the pathogenic microorganism, the specific gene of the pathogenic microorganism It may be a primer pair for amplifying.

The primer pair of the present invention is a primer pair for detecting food hazard microorganisms for detecting two or more food hazard microorganisms, and preferably a pair of primers capable of simultaneously detecting 10 specific genes of food hazard microorganisms, specifically SEQ ID NO: E. coli O157: H7 consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 1 and a primer comprising the nucleotide sequence of SEQ ID NO: 2 0157 : H7 ) primer pair for detection; Consisting of a primer comprising SEQ ID NO: 3 DNA sequence and the base sequence of primer SEQ ID NO: 4, including the Staphylococcus aureus (Staphylococcus aureus ) primer pairs for detection; Bacillus consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 5 and a primer comprising the nucleotide sequence of SEQ ID NO: 6 ( Bacillus cereus ) primer pairs for detection; Vibrio parahaemolyticus consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 7 and a primer comprising the nucleotide sequence of SEQ ID NO: 8 ( Vibrio parahaemolyticus ) primer pairs for detection; A pair of primers for detecting Listeria monocytogenes consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 9 and a primer comprising a nucleotide sequence of SEQ ID NO: 10; A pair of primers for detecting Y. entericolitica consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 11 and a primer comprising the nucleotide sequence of SEQ ID NO: 12; Consisting of a primer comprising the base sequence SEQ ID NO: 13 base sequence of the primers and SEQ ID NO: 14 containing the Clostridium flops presence (Clostridium perfringens ) primer pairs for detection; Consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 16 and a primer comprising the nucleotide sequence of SEQ ID NO: 15 Salmonella spp. (Salmonella spp . ) Primer pair for detection; Shigella consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 17 and a primer comprising the nucleotide sequence of SEQ ID NO: 18 spp . ) Primer pair for detection; And a primer comprising a nucleotide sequence of SEQ ID NO: 19 and a primer comprising a nucleotide sequence of SEQ ID NO: 20 ( Camhylobacter jejuni) jejuni ) may be one or more primer pairs selected from the group consisting of a primer pair for detection.

Since the primer pairs have different sizes and melting temperatures (Tm), that is, temperatures at which the double strand of DNA is separated into one strand, the specific genes of the 10 pathogenic microorganisms can be detected simultaneously.

In addition, the present invention E. coli O157 ( E. coli O157 : H7 ), Staphylococcus aureus), Bacillus cereus (Bacillus cereus ), Vibrio parahemolyticus parahaemolyticus ), Listeria monocytogenes , Yersinia entericolitica), Clostridium flops presence (Clostridium perfringens ), Salmonella spp. , and Shigella strains spp . ) Or each strain specific PCR primer pair capable of detecting Camphylobacter jejunii . The combination of specific PCR primer pairs for each strain to be detected is shown in Table 1 below.

microbe No direction Primer sequence (5 '→ 3') SEQ ID NO: E. coli O157 : H7 E1 Forward direction GAT AGA CTT TTC GAC CCA ACA AAG One E2 Reverse CCG ATG TGG TCCCCT GAG 2 101 E3 Reverse TTG CTC AAT AAT CAG ACGAAG ATG 21 208 E4 Reverse CGT TGT CAC ACC GGG CAC TGA 22 304 E5 Reverse CCC CAC TCT GAC ACC ATC CT 23 625 S. aureus SA1 Forward direction AAT TTA ACA GCT AAA GAG TTT GGT 3 SA2 Reverse TTC CCA CTA AAT GTG TTT CAT AA 24 122 SA3 Reverse TTC ATT AAA GAA AAA GTG TAC GAG 25 265 SA4 Reverse ACG TAT CTT CCA TGA ATG ATC TAA 4 622 B. cereus B1 Forward direction GAC TAC ATT CAC GAT TAC GCA GAA 5 B2 Reverse GTA GAC CAA TTG TTG CTT GTA ATG G 26 164 B3 Reverse CTA TGC TGA CGA GCT ACA TCC ATA 27 303 B4 Reverse TTC CTA AAC TTG CAC CAT CTC GG 6 640 V. parahaemolyticus V1 Forward direction CTC ATT TGT ACT GTT GAA CGC CTA 7 V2 Reverse AGG CGT CAT TGT TAT CAG AAG C 28 91 V3 Reverse TCA TGA GCA GAG GCG TCA TTG 29 101 V4 Reverse TAC TAC TGG CGC TTC TGG TTC 8 167 V5 Reverse AAT AGA AGG CAA CCA GTT GTT GAT 30 375 V6 Reverse GAC TGC CAT TCA TTT GAT GAT AGC 31 688 L. monocytogenes L1 Forward direction CTG GCA CAA AAT TAC TTA CAA CGA 9 L2 Reverse TGG AGT GCT TAC TGC TTT GTC AG 32 83 L3 Reverse AAC TAC TGG AGC TGC TTG TTT TTC 10 451 L4 Reverse GTT ACC ACC CCA TGA ATA AGC 33 800 Y. enterocolitica Y1 Forward direction AAT ACC GCA TAA CGT CTT CG 11 Y2 Reverse CTT CTT CTG CGA GTA ACG TC 12 330 Y3 Reverse CGG TAT TCC TCC AGA TCT CTA C 34 551 C. perfringens C1 Forward direction TGA TAT GTC CAA AAA TGA ACC AGA 13 C2 Reverse TTG TGA TTC CCC TGT GTC AGG 14 220 C3 Reverse TGC CAT TCA TAT CTA GCT AAT GCT 35 260 C4 Reverse TTT CCT TTC TTC TGC AAA AGT CTC 36 402 C5 Reverse CCT GCT GTT CCT TTT TGA GAG 37 630 Salmonella sp . SAL1 Forward direction GAA TCC TCA GTT TTT CAA CGT TTC 15 SAL2 Reverse CCA GAC GAA AGA GCG TGG TAA 38 60 SAL3 Reverse GTA TGC CCG GTA AAC AGA TGA GT 39 284 SAL4 Reverse TAG CCG TAA CAA CCA ATA CAA ATG 16 678 Shigella sp . SH1 Forward direction CAG GCA TGA AAT TAA GCC TG 17 SH2 Reverse CTG CAT TCT GGC AAT CTC TTC ACA 40 110 SH3 Reverse GCA TCT GAC AGC AAT AGT ACA 41 355 SH4 Reverse TGT ACA TCG ATA ATA CCA AAC 18 445 C. jejuni CA1 Forward direction ACT TCT TTA TTG CTT GCT GC 19 CA2 Reverse GCC ACA ACA AGT TAA AGA AGC 20 303

In detail, the combination of specific PCR primers for each strain to be detected in Table 1, which can detect microorganisms for each food, is as follows.

A pair of primers capable of detecting a specific gene of E. coli O157: H7 of the present invention, specifically, E. coli O157: H7 consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 1 and a primer comprising a nucleotide sequence of SEQ ID NO: 2 Primer pairs for; A primer pair for detecting Escherichia coli O157: H7 consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 1 and a primer comprising a nucleotide sequence of SEQ ID NO: 21; A primer pair for detecting Escherichia coli O157: H7 consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 1 and a primer comprising a nucleotide sequence of SEQ ID NO: 22; It may be a primer pair for detecting E. coli O157: H7 consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 1 and a primer comprising a nucleotide sequence of SEQ ID NO: 23.

A pair of primers capable of detecting a specific gene of Staphylococcus aureus of the present invention, specifically, Staphylococcus consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 3 and a primer comprising a nucleotide sequence of SEQ ID NO: 4 Primer pairs for detecting aureus; A primer pair comprising a base pair of SEQ ID NO: 3 and a primer pair comprising a base sequence of SEQ ID NO: 3 and a primer pair comprising a primer comprising SEQ ID NO: 3 and a primer comprising SEQ ID NO: 24; It may be a primer pair for detecting Staphylococcus aureus consisting of a primer comprising a sequence.

A primer pair for detecting a specific gene of Bacillus cereus of the present invention, specifically, a primer for detecting Bacillus cereus, comprising a primer comprising a nucleotide sequence of SEQ ID NO: 5 and a primer comprising a nucleotide sequence of SEQ ID NO: 6 pair; Bacillus cereus detection primer pair consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 5 and a primer comprising the nucleotide sequence of SEQ ID NO: 26 and a primer comprising the nucleotide sequence of SEQ ID NO: 5 and a nucleotide sequence of SEQ ID NO: 27 It may be a primer pair for detecting Bacillus cereus consisting of a primer.

A pair of primers capable of detecting a specific gene of Vibrio parahemoliticus of the present invention, specifically, a vibrio parahe consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 7 and a primer comprising a nucleotide sequence of SEQ ID NO: 8 Primer pairs for detecting moltycus; A pair of primers for detecting Vibrio parahaemolyticus consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 7 and a primer comprising a nucleotide sequence of SEQ ID NO: 28; A pair of primers for detecting Vibrio parahaemolyticus consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 7 and a primer comprising a nucleotide sequence of SEQ ID NO: 29; A primer pair comprising a base pair of SEQ ID NO: 7 and a primer pair comprising a base sequence of SEQ ID NO: 7 and a primer pair comprising a primer comprising SEQ ID NO: 7 and a primer comprising SEQ ID NO: 30; It may be a pair of primers for detecting Vibrio parahemolyticus consisting of a primer comprising a sequence.

A primer pair capable of detecting a specific gene of Listeria monocytogenes of the present invention, specifically, a Listeria monocytogege consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 9 and a primer comprising a nucleotide sequence of SEQ ID NO: 10 Primer pair for detection of ness; A primer pair comprising a primer for detecting Listeria monocytogenes consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 9 and a primer comprising the nucleotide sequence of SEQ ID NO: 32, and a base sequence of SEQ ID NO: 33 It may be a primer pair for detecting Listeria monocytogenes consisting of a primer comprising a.

A primer pair capable of detecting a specific gene of Clostridium perfringens of the present invention, specifically Clostridium consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 13 and a primer comprising a nucleotide sequence of SEQ ID NO: 14 Primer pairs for perpresence detection; A pair of primers for detecting Clostridium perpresence consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 13 and a primer comprising a nucleotide sequence of SEQ ID NO: 35; A primer pair comprising a base pair of SEQ ID NO: 13 and a primer pair comprising a base sequence of SEQ ID NO: 13 and a primer pair comprising a primer comprising SEQ ID NO: 13 and a primer comprising SEQ ID NO: 36; It may be a primer pair for detecting Clostridium perfringence consisting of a primer comprising a sequence.

A primer pair for detecting a specific gene of Salmonella spp. Of the present invention, specifically, a pair of primers for detecting Salmonella spp. Comprising a primer comprising a nucleotide sequence of SEQ ID NO: 15 and a primer comprising a nucleotide sequence of SEQ ID NO: 16; A primer pair for detecting Salmonella spp. Comprising a primer comprising the nucleotide sequence of SEQ ID NO: 15 and a primer comprising the nucleotide sequence of SEQ ID NO: 38, and a primer comprising the nucleotide sequence of SEQ ID NO: 39, and a primer comprising the nucleotide sequence of SEQ ID NO: 15. It may be a primer pair for detecting Salmonella spp. Consisting of primers.

A primer pair for detecting a specific gene of Shigella spp. Of the present invention, specifically, a primer for detecting Shigella spp. Consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 17 and a primer comprising a nucleotide sequence of SEQ ID NO: 18 pair; A primer pair comprising a primer containing a nucleotide sequence of SEQ ID NO: 17 and a primer pair comprising a nucleotide sequence of SEQ ID NO: 40, a primer comprising a nucleotide sequence of SEQ ID NO: 41, and a base sequence of SEQ ID NO: 41 It may be a primer pair for detecting Shigella bacteria consisting of primers.

* A pair of primers for detecting E. coli O157: H7 consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 1 and a primer comprising the nucleotide sequence of SEQ ID NO: 2, amplifies the specific gene of the E. coli O157: H7 strain to about 101 bp and Tm The value represents 79 ° C.

SEQ ID NO: 1 (E1): 5'-GATAGACTTTTCGACCCAACAAAG-3 '

SEQ ID NO: 2 (E2): 5'-CCGATGTGGTCCCCTGAG-3 '

The primer pair for detecting E. coli O157: H7 may be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

A primer pair for detecting Staphylococcus aureus consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 3 and a primer comprising the nucleotide sequence of SEQ ID NO: 4 amplifies a specific gene of Staphylococcus aureus at about 622 bp. Tm value is 80.9 ° C.

SEQ ID NO: 3 (SA1): 5'-AATTTAACAGCTAAAGAGTTTGGT-3 '

SEQ ID NO: 4 (SA4): 5'-ACGTATCTTCCATGAATGATCTAA-3 '

The primer pair for detecting Staphylococcus aureus may be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

A primer pair for detecting Bacillus cereus, comprising a primer comprising the nucleotide sequence of SEQ ID NO: 5 and a primer comprising the nucleotide sequence of SEQ ID NO: 6, amplifies a specific gene of Bacillus cereus to about 640 bp and Tm value is 82.7 ° C. Indicates.

SEQ ID NO: 5 (B1): 5'-GACTACATTCACGATTACGCAGAA-3 '

SEQ ID NO: 6 (B4): 5'-TTCCTAAACTTGCACCATCTCGG-3 '

The primer pair for detecting Bacillus cereus may be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

A pair of primers for detecting Vibrio parahaemolyticus, consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 7 and a primer comprising the nucleotide sequence of SEQ ID NO: 8, amplifies a specific gene of Vibrio parahaemolyticus at about 167 bp. Tm value is 82.7 ° C.

SEQ ID NO: 7 (V1): 5'-CTCATTTGTACTGTTGAACGCCTA-3 '

SEQ ID NO: 8 (V4): 5'-TACTACTGGCGCTTCTGGTTC-3 '

The Vibrio parahemolyticus primer pair may be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

A primer pair for detecting Listeria monocytogenes comprising a primer comprising the nucleotide sequence of SEQ ID NO: 9 and a primer comprising the nucleotide sequence of SEQ ID NO: 10, amplifies a specific gene of Listeria monocytogenes to about 451 bp and Tm value is 82.3 ° C.

SEQ ID NO: 9 (L1): 5'-CTGGCACAAAATTACTTACAACGA-3 '

SEQ ID NO: 10 (L3): 5'-AACTACTGGAGCTGCTTGTTTTTC-3 '

The primer pair for detecting Listeria monocytogenes may be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

A primer pair for detecting thermocinta entericholicica, comprising a primer comprising a nucleotide sequence of SEQ ID NO: 11 and a primer comprising a nucleotide sequence of SEQ ID NO: 12, amplifies the gene of Thermocinia entericholicica to about 330 bp Tm value represents 88 degreeC.

SEQ ID NO: 11 (Y1): 5'-AATACCGCATAACGTCTTCG-3 '

SEQ ID NO: 12 (Y2): 5'-CTTCTTCTGCGAGTAACGTC-3 '

The pair of primers for detecting thermocinia entericitis can be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

A primer pair for detecting Clostridium perfringens, comprising a primer comprising the nucleotide sequence of SEQ ID NO: 13 and a primer comprising the nucleotide sequence of SEQ ID NO: 14, amplifies a specific gene of Clostridium perfringence to about 220 bp. Tm value is 78 ° C.

SEQ ID NO: 13 (C1): 5'-TGATATGTCCAAAAATGAACCAGA-3 '

SEQ ID NO: 14 (C2): 5'-TTGTGATTCCCCTGTGTCAGG-3 '

The primer pair for detecting Clostridium perpresence may be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

A primer pair for detecting a Salmonella strain comprising a primer comprising the nucleotide sequence of SEQ ID NO: 15 and a primer comprising the nucleotide sequence of SEQ ID NO: 16, amplified a specific gene of the Salmonella strain to about 678 bp and the Tm value was 86.7 ° C. Indicates.

SEQ ID NO: 15 (SAL1): 5'-GAATCCTCAGTTTTTCAACGTTTC-3 '

SEQ ID NO: 16 (SAL4): 5'-TAGCCGTAACAACCAATACAAATG-3 '

The primer pair for detecting the strain of genus Salmonella may be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

A primer pair for detecting Shigella genus consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 17 and a primer comprising the nucleotide sequence of SEQ ID NO: 18, amplifies a specific gene of the Shigella genus strain to about 445 bp and the Tm value is 80.8 ° C.

SEQ ID NO: 17 (SH1): 5'-CAGGCATGAAATTAAGCCTG-3 '

SEQ ID NO: 18 (SH4): 5'-TGTACATCGATAATACCAAAC-3 '

The primer pair for detecting the Shigella genus strain may be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

A primer pair for detecting Campylobacter jejuni consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 19 and a primer comprising the nucleotide sequence of SEQ ID NO: 20, amplifies a specific gene of Campylobacter jejuni to about 303 bp and Tm value is 80.7 ° C. Indicates.

SEQ ID NO: 19 (CA1): 5'-ACTTCTTTATTGCTTGCTGC-3 '

SEQ ID NO: 20 (CA2): 5'-GCCACAACAAGTTAAAGAAGC-3 '

The primer pair for detecting Campylobacter jejuni may be used in a conventional polymerase chain reaction, and preferably used in a conventional polymerase chain reaction or a real-time polymerase chain reaction.

In addition, the present invention relates to a method for detecting food hazard microorganisms using a primer pair for detecting food hazard microorganisms of the present invention, preferably by using a polymerase chain reaction (PCR) using a primer for detecting food hazard microorganisms. It may be related to a food hazard microorganism detection method comprising the step of detecting a food hazard microorganism.

In another embodiment of the present invention, the present invention provides a food hazard microorganism detection kit comprising the polymerase chain reaction primer, reaction buffer and polymerase for detecting the food hazard microorganisms.

In the detection method and detection kit of the present invention, each of the food harmful microorganisms can be detected by using the primer pair for the one food harmful microorganism. A primer pair capable of detecting each pathogenic microorganism using the primer pair for the one pathogenic microorganism may use one or more selected from the group consisting of the primer pairs in Table 1 according to the type of each microorganism detected.

In addition, by performing a multiplex polymerase chain reaction (Multiplex Polymerase Chain Reaction) using a pair of primers that can detect the specific gene of the microorganisms for the two or more foods in Table 2 in the detection method and detection kit of the present invention, More than one species of food hazard can be detected simultaneously.

microbe No direction Primer sequence (5 '→ 3') SEQ ID NO: E. coli O157 : H7 E1 Forward direction GAT AGA CTT TTC GAC CCA ACA AAG One E2 Reverse CCG ATG TGG TCCCCT GAG 2 S. aureus SA1 Forward direction AAT TTA ACA GCT AAA GAG TTT GGT 3 SA4 Reverse ACG TAT CTT CCA TGA ATG ATC TAA 4 B. cereus B1 Forward direction GAC TAC ATT CAC GAT TAC GCA GAA 5 B4 Reverse TTC CTA AAC TTG CAC CAT CTC GG 6 V. parahaemolyticus V1 Forward direction CTC ATT TGT ACT GTT GAA CGC CTA 7 V4 Reverse TAC TAC TGG CGC TTC TGG TTC 8 L. monocytogenes L1 Forward direction CTG GCA CAA AAT TAC TTA CAA CGA 9 L3 Reverse AAC TAC TGG AGC TGC TTG TTT TTC 10 Y. enterocolitica Y1 Forward direction AAT ACC GCA TAA CGT CTT CG 11 Y2 Reverse CTT CTT CTG CGA GTA ACG TC 12 C. perfringens C1 Forward direction TGA TAT GTC CAA AAA TGA ACC AGA 13 C2 Reverse TTG TGA TTC CCC TGT GTC AGG 14 Salmonella sp . SAL1 Forward direction GAA TCC TCA GTT TTT CAA CGT TTC 15 SAL4 Reverse TAG CCG TAA CAA CCA ATA CAA ATG 16 Shigella sp . SH1 Forward direction CAG GCA TGA AAT TAA GCC TG 17 SH4 Reverse TGT ACA TCG ATA ATA CCA AAC 18 C. jejuni CA1 Forward direction ACT TCT TTA TTG CTT GCT GC 19 CA2 Reverse GCC ACA ACA AGT TAA AGA AGC 20

The food-hazard microorganism detection primer for detecting two or more food-hazard microorganisms may be a primer pair selected from two or more from the group consisting of primers capable of simultaneously detecting the specific genes of the 10 food-hazard microorganisms. Primer pairs selected from two or more species from the group consisting of primer pairs capable of simultaneously detecting the specific genes of the ten harmful food microorganisms are each strain specific primer pair consisting of the primers set forth in SEQ ID NOs: 1 to 20 shown in Table 2 above. Can be.

Preferably, the primer pair capable of simultaneously detecting the specific genes of the 10 pathogenic microorganisms is E. coli O157: H7 (a primer comprising a nucleotide sequence of SEQ ID NO: 1 and a primer comprising a nucleotide sequence of SEQ ID NO: 2). E. coli 0157 : H7 ) primer pair for detection; Consisting of a primer comprising SEQ ID NO: 3 DNA sequence and the base sequence of primer SEQ ID NO: 4, including the Staphylococcus aureus (Staphylococcus aureus ) primer pairs for detection; Bacillus consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 5 and a primer comprising the nucleotide sequence of SEQ ID NO: 6 ( Bacillus cereus ) primer pairs for detection; Vibrio parahaemolyticus consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 7 and a primer comprising the nucleotide sequence of SEQ ID NO: 8 ( Vibrio parahaemolyticus ) primer pairs for detection; Listeria monocytogenes consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 9 and a primer comprising the nucleotide sequence of SEQ ID NO: 10 ( Listeria monocytogenes ) primer pairs for detection; A pair of primers for detecting Y. entericolitica consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 11 and a primer comprising the nucleotide sequence of SEQ ID NO: 12; And a primer pair for detecting Clostridium perfringens consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 13 and a primer comprising a nucleotide sequence of SEQ ID NO: 14; Consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 16 and a primer comprising the nucleotide sequence of SEQ ID NO: 15 Salmonella spp. (Salmonella spp . ) Primer pair for detection; Shigella consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 17 and a primer comprising the nucleotide sequence of SEQ ID NO: 18 spp . ) Primer pair for detection; And a primer comprising a nucleotide sequence of SEQ ID NO: 19 and a primer comprising a nucleotide sequence of SEQ ID NO: 20 ( Camhylobacter jejuni) jejuni ) It may be one or more selected from the group consisting of a primer pair for detection.

In the case of using the primer for the detection of food hazard microorganisms for the detection of two or more food hazard microorganisms of Table 2, since the multiple polymerase chain reaction can be performed under the same reaction conditions, the detection of two or more food hazard microorganisms may be possible at the same time. . The multiple polymerase chain reaction may be a polymerase chain reaction capable of simultaneously performing a polymerase chain reaction with respect to one or more template DNAs, preferably two or more template DNAs, by simultaneously using two or more primer pairs.

The food hazard microorganism detection method of the present invention may be related to a food hazard microorganism detection method comprising the step of detecting food hazard microorganisms by a polymerase chain reaction using the primer for detecting food hazard microorganisms of the present invention.

The detection method is a food microorganism of the 10 species, namely E. coli O157 ( E. coli O157: H7), Staphylococcus aureus), Bacillus cereus (Bacillus cereus ), Vibrio parahemolyticus parahaemolyticus), L. monocytogenes to Ness (Listeria monocytogenes ), Yersinia entericolitica), Clostridium flops presence (Clostridium perfringens), Salmonella spp (Salmonella spp . ), Shigella spp. And Camphylobacter jejunii for detecting one food microorganism selected from the group consisting of, preferably according to the type of each detection microorganism Amplifying DNA by performing a polymerase chain reaction using at least one selected from the group consisting of primer pairs of 1; And using the amplified DNA may be a detection method comprising the step of confirming the presence of food hazard microorganisms, using the amplified DNA to determine the presence of food hazard microorganisms amplified DNA product The size of the amplified DNA product may be performed by electrophoresis. Confirmation of the size of the amplified DNA product may be to confirm whether or not the size of the expected DNA product when amplifying using the primers used, preferably the size of the amplification products of Table 1.

The polymerase chain reaction may be a conventional polymerase chain reaction, preferably a conventional polymerase chain reaction (Conventional PCR) or a real time polymerase chain reaction (Real time PCR), more preferably The conventional polymerase chain reaction refers to a conventional polymerase chain reaction in which a step of amplifying a specific DNA and additionally performing a step of checking whether a specific DNA is amplified is performed. The step of confirming the amplification of the specific DNA may be performed by a method of confirming the size of the DNA product amplified by electrophoresis, etc. The real-time polymerase chain reaction is preferably a real-time polymerization using SYBR Green I It may be an enzyme chain reaction.

In addition, when using two or more selected from the group consisting of the primer pairs in Table 1 according to the type of each detection microorganism, multiple polymerase chain reaction can be used.

When the polymerase chain reaction is a conventional polymerase chain reaction, the step of confirming the presence of microorganisms for food by using the amplified DNA comprises electrophoresis of the amplified DNA product to the size of the amplified DNA product This can be done by checking.

When the polymerase chain reaction is a real-time polymerase chain reaction, the step of confirming the presence of microorganisms for food using the amplified DNA is the degree of fluorescence, that is, fluorescence resonance energy transfer (FRET) It can be carried out by a method for measuring, and may be carried out by a method comprising the step of identifying the temperature (melting temperature) in which the double helix of the amplified DNA product is separated.

When the detection method is for detecting two food harmful microorganisms selected from the group consisting of the 10 food harmful microorganisms, it is preferably multiplexed using two or more selected from the group consisting of the primer pairs of Table 2 above. Amplifying DNA by performing a polymerase chain reaction; And using the amplified DNA may be a detection method comprising the step of confirming the presence of food hazard microorganisms, using the amplified DNA to determine the presence of food hazard microorganisms amplified DNA product The size of the amplified DNA product may be performed by electrophoresis. Confirmation of the size of the amplified DNA product may be to confirm whether or not the size of the expected DNA product when amplifying using the primers used, preferably the size of the amplification products of Table 1.

When the polymerase chain reaction is a conventional polymerase chain reaction, the step of confirming the presence of microorganisms for food by using the amplified DNA comprises electrophoresis of the amplified DNA product to the size of the amplified DNA product This can be done by checking.

When the polymerase chain reaction is a real-time polymerase chain reaction, the step of confirming the presence of microorganisms for food using the amplified DNA is the degree of fluorescence, that is, fluorescence resonance energy transfer (FRET) It can be carried out by a method for measuring, and may be carried out by a method comprising the step of identifying the temperature (melting temperature) in which the double helix of the amplified DNA product is separated.

The detection kit may include conventional components of the microbial detection kit using the primer pair for detecting the microorganism and a polymerase chain reaction. The conventional components may be reaction buffer, Taq DNA polymerase, dNTP and MgCl ₂ , and the like, and may further include SYBR Green I for real-time polymerase chain reaction using SYBR Green I.

As a preferred example of the microorganism detection kit for the food of the present invention, the detection kit of the present invention is (i) at least one selected from the group consisting of the primers of each strain of Table 1 or in the group consisting of the primers described in SEQ ID NOs: 1 to 20 At least one strain specific PCR primer pair selected; (ii) 2X PCR Master Mix comprising Taq DNA polymerase and SYBR Green I; And (iii) template DNA, that is, sample DNA extracted from a substance to be measured for which food-hazard microorganisms can be found.

The polymerase chain reaction can be carried out using a conventional polymerase chain reaction device, the polymerase chain reaction device is preferably a real-time polymerase chain reaction device, thermal block polymerase chain reaction (Thermal Block PCR) device, a micro polymerase chain reaction (Micro PCR) may be a device, more preferably a real-time polymerase chain reaction device. For example, the 7700 real-time polymerase chain reaction device (ABI, USA) or TMC-1000 real-time polymerase chain reaction device (Samsung Techwin, South Korea), but is not limited thereto.

The template DNA of the polymerase chain reaction, which is a target sample used in the microorganism detection method, is any one selected from the group consisting of all substances in which microorganisms can be found, preferably food, feed, and specimens, more preferably food. It may be extracted from.

Extraction of the template DNA, that is, sample DNA, may be performed by a conventional DNA extraction method, and the DNA extraction method is preferably an alkaline extraction method, a hydrothermal extraction method, a column extraction method, or a phenol / chloroform (Phenol / Chloroform) extraction method, and more preferably, an alkaline extraction method. In addition, before performing the DNA extraction method, a step in which the pathogenic microorganisms can be found, that is, a target material or a sample, is added to sterile water or saline and crushed using a homogenizer, and then the filtrate is taken. Can be.

As one specific example of the present invention, the extraction of the sample DNA may include collecting a target sample suspected of being contaminated with food hazard microorganisms; This may be carried out by sterile water or physiological saline, preferably 0.85% saline, followed by an extraction method comprising treating proteinase K and recovering the pellet and performing DNA extraction. .

In addition, the column extraction method as a specific example of the present invention

i) taking 1 ml of shake culture and transferring it to a 1.5 ml tube and centrifuging for 5 minutes at 12,000 rpm; ii) After discarding the supernatant, pellets were added with 300 ul of lysis A buffer (Food & Feed DNA Extraction kit, KoGeneBioTech) and 30 ul of lysis B buffer (Food & Feed DNA Extraction kit, KoGeneBioTech). Dissolving; iii) reacting the lysis buffer and the pellets in a 65 ° C. water bath for 1 hour; iv) adding the same amount (330 ul) of chloroform as lysis buffer of step ii) and performing vortexing; v) centrifuging the mixed solution at 12,000 rpm for 10 minutes, taking 200 ul of supernatant into a new 1.5 ml tube, adding and mixing 200 ul of binding buffer and 200 ul of isopropyl alcohol; vi) taking 600 ul of the mixed solution into a column tube and centrifuging for 2 minutes at 12,000 rpm; vii) emptying the collection tube and adding 600 ul of Wash buffer and centrifuging for 2 minutes at 12,000 rpm; viii) emptying the collection tube, adding 500 ul of Wash buffer, and centrifuging at 2,000 rpm for 2 minutes; ix) centrifuging at 12,000 rpm for 3 minutes after emptying the collection tube and x) adding 150 ul of TE buffer and centrifuging at 8,000 rpm for 3 minutes to collect the DNA eluate.

In addition, as a specific example of the present invention the phenol / chloroform extraction method

i) transferring 1 ml of the bacterial culture to a 1.5 ml tube and centrifuging for 5 minutes at 12,000 rpm; ii) after discarding the supernatant, suspending the pellet by adding 467 ul of TE buffer to the pellet; iii) further 30 ul of 10% SDS, 3 ul of proK (20 mg / ul) and reacted at 65 ° C. for 1 hour; iv) adding 500 ul of phenol / chloroform / isoamyl alcohol (phenol: chloroform: isoamyl alcohol) at 25: 24: 1 and performing vortex; v) The mixture was centrifuged at 12,000 rpm for 10 minutes, then 400 ul of supernatant was taken up in a new 1.5 ml tube, 40 ul of 3M sodium acetate and 800 ul of 100% ethanol (EtOH). Adding and mixing; vi) centrifuging the mixture at 12,000 rpm for 10 minutes, discarding the supernatant and adding 500 ul of 75% ethanol (75% cold EtOH); vii) centrifuging the mixed solution at 12,000 rpm for 10 minutes, removing the supernatant, drying the pellets, and viii) dissolving the pellet by adding 150 ul of TE buffer or sterile water to elution. It may be to include.

The drying of the pellet of step vii) may be preferably performed so that the pellet is not completely dried, and the extracted DNA may be used in a PCR reaction by measuring absorbance and calculating a concentration.

The polymerase chain reaction mixture may be prepared in an appropriate composition according to the polymerase chain reaction device. For example, in the case of ABI 7700, the composition of the polymerase chain reaction mixture may be as shown in Table 3, and in the case of Samsung TMC-1000, the composition of the polymerase chain reaction mixture may be as shown in Table 4 below. It is not limited to this.

 SYBR Green I ABI Real-Time Polymerase Chain Reaction Mixture Composition density Usage (ul) Mold (10ng / ul) 5 ng 5 Primer (F, R) 5 pmole/ul 20 pmole 4 50X ROX - 0.4 2X SYBR Green I Premix Ex Taq - 10 D.W. - 0.6 Sum - 20

SYBR Green I TMC-1000 PCR Mixture Composition density Usage (ul) Mold (10ng / ul) 4 ng 4 Primer (F, R) 10 pmole/ul 20 pmole 2 2X SYBR Green I Premix Ex Taq - 6 Sum 12

The reaction conditions of the polymerase chain reaction may be selected according to the type of the polymerase chain reaction, the type of the polymerase chain reaction device and the type of primers, or may be appropriately modified conditions appropriately selected. The type of polymerase chain reaction may be, for example, real-time polymerase chain reaction, conventional polymerase chain reaction, nested polymerase chain reaction, multiple polymerase chain reaction, single polymerase chain reaction, or micro polymerase chain reaction. have. For example, in the case of ABI 7700, the reaction conditions of the polymerase chain reaction may be as shown in Table 5 below. In the case of Samsung TMC-1000, the reaction conditions of the polymerase chain reaction may be as shown in Table 6 below. It is not limited to this.

SYBR Green I ABI Real-Time Polymerase Chain Reaction Running Condition Temperature Cycle number Denaturation 94 ℃ / 5 min One Amplification 94 ℃ / 30 sec 35 60 ℃ / 30 sec 72 ℃ / 30 sec Final extension 72 ℃ / 5 min One Analysis Denaturation 94 ℃ / 2 mim One Annealing 60 ℃ / 15 sec One Data collection 60 ℃ to 94 ℃ 19 min 59 sec Denaturation 94 ℃ / 15 sec One

SYBR Green I TMC-1000 PCR Running Condition step Temperature Cycle number Denaturation 94 ℃ / 10 min One Amplification 94 ℃ / 15 sec 50 55 ℃ / 15 sec 72 ℃ / 30 sec Final extension 72 ℃ / 5 min One Analysis Data collection 60 ℃ to 94 ℃ Ramp rate 0.2 ℃ / sec

The detection method of the present invention can efficiently detect specific pathogenic microorganisms, and in particular, can increase the existing detection limit of about 10 ⁵ CFU / ㎖ to about 100 to 10 CFU / ㎖, depending on the type of primer pair In addition, two or more pathogenic microorganisms can be efficiently detected simultaneously.

As described above, the 10 pairs of primer pairs for detecting pathogenic microorganisms obtained through the present invention are Salmonella spp., Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio parahemoliticus, Thermocinia ente Rapidly and accurately amplify specific genes of each of Richolika, Shigella, Bacillus cereus, Campylobacter jejuni, and Clostridium enterolytica, and have a detection limit of 100 to 10 CFU / ml. Therefore, it is possible to perform a rapid epidemiological investigation that can diagnose the infection of pathogenic microorganisms within 5 hours using the primer pair. In addition, it is possible to simultaneously analyze the three groups using the primers presented in the present invention, and commercially available products for detecting microorganisms for food using single or multiple polymerase chain reactions based on this are very effective industrially. something to do.

And the result of the: (H7 Escherichia coli O157.) Conventional polymerase chain reaction (Conventional PCR) using the PCR primer set, a variety of production with respect to: Figure 1 E. coli O157 H7

2 is a real-time PCR result using a PCR primer pair for detecting O157: H7 ( Esherichia coli O157: H7) selected through a conventional polymerase chain reaction .

3 is a result of performing a conventional polymerase chain reaction (Conventional PCR) using a variety of PCR primers prepared for Staphylococcus aureus ( Staphylococcus aureus ),

Figure 4 is Staphylococcus selected by the conventional polymerase chain reaction ( Staphylococcus) aureus ) Real-Time PCR using a PCR primer pair for detection.

5 is a result of performing a conventional polymerase chain reaction (Conventional PCR) using a variety of PCR primers produced for Bacillus cereus ( Bacillus cereus ),

Figure 6 Bacillus cereus ( Bacillus) screened through a conventional polymerase chain reaction cereus ) real-time PCR using a PCR primer pair for detection,

Figure 7 is Vibrio parahemoliticus ( Vibrio parahaemolyticus ) is a result of performing a conventional PCR using a set of various PCR primers,

8 is Vibrio parahemoliticus screened by conventional polymerase chain reaction ( Vibrio). parahaemolyticus ) real-time PCR using PCR primer pairs for detection.

9 is Listeria monocytogenes ( Listeria) monocytogenes ) using a set of PCR primers prepared by the conventional PCR (Conventional PCR) results,

10 is Listeria monocytogenes screened through conventional polymerase chain reaction ( Listeria) monocytogenes ) real-time PCR using a PCR primer pair for detection,

FIG. 11 shows Yersinia Conventional PCR was performed using various PCR primer sets prepared for entericolitica ).

12 is Yersinia screened through thermopolymerase chain reaction. entericolitica ) real-time PCR using a PCR primer pair for detection,

FIG. 13 shows the results of a conventional PCR using a set of various PCR primers prepared for Clostridium perfringens .

14 is selected through the Conventional polymerase chain reaction Clostridium flops presence (Clostridium perfringens ) real-time PCR using a PCR primer pair for detection,

Figure 15 is Salmonella genus strain ( Salmonella is a result of performing a conventional polymerase chain reaction (PCR) using a variety of PCR primers prepared for spp ),

Figure 16 Salmonella genus strain ( Salmonella) selected through the conventional polymerase chain reaction spp ) Real-Time PCR using a PCR primer pair for detection.

Figure 17 is Shigella genus strain ( Shigella spp . Is a result of performing a conventional PCR using a variety of PCR primers prepared for),

18 is a Shigella genus strain ( Shigella) selected through a conventional polymerase chain reaction. spp . ) Real-Time PCR using a PCR primer pair for detection,

19 is a result of performing a conventional polymerase chain reaction (Conventional PCR) using a variety of PCR primer set produced for Camphylobacter jejunii ,

20 shows Campylobacter jejuni ( Camhylobacter) screened through conventional polymerase chain reaction. jejunii ) real-time PCR using a PCR primer pair for detection,

21 is a result of measuring the change in Tm value according to the GC content change using a 60 bp PCR primer for detecting the genus Salmonella,

22 is a schematic diagram showing the difference in each Tm value for simultaneous analysis of five food poisoning bacteria,

Figure 23 is a schematic diagram showing the difference in each Tm value for simultaneous analysis of four food poisoning bacteria,

24 is a result of a real-time polymerase chain reaction performed for simultaneous analysis of five food poisoning bacteria using SYBR Green I.

25 is a result of a real-time polymerase chain reaction carried out for the simultaneous analysis of four food poisoning bacteria using SYBR Green I,

Fig. 26 shows the results of E. coli O157: H7 PCR detection in human contaminated food,

27 is a result of detection of ABI real-time polymerase chain reaction for detecting E. coli O157: H7 in human contaminated food,

28 is a result of detecting TMC-1000 real-time polymerase chain reaction for detecting E. coli O157: H7 in human contaminated food,

Figure 29 shows the results of Staphylococcus aureus PCR detection in human contaminated food,

30 shows the results of ABI real-time polymerase chain reaction for the detection of Staphylococcus aureus in human contaminated food,

Fig. 31 shows the results of Vibrio parahemolyticus PCR detection in human contaminated food,

32 is a result of detection of ABI real-time polymerase chain reaction for the detection of Vibrio parahemolyticus in human contaminated food,

33 is a result of detecting TMC-1000 real-time polymerase chain reaction for the detection of Vibrio parahemoliticus in human contaminated food,

34 shows the results of Listeria monocytogenes PCR detection in human contaminated food,

35 shows the results of ABI real-time polymerase chain reaction for the detection of Listeria monocytogenes in human contaminated food,

36 is a result of detecting TMC-1000 real-time polymerase chain reaction for detecting Listeria monocytogenes in human contaminated food,

37 shows the results of PCR detection of Salmonella spp. In human contaminated food,

38 shows the results of ABI real-time polymerase chain reaction for the detection of Salmonella spp. In human contaminated food,

39 shows Bacillus cereus PCR detection results in human contaminated food,

40 is a result of ABI real-time polymerase chain reaction for the detection of Bacillus cereus in human contaminated food,

Fig. 41 shows the results of PCR detection of thermocinnia enterolytica in human contaminated food;

42 is a result of detection of ABI real-time polymerase chain reaction for detection of thermocinnia enterolytica in human contaminated food,

FIG. 43 shows the results of TMC-1000 Real-Time Polymerase Chain Reaction for the detection of thermocinia entericulitis in human contaminated food,

44 is a result of PCR detection of Shigella strains in human contaminated food,

45 is a result of ABI real-time polymerase chain reaction for the detection of Shigella genus strains in human contaminated food.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example

Example  1: Escherichia coli O157 : H7

PCR primers for detecting pathogenic microorganisms designed and synthesized to detect Escherichia coli O157: H7 pathogenic bacteria are shown in Table 7 below.

direction Primer sequence (5 '→ 3') Size of amplification product Forward GAT AGA CTT TTC GAC CCA ACA AAG Reverse CCG ATG TGG TCC CCT GAG 101 GCC CTC GTA TAT CCA CAG CA 158 TTG CTC AAT AAT CAG ACGAAG ATG 208 TTG CCG TAT TAA CGA ACC CG 245 CGT TGT CAC ACC GGG CAC TGA 304 GAC ACG TTG CAG AGT GGT ATA ACT 349 GAA CTC CAT TAA CGC CAG ATA TGA T 424 AAG CGT AAG GCT TCT GCT GTG 501 CCC AGT TCA GAG TGA GGT CCA 596 CCC CAC TCT GAC ACC ATC CT 625 GAT GAT GGC AAT TCA GTA TAA CGG 719 TCA TTC CAC GGC GCG AAC 750

PCR results using the various primer sets of Table 7 prepared for E. coli O157: H7 are shown in FIG. 1.

PCR was performed using chromosomal DNA of Escherichia coli 0157: H7 as a template, and the DNA was extracted by the following Colum extraction method.

In the Colum extraction method, first, 1 ml of shake culture cultured with E. coli O157: H7 was taken for 8 hours, transferred to a 1.5 ml tube, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was discarded. Pellets were dissolved by adding lysis A buffer (Food & Feed DNA Extraction kit, KoGeneBioTech, Korea) and 30 ul lysis B buffer (Food & Feed DNA Extraction kit, KoGeneBioTech, Korea). After melting the pellet, the reaction was carried out for 1 hour in a 65 ℃ water bath, the same amount (330 ul) of chloroform (chloroform) and vortex (vortex) after the addition of the lysis buffer, The mixture was centrifuged at 12,000 rpm for 10 minutes. After performing the centrifugation, 200 ul of the supernatant was taken and placed in a new 1.5 ml tube. After adding and mixing 200 ul of binding buffer and 200 ul of isopropyl alcohol, 600 ul of the mixed solution was added to a column tube (column tube, Cosmojintech, Korea), centrifuged at 12,000 rpm for 2 minutes, empty the collection tube, add 600 ul of wash buffer, centrifuge for 2 minutes at 12,000 rpm, empty the collection tube, and wash 500 ul. After adding the buffer and centrifuged at 2,000 rpm for 2 minutes, the collection tube was emptied and centrifuged at 12,000 rpm for 3 minutes. After performing the centrifugation, 150 ul of TE buffer was added and centrifuged at 8,000 rpm for 3 minutes to collect DNA eluate.

The polymerase chain reaction was performed under the composition of Table 8 and the conditions of Table 9.

Composition of Polymerase Chain Reaction Composition density Usage (ul) Mold (10ng / ul) 5 ng 5 Primer (F, R) 5 pmole/ul 20 pmole 4 50X ROX 0.4 2X SYBR Green IPremix Ex Taq 10 D.W. 0.6 Sum 20

Polymerase Chain Reaction Running Condition Temperature Cycle number Denaturation 94 ℃ / 5 min One Amplification 94 ℃ / 30 sec 35 60 ℃ / 30 sec 72 ℃ / 30 sec Final extension 72 ℃ / 5 min One Analysis Denaturation 94 ℃ / 2 mim One Annealing 60 ℃ / 15 sec One Data collection 60 ℃ to 94 ℃ 19 min 59 sec Denaturation 94 ℃ / 15 sec One

The M of FIG. 1 is 100bp Size marker, lanes 1 and 2 were PCR with 101bp primer set, lanes 3 and 4 were PCR with 158bp primer set, lanes 5 and 6 were PCR with 208bp primer set and lanes 7 and 8 Were PCR with 245 bp primer set, lanes 9 and 10 were PCR with 304 bp primer set, lanes 11 and 12 were PCR with 349 bp primer set, lanes 13 and 14 were PCR with 424 bp primer set, and lanes 15 and 16 were 501 bp PCR with primer set, lanes 17 and 18 were PCR with 596bp primer set, lanes 19 and 20 were PCR with 625bp primer set, lanes 21 and 22 were PCR with 719bp primer set, lanes 23 and 24 were 750bp primer set By PCR.

In addition, in the odd lane of FIG. 1, Chromosomal DNA extracted from E. coli KCTC1457 was used as a template, and in the even lane, Chromosomal DNA extracted from E. coli O157: H7 was used as a template.

As shown in FIG. 1, in the case of primer pairs of 101 bp, 208 bp, 304 bp, and 625 bp in size, PCR products were able to confirm normal PCR reaction, but 158 bp, 349 bp, 596 bp, 719 bp, 750 The primer pair of bp was judged to be unsuitable because nonspecific PCR products were observed even when using E. coli KCTC1457 as a template. In addition, primer pairs of 245 bp, 424 bp and 501 bp were confirmed that no amplification products were observed when E. coli O157: H7 was used as a template. Therefore, primer pairs were finally selected that could amplify 101 bp, 208 bp, 304 bp, and 625 bp sizes.

Real-time polymerase chain reaction was performed on the selected four primer pairs, and the results are shown in FIG. 2.

The real-time polymerase chain reaction was performed under the composition of Table 10 and the conditions of Table 11.

Composition of Polymerase Chain Reaction Composition density Usage (ul) Mold (10ng / ul) 5 ng 5 Primer (F, R) 5 pmole/ul 20 pmole 4 50X ROX 0.4 2X SYBR Green I Premix Ex Taq 10 D.W. 0.6 Sum 20

Polymerase Chain Reaction Running Condition Temperature Cycle number Denaturation 94 ℃ / 5 min One Amplification 94 ℃ / 30 sec 35 60 ℃ / 30 sec 72 ℃ / 30 sec Final extension 72 ℃ / 5 min One Analysis Denaturation 94 ℃ / 2 mim One Annealing 60 ℃ / 15 sec One Data collection 60 ℃ to 94 ℃ 19 min 59 sec Denaturation 94 ℃ / 15 sec One

As shown in A and C of FIG. 2, primers capable of amplifying 101 bp and 304 bp sizes showed a normal reaction without generating peaks in E. coli KCTC1457, while 208 bp sizes were shown in B. Primer capable of amplifying showed the same Tm value as E. coli O157: H7. In addition, as shown in D, primers capable of amplifying the 625 bp size were found to be inappropriate because the reaction in the primer dimer.

Therefore, primers capable of amplifying the 101 bp and 304 bp sizes were selected as primer pairs usable for the SYBR Green I system of E. coli O157: H7. The Tm value of the 101 bp is 79.2 ℃, Tm value of 304 bp is 83.4 ℃.

Example  2 : Staphylococcus Aureus ( Staphylococcus aureus )

To detect the Staphylococcus aureus pathogenic bacteria, PCR primers designed and synthesized for detecting pathogenic microorganisms are shown in Table 12 below.

Preparation of Staphylococcus aureus primer direction Primerr sequence (5 '→ 3') Size of amplification product Forward AAT TTA ACA GCT AAA GAG TTT GGT Reverse TTC CCA CTA AAT GTG TTT CAT AA 122 TAA AAA TAC TTG AAC ACT TTC AT 200 TTC ATT AAA GAA AAA GTG TAC GAG 265 TAT CAA AGA ACC AAC CAT TAC C 388 CTT TAT GGA ATC CAG TAT CTT CAA 430 ACG TAT CTT CCA TGA ATG ATC TAA 517 ACG TAT CTT CCA TGA ATG ATC TAA 622

Using the various primer sets of Table 12 prepared for Staphylococcus aureus, PCR was performed by the method described in Example 1, and the results are shown in FIG. 3.

In Figure 3 M is 100bp Size marker, lanes 1 and 2 were PCR with a 122bp primer set, lanes 3 and 4 were PCR with a 200bp primer set, lanes 5 and 6 were PCR with a 265bp primer set and lanes 7 and 8 were PCR with 388bp primer set, lanes 9 and 10 were PCR with 430bp primer set, lanes 11 and 12 were PCR with 517bp primer set, and lanes 13 and 14 were PCR with 622bp primer set.

In addition, in the odd lane of FIG. 3, Staphylococcus Cohnii KCTC3574 was used, and for even lanes, Chromosomal DNA extracted from Staphylococcus aureus KCTC1927 was used as a template.

As shown in FIG. 3, primer pairs having a size of 122 bp, 265 bp, and 622 bp of PCR products showed a normal PCR reaction without nonspecific amplification, and 200 bp, 388 bp, and 517 bp primer pairs were Staphylococcus. Even when cohnii KCTC3574 was used as a template, it was judged to be unsuitable because nonspecific PCR products were observed. In addition, no primer product was observed in the 430 bp primer pair. Therefore, three primer pairs of 122 bp, 265 bp, and 622 bp were selected as the detection primers for Staphylococcus aureus.

Using the three primer pairs selected above, a real-time polymerase chain reaction was performed by the method described in Example 1, and the results are shown in FIG. 4.

As shown in A of FIG. 4, a primer pair capable of amplifying a 122 bp size showed weak amplification of a primer dimmer, and as shown in B, a primer capable of amplifying a 265 bp size Pairs showed nonspecific amplification. As shown in C, the primer pair capable of amplifying the 622 bp size was subjected to normal reaction without primer dimer or nonspecific reaction, and was selected as a primer pair usable for the SYBR Grenn I system by measuring the Tm value at 80.8 ° C.

Example  3: Bacillus Cereus ( Bacillus cereus )

In order to detect Bacillus cereus pathogenic bacteria, PCR primers designed and synthesized for detecting pathogenic microorganisms are shown in Table 13 below.

Preparation of Bacillus cereus primer direction Primerr sequence (5 '→ 3') Size of amplification product Forward GAC TAC ATT CAC GAT TAC GCA GAA Reverse GAA CTG AAT AGC GTG TAA GTC T 130 GTA GAC CAA TTG TTG CTT GTA ATG G 164 CTA TGC TGA CGA GCT ACA TCC ATA 303 AAA TAC ACA TGC AAA TGC TCC G 389 AAT CCT TTC AAA GGT AGA AGA CC 441 TCC CGC TTG CCT TTT CAT AC 520 TTC CTA AAC TTG CAC CAT CTC GG 640 CTA AGT CTC ATT TTG TCG CCC T 800

5 shows the results of PCR performed by the method described in Example 1 using various primer sets of Table 13 prepared for Bacillus cereus.

5 M is 100bp Size marker, lanes 1 and 2 were PCR with a 164bp primer set, lanes 3 and 4 were PCR with a 130bp primer set, lanes 5 and 6 were PCR with a 303bp primer set, and lanes 7 and 8 were PCR with 389bp primer set, lanes 9 and 10 were PCR with 441bp primer set, lanes 11 and 12 were PCR with 520bp primer set, lanes 13 and 14 were PCR with 640bp primer set, lanes 15 and 16 were 800bp primer PCR was performed as a set.

In addition, in the odd lane of FIG. 5, Bacillus Chromosomal DNA extracted from subtilis KCTC3013 was used as a template, and Chromosomal DNA extracted from Bacillus cereus KCTC1661 was used as a template for even lanes.

As shown in FIG. 5, primer pairs having 164 bp, 303 bp, and 640 bp PCR products showed a normal PCR reaction without nonspecific amplification, but primers having 130 bp, 389 bp, and 520 bp sizes of PCR products. Bacillus Even when subtilis KCTC3013 was used as a template, non-specific PCR products were observed, which was judged to be unsuitable. In addition, primer pairs of 441 bp and 800 bp in size of the PCR product were not observed at all. Therefore, three primer pairs of 164 bp, 303 bp and 640 bp were selected as the detection primers for Bacillus cereus.

Using the three primer pairs selected above, a real-time polymerase chain reaction was performed by the method described in Example 1, and the results are shown in FIG. 6.

As shown in A and B of FIG. 6, primer pairs capable of amplifying 164 bp and 303 bp sizes are Bacillus. Subtilis KCTC3013 and Bacillus cereus KCTC1661 exhibited the same Tm value and were amplified and determined to be inappropriate primers for the system. As shown in C, primer pairs capable of amplifying the 640 bp size showed normal amplification and were selected as primer pairs available for the SYBR Grenn I system, with a Tm value of 82.7 ° C.

Example  4: Vibrio parahemoliticus ( Vibrio parahaemolyticus )

PCR primers designed and synthesized for detecting pathogenic microorganisms to detect Vibrio parahemolyticus pathogenic bacteria are shown in Table 14 below.

Fabrication of Vibrio Parahemolyticus Primer direction Primerr sequence (5 '→ 3') Size of amplification product Forward CTC ATT TGT ACT GTT GAA CGC CTA Reverse AGG CGT CAT TGT TAT CAG AAG C 91 TCA TGA GCA GAG GCG TCA TTG 101 TAC TAC TGG CGC TTC TGG TTC 167 CAG CAG TAC GCA AAT CGG TAG TAA 267 AAT AGA AGG CAA CCA GTT GTT GAT 375 TGT CCG CCA GTG GCA AT 456 TGG ATC ATT TTG ACC TGC GAA A 543 AGA TGC TAC TTT AAT CTT CAT GCT GG 593 GAC TGC CAT TCA TTT GAT GAT AGC 688

Using the primer set of Table 14 prepared for Vibrio parahemolyticus, the results of PCR by the method described in Example 1 is shown in FIG.

In Figure 7, M is 100bp size marker, lanes 1 and 2 were PCR with 91bp primer set, lanes 3 and 4 were PCR with 101bp primer set, lanes 5 and 6 were PCR with 167bp primer set, and lanes 7 and 8 were PCR with 304 bp primer set, lanes 9 and 10 were PCR with 375 bp primer set, lanes 11 and 12 were PCR with 456 bp primer set, lanes 13 and 14 were PCR with 543 bp primer set, lanes 15 and 16 with 688 bp primer PCR was performed as a set.

In the case of odd-numbered lane of Figure 7 Vibrio We used the Chromosomal DNA extracted from alginolyticus KCCM40513 as a template, in the case of even-numbered lane was used for the Chromosomal DNA extracted from Vibrio para H. Molina T KCCM41654 carcass as a template.

As shown in FIG. 7, primer pairs having a PCR product of 91 bp, 101 bp, 167 bp, 375 bp, and 688 bp showed a normal PCR reaction without non-specific amplification, but the PCR product had a size of 304 bp. Vibrio Even when the alginolyticus KCCM40513 as the template, being a non-specific PCR product observed was judged unsuitable. In addition, primers having a size of 456 bp of PCR product were not observed at all. Therefore, primer pairs of 91 bp, 101 bp, 167 bp, 375 bp and 688 bp were selected.

Using the selected five primer pairs, a real-time polymerase chain reaction was performed by the method described in Example 1, and the results are shown in FIG. 8.

As shown in A, B, and D of FIG. 8, primer pairs capable of amplifying 91 bp, 101 bp, and 375 bp sizes are Vibrio. The amplicon was amplified in both alginolyticus KCCM40513 and Vibrio parahemolyticus KCCM41654, and showed the same Tm value, whereas primer pairs capable of amplifying 167 bp and 688 bp sizes, as shown in C and E, were normal. The amplification was shown with primer pairs available for the SYBR Grenn I system, showing Tm values of 84.9 ° C. and 85.5 ° C., respectively.

Example  5: Listeria  Monocytogenes ( Listeria monocytogenes )

In order to detect Listeria monocytogenes pathogenic bacteria, PCR primers designed and synthesized for detecting pathogenic microorganisms are shown in Table 15 below.

Preparation of Listeria monocytogenes primer direction Primerr sequence (5 '→ 3') Size of amplification product Forward CTG GCA CAA AAT TAC TTA CAA CGA Reverse TGG AGT GCT TAC TGC TTT GTC AG 83 TGC TAC TTT AGG CGC AGG TGT 202 CCA TAA AGC CCA AAT AGT GTC ACC 271 CAT AAT GTC TTG AAC AGA AAC ACC G 304 TTT CAC TTC TGC TTT TGG AGT AGC 400 AAC TAC TGG AGC TGC TTG TTT TTC 451 AGG TGC TGT TTG TTG CTG TGT AG 504 GCA GGA GCT GGT TTT GCA G 539 GTT ACC ACC CCA TGA ATA AGC 800

PCR was carried out by the method described in Example 1, using a set of nine pairs of primers in combination with one forward primer and nine different reverse primers as shown in Table 15 prepared for Listeria monocytogenes. The results are shown in FIG.

In Figure 9, M is a 100bp size marker, lanes 1 and 2 were PCR with 83bp primer set, lanes 3 and 4 were PCR with 202bp primer set, lanes 5 and 6 were PCR with 271bp primer set, and lanes 7 and 8 were PCR with 304 bp primer set, lanes 9 and 10 were PCR with 400 bp primer set, lanes 11 and 12 were PCR with 451 bp primer set, lanes 13 and 14 were PCR with 539 bp primer set, lanes 15 and 16 were 800 bp primer PCR was performed as a set.

In the case of odd-numbered lane of Figure 9 Listeria Chromosomal DNA extracted from grayi ATCC25400 was used as a template and Chromosomal DNA extracted from Listeria monocytogenes ATCC86152 was used as a template for even lanes.

As shown in FIG. 9, primer pairs having 83 bp, 451 bp, and 800 bp of PCR product showed a normal PCR reaction without nonspecific amplification, but primers having a size of 271 bp and 271 bp of PCR product were Listeria. Even when grayi ATCC25400 was used as a template, it was judged to be unsuitable because nonspecific PCR products were observed. In addition, primers having a size of 400 bp of PCR products were not observed at all. In addition, primers having a size of 202 bp of PCR product were eliminated from primer selection because the size of PCR product was observed unexpectedly. Therefore, primer pairs of 83 bp, 451 bp and 800 bp were selected.

Using the three primer pairs selected above, a real-time polymerase chain reaction was performed by the method described in Example 1, and the results are shown in FIG. 10.

As shown in FIG. 10A, a pair of primers capable of amplifying 83 bp size is Listeria. The grayi ATCC25400 showed the same Tm value and peak, which was determined to be inappropriate. As shown in C, primer pairs capable of amplifying the 800 bp size were judged to be inappropriate due to weak peaks of the primer dimer. In this case, remaking the primer can remove the dimer. As shown in B, primer pairs capable of amplifying the 451 bp size showed normal amplification, were selected as primer pairs usable for the SYBR Green I system, and showed a Tm value of 82.3 ° C.

Example  6: Heat sinia  Entericulica ( Yersinia entericolitica )

PCR primers designed and synthesized for detecting pathogenic microorganisms are described in Table 16 below to detect thermocintic entericitis pathogenic bacteria.

Preparation of Thermosini Entericulica Primer direction Primerr sequence (5 '→ 3') Size of amplification product Forward AAT ACC GCA TAA CGT CTT CG Reverse AGG CGT CAT TGT TAT CAG AAG C 91 GAT TGC GTC AGA AGT CGT CG 150 GCA AAT CGG TAG TAA TAG TGC C 212 CTT CTT CTG CGA GTA ACG TC 330 CGG TAT TCC TCC AGA TCT CTA C 551 GAC TGC CAT TCA TTT GAT GTA AGC 702

11 shows the results of PCR performed by the method described in Example 1, using the primer set of Table 16 prepared for Thermocinia entericitis.

In Figure 11, M is a 100bp size marker, lanes 1 and 2 were PCR with a 91bp primer set, lanes 3 and 4 were PCR with a 150bp primer set, lanes 5 and 6 were PCR with a 212bp primer set, and lanes 7 and 8 were PCR was performed with 330 bp primer set, lanes 9 and 10 were PCR with 551 bp primer set, and lanes 11 and 12 were PCR with 702 bp primer set.

In addition, Citrobacter in the odd lane of FIG. freundii Chromosomal DNA extracted from KCCM11931 was used as a template, and in even lanes, Chromosomal DNA extracted from Thermocinia entericitis KCCM41657 was used as a template.

As shown in FIG. 11, the primers showing 91 bp, 330 bp, and 551 bp of PCR products showed normal PCR reactions without non-specific amplification, but the primers of 150 bp and 212 bp in size of the PCR product. Citrobacter freundii Even when KCCM11931 was used as a template, it was judged to be unsuitable because nonspecific PCR products were observed. In addition, no PCR product was observed in the primer for amplification of 702 bp in PCR product size. Therefore, primer pairs of 330 bp and 551 bp were selected.

Using the two primer pairs selected above, a real-time polymerase chain reaction was performed by the method described in Example 1, and the results are shown in FIG. 12.

As shown in FIG. 12, primer pairs capable of amplifying 330 bp and 551 bp sizes showed normal amplification, and were selected as primer pairs usable for the SYBR Grenn I system, and Tm values were 88.5 ° C. and 88.4 ° C., respectively. .

Example  7: Clostridium  Per Presence ( Clostridium perfringens )

PCR primers designed and synthesized for detecting pathogenic microorganisms to detect Clostridium perfringens pathogenic bacteria are shown in Table 17 below.

Fabrication of Clostridium perfringence primer direction Primerr sequence (5 '→ 3') Size of amplification product Forward TGA TAT GTC CAA AAA TGA ACC AGA Reverse TAA GTA GAA CCT AAT TGA AGC 90 TTG TGA TTC CCC TGT GTC AGG 220 TGC CAT TCA TAT CTA GCT AAT GCT 260 TAG TGC ATA GCT TCT CCA AGA TAG A 309 TTT CCT TTC TTC TGC AAA AGT CTC 402 TCC CAA TCA TCC CAA CTA TGA CT 578 CCT GCT GTT CCT TTT TGA GAG 630 TCA CCA CTA GTT GAT ATG TAA GCT 728

13 shows the results of PCR by the method described in Example 1 using the primer set of Table 17 prepared for Clostridium perfringens.

In Figure 13 M is 100bp Size marker, lanes 1 and 2 were PCR with 90bp primer set, lanes 3 and 4 were PCR with 220bp primer set, lanes 5 and 6 were PCR with 260bp primer set, and lanes 7 and 8 were PCR with 309 bp primer set, lanes 9 and 10 were PCR with 402 bp primer set, lanes 11 and 12 were PCR with 578 bp primer set, lanes 13 and 14 were PCR with 630 bp primer set, lanes 15 and 16 with 728 bp PCR was done.

In addition, Camphylobacter in the odd lane of FIG. Chromosomal DNA extracted from jejuni (Kozen Biotech, Korea) was used as a template, and Chromosomal DNA extracted from Clostridium perfringens (KCCM41773, Kozen Biotech, Korea) was used as a template for even lanes.

As shown in FIG. 13, primers showing 90 bp, 220 bp, 260 bp, 402 bp, and 630 bp of PCR product size showed a normal PCR reaction without nonspecific amplification, but the PCR product size was 578 bp. , 728 bp primer was judged to be unsuitable even when Camphylobacter jejuni was used as a template. In addition, no PCR product was observed in the amplification primer having a size of 309 bp PCR product. Therefore, primer pairs of 220 bp, 260 bp, 402 bp and 630 bp were selected.

Using the four primer pairs selected above, a real-time polymerase chain reaction was performed by the method described in Example 1, and the results are shown in FIG. 14.

As shown in FIG. 14, only a primer pair capable of amplifying a size of 260 bp is Camphylobacter A pair of primers capable of amplifying 220 bp, 402 bp, and 630 bp sizes except for jejuni and Clostridium perfringens showed the same peak at the same Tm value, and were selected as primer pairs available for the SYBR Grenn I system. Tm values were 78.0 ° C, 80.2 ° C and 80.7 ° C, respectively.

Example  8: Salmonella spp. Salmonella spp .)

PCR primers designed and synthesized to detect pathogenic bacteria of the genus Salmonella strains are listed in Table 18 below.

Preparation of Salmonella Genus Strain Primer direction Primerr sequence (5 '→ 3') Size of amplification product Forward GAA TCC TCA GTT TTT CAA CGT TTC Reverse CCA GAC GAA AGA GCG TGG TAA 60 GAA GCC CGA ACG TGG CGA 137 GTA TGC CCG GTA AAC AGA TGA GT 284 AAA GGA ACC GTA AAG CTG GCT 330 GGG TCA TCC CCA CCG AAA TAC 424 GAT CTG GGC GAC AAG ACC A 551 TAG CCG TAA CAA CCA ATA CAA ATG 678 TAC GTT TTG CTT CAC GGA ATT TAA 787

Using the primer set of Table 18 prepared for the strain of genus Salmonella, the results of PCR by the method described in Example 1 is shown in Figure 15.

15 M is 100bp size marker, lanes 1 and 2 were PCR with 60bp primer set, lanes 3 and 4 were PCR with 137bp primer set, lanes 5 and 6 were PCR with 284bp primer set, and lanes 7 and 8 were PCR with 330 bp primer set, lanes 9 and 10 were PCR with 424 bp primer set, lanes 11 and 12 were PCR with 551 bp primer set, lanes 13 and 14 were PCR with 678 bp primer set, lanes 15 and 16 with 787 bp PCR was done.

In the case of odd-numbered lane of Fig. 15 is a Shigella Chromosomal DNA extracted from sonnei KCCM41282 was used as a template, and Chromosomal DNA extracted from Salmonella enteritidis KCCM12021 was used as a template for even lanes.

As shown in FIG. 15, primers showing 60 bp, 284 bp, and 678 bp of PCR products showed a normal PCR reaction without nonspecific amplification, but primers of 137 bp, 330 bp, and 551 bp of PCR products were shown. IsShigella sonnei Even when KCCM41282 was used as a template, it was judged to be unsuitable because nonspecific PCR products were observed. In addition, no PCR product was observed in the amplification primer having a PCR product size of 424 bp. Therefore, primer pairs of 60 bp, 284 bp and 678 bp were selected.

Using the three primer pairs selected above, a real-time polymerase chain reaction was performed by the method described in Example 1, and the results are shown in FIG. 16.

As shown in FIG. 16, in the case of a primer pair capable of amplifying a 60bp size, Shigella sonnei KCCM41282 and Salmonella Both enteritidis KCCM12021 showed the same peak at the same Tm value, and in the case of primer pairs capable of amplifying a 284bp size, Shigella sonnei KCCM41282 showed no amplification products, but Salmonella The peak amplification of enteritidis KCCM12021 was weak, the Tm value was 86.8 ℃, and Shigella for the primer pair that could amplify the 678bp size. In case of sonnei KCCM41282, an amplification product was observed, but the amplification of the peak was weak and it was negligible because it appeared below 75 ° C. Since the Tm value was 86.7 ° C, it was selected as a primer available for the SYBR Green I system.

Example  9: Shigella genus strain ( Shigella spp . )

PCR primers designed and synthesized to detect pathogenic bacteria of the genus Shigella are listed in Table 19 below.

Preparation of Shigella Strain Primer direction Primerr sequence (5 '→ 3') Size of amplification product Forward GAA TCC TCA GTT TTT CAA CGT TTC Reverse CTG CAT TCT GGC AAT CTC TTC ACA 110 TTC AAA CTT CCC CCA ACA AGA 201 GCA TCT GAC AGC AAT AGT ACA 355 TGT ACA TCG ATA ATA CCA AAC 445

17 shows the results of PCR by the method described in Example 1 using the primer set of Table 19 prepared for Shigella genus.

In Figure 17, M is a 100bp size marker, lanes 1 and 2 were PCR with 110bp primer set, lanes 3 and 4 were PCR with 201bp primer set, lanes 5 and 6 were PCR with 355bp primer set, and lanes 7 and 8 were PCR was performed with a 445 bp primer set.

In the case of odd-numbered lane of Fig. 17 Salmonella Shigella for typhimurium ATCC19585 and for even lanes Chromosomal DNA extracted from flexneri ATCC12022 was used as a template.

As shown in FIG. 17, primers showing the size of the PCR product 110bp, 355bp, 445bp showed a normal PCR reaction without non-specific amplification, but primers with the size of the PCR product 110bp primer were Salmonella. Even when typhimurium ATCC19585 was used as a template, non-specific PCR products were observed and judged to be unsuitable. Therefore, primer pairs of 110 bp, 355 bp, and 445 bp were selected.

Using the three primer pairs selected above, a real-time polymerase chain reaction was performed by the method described in Example 1, and the results are shown in FIG. 18.

As shown in FIG. 18, in the case of a primer pair capable of amplifying a 355 bp size, Salmonella A weak but peak peak was observed in the typhimurium ATCC19585, while the dimer of the primer appeared, whereas the amplification primers with the size of the PCR products 110 bp and 445 bp were Shigella. Only a single peak was found in flexneri ATCC12022, and the Tm value of the amplification primer of 445 bp was 80.9 ° C. Thus, 445 bp amplification primer pairs were selected as primer pairs available for the SYBR Grenn I system.

Example  10: Campylobacter  Jeju Camphylobacter jejuni )

In order to detect the Campylobacter jejuni pathogenic bacteria, PCR primers designed and synthesized for detecting pathogenic microorganisms are shown in Table 20 below.

Preparation of Campylobacter jejuni strain primer direction Primerr sequence (5 '→ 3') Size of amplification product Forward ACT TCT TTA TTG CTT GCT GC Reverse GCC ACA ACA AGT TAA AGA AGC 303

19 shows the results of PCR by the method described in Example 1 using the primer set of Table 20 prepared for Campylobacter jejuni.

Figure 19 M is 100bp Size marker, lane 1 is Listeria monocytogenes ATCC19113, lane 2 is Campylobacter jejuni, lane 3 is Salmonella choleraesuis KCCM41035, lane 4 is E. coli KCTC1457, lane 5 is E. coli KCTC1457, lane 6 is Staphylococcus aureus KCTC1916, lane 7 is Bacillus cereus KCTC1661, lane 8 is Salmonella typhimurium KCTC2421, and lane 9 is Staphylo Caucasus Aureus KCTC1927, Lane 10 is Shigella boydii ATCC12034 and lane 11 used Vibrio parahemolyticus ATCC11965.

As a result, only the Campylobacter jejuni was selected by showing a specific 303 bp band and a normal PCR reaction without nonspecific amplification.

Using the primer pairs of Table 20, the real-time polymerase chain reaction was repeated three times by the method described in Example 1, and the results are shown in FIG.

As shown in FIG. 20, in the case of the primer pairs of the selected Table 20, it was confirmed that the Tm value was 80.7 ° C. Thus, the primer pairs of Table 20 selected above were selected as primer pairs usable for the SYBR Grenn I system.

Example  11: Primer modification On by Tm  Change in value

To determine whether GC tailing of primers affects Tm value in the SYBR Green I system, real-time polymerase was added by randomly adding 4 or 8 GCs to a 60 bp primer to detect Salmonella strains. The chain reaction was carried out and the results are shown in FIG. 21 and Table 21 below.

The composition, reaction conditions, and sample DNA of the polymerase chain reaction mixture used the sample DNA of Example 8, the composition of Table 10 of Example 1, and the conditions of Table 11.

Changes in Tm Values According to GC Tailing Changes Using 60bp Primer for Salmonella Strain Detection direction Primerr sequence (5 '→ 3') Tm value A Clove Room GAA TCC TCA GTT TTT CAA CGT TTC 78.4 Reverse CCA GAC GAA AGA GCG TGG TAA B Forward direction GCGC GAA TCC TCA GTT TTT CAA CGT TTC 80.6 Reverse GCGC CCA GAC GAA AGA GCG TGG TAA C Forward direction GCGCGCGC GAA TCC TCA GTT TTT CAA CGT TTC 82.5 Reverse GCGCGCGC CCA GAC GAA AGA GCG TGG TAA

As shown in FIG. 21 and Table 21, the Tm value of the primer pair without GC was 78.4 ° C., and the Tm value of the primer pair with 4 GCs was 80.6 ° C., and the primer with 8 GCs was shown. The Tm value of the pair was found to be 82.5 ° C.

From the above results, as the GC increases, the temperature of Tm increases, and when one GC (2 bp additional tailing) is added, the average Tm value increases by about 0.5 ° C. Thus, through GC tailing Controlling the Tm value of the primer was found to be invalid.

Example  12: SYBR Green  I system  Optimum for the application primer Selection of

Table 22 shows the Tm values for the amplicons amplified using the respective optimal primer pairs designed for 10 pathogenic microorganisms. The primers set forth in SEQ ID NOS: 1-20 in Table 22 indicate that they are optimal primer pairs in both single PCR and multiplex PCR.

SYBR Green I system Tm values for various primer pairs available microbe No primer sequence Size of amplification product Tm SEQ ID NO: E. coli O157 : H7 E1 GAT AGA CTT TTC GAC CCA ACA AAG One E2E3E4E5 CCG ATG TGG TCCCCT GAGTTG CTC AAT AAT CAG ACGAAG ATGCGT TGT CAC ACC GGG CAC TGACCC CAC TCT GAC ACC ATC CT 101 208304625 7983.483.486.2 2212223 S. aureus SA1 AAT TTA ACA GCT AAA GAG TTT GGT 3 SA2SA3SA4 TTC CCA CTA AAT GTG TTT CAT AATTC ATT AAA GAA AAA GTG TAC GAGACG TAT CTT CCA TGA ATG ATC TAA 122265622 80.681.380.9 24254 B. cereus B1 GAC TAC ATT CAC GAT TAC GCA GAA 5 B2B3B4 GTA GAC CAA TTG TTG CTT GTA ATG GCTA TGC TGA CGA GCT ACA TCC ATATTC CTA AAC TTG CAC CAT CTC GG 164303640 80.282.182.7 26276 V. parahaemolyticus V1 CTC ATT TGT ACT GTT GAA CGC CTA 7 V2V3V4V5V6 AGG CGT CAT TGT TAT CAG AAG CTCA TGA GCA GAG GCG TCA TTGTAC TAC TGG CGC TTC TGG TTCAAT AGA AGG CAA CCA GTT GTT GATGAC TGC CAT TCA TTT GAT GAT AGC 91101167375688 81.281.784.985.585.5 282983031 L. monocytogenes L1 CTG GCA CAA AAT TAC TTA CAA CGA 9 L2L3L4 TGG AGT GCT TAC TGC TTT GTC AGAAC TAC TGG AGC TGC TTG TTT TTCGTT ACC ACC CCA TGA ATA AGC 83451800 78.682.382.8 321033 Y. enterocolitica Y1 AAT ACC GCA TAA CGT CTT CG 11 Y2Y3 CTT CTT CTG CGA GTA ACG TCCGG TAT TCC TCC AGA TCT CTA C 330551 8888 1234 C. perfringens C1 TGA TAT GTC CAA AAA TGA ACC AGA 13 C2C3C4C5 TTG TGA TTC CCC TGT GTC AGGTGC CAT TCA TAT CTA GCT AAT GCTTTT CCT TTC TTC TGC AAA AGT CTCCCT GCT GTT CCT TTT TGA GAG 220260402630 7878.38080.6 14353637 Salmonella sp . SAL1 GAA TCC TCA GTT TTT CAA CGT TTC 15 SAL2SAL3SAL4 CCA GAC GAA AGA GCG TGG TAAGTA TGC CCG GTA AAC AGA TGA GTTAG CCG TAA CAA CCA ATA CAA ATG 60284678 78.285.686.7 383916 Shigella sp . SH1 CAG GCA TGA AAT TAA GCC TG 17 SH2SH3SH4 CTG CAT TCT GGC AAT CTC TTC ACAGCA TCT GAC AGC AAT AGT ACATGT ACA TCG ATA ATA CCA AAC 110355445 8180.880.8 404118 C. jejuni CA1 ACT TCT TTA TTG CTT GCT GC 19 CA2 GCC ACA ACA AGT TAA AGA AGC 303 80.7 20

Example 13: SYBR Multiplex Polymerase Chain Reaction Using Green I (Multiplex PCR using SYBR Green I)

13-1. multiple Polymerase Chain Reaction  System configuration Multiplex PCR system )

The primers identified as the optimal primers in Table 22 were divided into two groups. The 5-Plex PCR system divided into the first group was composed of Escherichia coli O157: H7, Staphylococcus aureus, Salmonella spp., Vibrio parahemolyticus and Listeria monocytogenes. The 4-Plex PCR system consisted of Bacillus cereus, Shigella genus strains, Thermocinella entericitis and Clostridium perfringens.

In addition, Campylobacter jejuni was designed to be used as a single polymerase chain reaction because the culture method of microaerobic bacteria is different from the above strains. Primer pairs for each group were selected so that the Tm value was 1.8 to 2 ° C. difference.

13-2. Group 1

As shown in Table 22 and FIG. 22, the 101 bp primer pair, which is the optimal primer for E. coli O157: H7, had a Tm of 79 ° C, the 208 bp and 304 bp primer pairs had a Tm of 83.4 ° C, and the 625 bp primer pair had a Tm of In the case of Staphylococcus aureus, the 122 bp primer pair had a Tm of 80.6 ° C, the 265 bp primer pair had a Tm of 81.3 ° C, and the 622 bp primer pair had a Tm of 80.9 ° C. .

In addition, in the Salmonella spp. Strain, the 60 bp primer pair had a Tm of 78.2 ° C, the 284 bp primer pair had a Tm of 85.6 ° C, and the 678 bp primer pair had a Tm of 86.7 ° C. For the 91 bp primer pair, the Tm was 81.2 ° C, the 101 bp primer pair was Tm 81.7 ° C, the 167 bp primer pair was Tm 84.9 ° C, and the 375 bp and 688 bp primer pair were Tm 85.5 ° C. .

In addition, in the case of Listeria monopsychogenes, the 83 bp primer pair had a Tm of 78.6 ° C, the 451 bp primer pair had a Tm of 82.3 ° C, and the 800 bp primer pair had a Tm of 82.8 ° C.

In order to develop a 5-multiplex PCR system, it is preferable that the Tm values differ by about 2 ° C. Based on the Tm value, E. coli O157: H7 uses a 101 bp primer pair having a Tm of 79 ° C. Woods has a 622 bp primer pair with a Tm of 80.9 ° C, a Salmonella strain has a 678 bp primer pair with a Tm of 86.7 ° C, and Vibrio parahemoliticus has a 167 bp primer pair with a Tm of 84.9 ° C. Cytogenes selected a primer pair of 451 bp with a Tm of 82.3 ° C., and the final selected primer sequences were specified in Table 23 below.

13-3. 2nd group

As shown in Table 22 and FIG. 23, the Bacillus cereus had a Tm of 80.2 ° C for the 164 bp primer pair, a Tm of 82.1 ° C for the 303 bp primer pair, and a Tm of 82.7 ° C for the 640 bp primer pair. In the Shigella genus strain, the 113 bp primer pair had a Tm of 81 ° C., and the 355 bp and 445 bp primer pairs had a Tm of 80.8 ° C.

In addition, the thermocinta entericulica had a Tm of 88 ° C. for both 330 bp and 551 bp primer pairs, and for Clostridium perfusion, the 220 bp primer pair had a Tm of 78 ° C. and the 260 bp primer pair The Tm was 78.3 ° C., the 402 bp primer pair had a Tm of 80 ° C., and the 630 bp primer pair had a Tm of 80.6 ° C.

In order to develop a 4-multiplex PCR system, it is preferable that the Tm value be more than 2 ° C. Based on the Tm value, Bacillus cereus has a 640 bp primer pair with a Tm of 82.7 ° C, and the strain of Shigella genus 445 bp primer pair was selected at 80.8 ° C, Thermocinia entericulica was selected as 330 bp primer pair at 88 ° C, and Clostridium perprisence was selected at 220 bp primer pair at 78 ° C. The final selected primer sequences are shown in Table 23 below.

Optimal Primer Pairs in SYBR Green I System Used for Simultaneous Analysis microbe Primerr sequence (5 '→ 3') SEQ ID NO: Size of amplification product Tm E. coli O157 : H7 F GAT AGA CTT TTC GAC CCA ACA AAG One 101 79 R CCG ATG TGG TCCCCT GAG 2 S. aureus F AAT TTA ACA GCT AAA GAG TTT GGT 3 622 80.9 R ACG TAT CTT CCA TGA ATG ATC TAA 4 B. cereus F GAC TAC ATT CAC GAT TAC GCA GAA 5 640 82.7 R TTC CTA AAC TTG CAC CAT CTC GG 6 V. parahaemolyticus F CTC ATT TGT ACT GTT GAA CGC CTA 7 167 84.9 R TAC TAC TGG CGC TTC TGG TTC 8 L. monocytogenes F CTG GCA CAA AAT TAC TTA CAA CGA 9 451 82.3 R AAC TAC TGG AGC TGC TTG TTT TTC 10 Y. enterocolitica F AAT ACC GCA TAA CGT CTT CG 11 330 88 R CTT CTT CTG CGA GTA ACG TC 12 C. perfringens F TGA TAT GTC CAA AAA TGA ACC AGA 13 220 78 R TTG TGA TTC CCC TGT GTC AGG 14 Salmonella spp F GAA TCC TCA GTT TTT CAA CGT TTC 15 678 86.7 R TAG CCG TAA CAA CCA ATA CAA ATG 16 Shigella spp F CAG GCA TGA AAT TAA GCC TG 17 445 80.8 R TGT ACA TCG ATA ATA CCA AAC 18 C. jejuni F ACT TCT TTA TTG CTT GCT GC 19 303 80.7 R GCC ACA ACA AGT TAA AGA AGC 20

13-4. SYBR green  With I multiplex PCR  Establish condition

To determine the minimum concentration of primers of the first group (5-plex) of Example 13-2 and the second group (4-plex) of Example 13-3, real-time polymerase chain reaction was performed. The results are shown in FIGS. 24 and 25.

The polymerase chain reaction was carried out using a 7700 real-time polymerase chain reaction apparatus (ABI, USA), the composition and reaction conditions of the polymerase chain reaction composition (Mixture), the composition of Table 3 and the conditions of Table 5 However, in order to establish the conditions of the multi-primers, the primers of the first group of Example 13-2 and the second group of Example 13-3 were diluted to 2 pmol / ul, and SYBR Sample DNA, that is, template DNA, used in the green I system was used in the same concentration of 10 ng / ul.

The results of the polymerase chain reaction are shown in FIGS. 24 and 25. In FIG. 24, 1 is E. coli O157: H7, 2 is Staphylococcus aureus, 3 is Listeria monocytogenes, 4 is Vibrio parahemolyticus, and 5 is Salmonella genus strain, ie Salmonella enteridis ( Salmonella) enteritidis ). In FIG. 25, 1 represents Clostridium perfringens, 2 represents Shigella genus strains, that is, Shigella flexneri , 3 represents Bacillus cereus, and 4 represents thermocinia entericulica.

As shown in FIGS. 24 and 25, each primer of each of the first group of Example 13-2 and the second group of Example 13-3 has a single Tm value equal to the expected value. Showed that simultaneous analysis is possible. More specifically, in FIG. 24, the E. coli O157: H7 had a Tm value of 79 ° C., a Staphylococcus aureus had a Tm value of 80.9 ° C., and Listeria monocytogenes had a Tm value of 82.3 ° C., and Vibrio para. The hemolyticus had a Tm value of 84.9 ° C. and the Salmonella genus, Salmonella enteritidis , had a Tm value of 86.7 ° C. In addition, in Fig. 25, the Clostridium perfringens had a Tm value of 78 ° C., a strain of Shigella genus, ie, Shigella flexneri , had a Tm value of 80.8 ° C., and Bacillus cereus had a Tm value of 82.7 ° C. The thermocinia entericulica had a Tm value of 88 ° C.

As can be seen in FIGS. 24 and 25, each primer pair showed only one peak, and the Tm values were shown as single Tm values which can be distinguished from each other.

Example 14 SYBR in Contaminated Food Green I PCR Detection of pathogenic microorganisms using the system

In order to directly detect whether a pathogenic microorganism is contaminated in food using SYBR Green I system, after artificially infecting a pathogenic microorganism with a specific food, it is appropriate for 12 hours or according to the type of food and pathogenic microorganism. The microorganisms were incubated for a modified time. The degree of detection of the cultured microorganisms was examined using the SYBR green I PCR system. The pathogenic microorganisms applied in Example 14 and the applied food groups are listed in Table 24 below.

Food Classes and Infected Pathogenic Microorganisms Group Pathogenic microorganisms Food Group i Escherichia coli O157: H7 Hamburger Patty Crushed Chicken Staphylococcus aureus Sundae Kimbab Vibrio parahemolyticus sea water shrimp Listeria monocytogenes salad Ice cream Salmonella genus strain Frozen chicken salad Group ii Bacillus cereus Miso Kochujang Thermostine Entertainment bottled water milk Shigella genus strain Spring water oyster

The experiment to confirm the detection degree of the microorganism was extracted from the sample DNA from the food, and then carried out a polymerase chain reaction using ABI 7700 or Samsung TMC-1000 (Samsung Tech One, Korea), polymerase chain reaction Composition and reaction conditions of the composition for the (Mixture) was according to Tables 3 to 6. Extraction of the sample DNA was performed in the following manner.

After taking 25 g from the food sample of Table 24, inoculated in 225 ml strain seed medium and incubated for 8 hours, 1 ml of the culture solution was placed in a 1.5 ml tube and centrifuged at 12,000 rpm for 5 minutes. The supernatant was removed from the centrifuged culture and the precipitate was suspended in 100ul of DNA free waater, and then heat-treated at 95 ° C. for 20 minutes and re-centrifuged at 10,000 rpm for 10 minutes. DNA was recovered from the centrifuged mixture and the sample DNA was extracted.

14-1. Escherichia coli O157 : H7  ( primer  101 bp )

Conventional polymerase chain reaction and real-time polymerase chain reaction were performed on the food groups contaminated with E. coli O157: H7 using primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. It was performed using a 7700 real-time polymerase chain reaction device and TMC-1000 real-time polymerase chain reaction device, each result is shown in Figure 26 to 28. Each lane of FIG. 26 is as follows.

Lane M: Size marker

N: negative control (does not contain DNA)

1: Escherichia coli O157: H7 105 CFU / mL-Hamburger Patty

2: Escherichia coli O157: H7 105 CFU / mL-ground chicken

3: Staphylococcus aureus KCTC1927 105 CFU / mL-Sundae

4: Staphylococcus aureus KCTC1927 105 CFU / mL-Gimbap

5: Vibrio parahaemolyticus KCCM41654 105 CFU / mL-seawater

6: Vibrio parahaemolyticus KCCM41654 105 CFU / ml-Shrimp

7: Listeria monocytogenes ATCC86152 105 CFU / mL-Salad

8: Listeria monocytogenes ATCC86152 105 CFU / ml-Ice Cream

9: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Frozen Chicken

10: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Salad

11: Bacillus cereus KCTC1661 105 CFU / mL-Miso

12: Bacillus cereus KCTC1661 105 CFU / mL-Gochujang

13: Thermoscene Entericulica KCCM41657 105 CFU / mL-Bottled water

14: Thermocinia Entericulica KCCM41657 105 CFU / mL-Milk

15: Shigella Flexneri ATCC12022 105 CFU / mL-Eating Spring Water

16: Shigella Flexnery ATCC12022 105 CFU / mL-Oyster

P: positive control (E. coli 0157: H7 105 CFU / mL)

As shown in FIG. 26, a band of the correct size was identified only in the food inoculated with E. coli O157: H7 and the positive control group, and as shown in FIG. 27, the product of SYBR green I PCR using the ABI 7700 device. The Tm value was found to be a Tm value of 79.1 ° C. More specifically, the Tm value of the E. coli O157: H7 PCR product showed 79.2 ° C in the analysis results of the primary food, and 79.1 ° C in the analysis results of the secondary food. From the results, it was confirmed that the error range of the SYBR Green I system using the ABI 7700 is ± 0.2 ℃, when using the TMC-1000 the error range, that is, the difference in the Tm value between the primary and secondary food ± 1 ℃ Was confirmed.

14-2. Staphylococcus  Aureus (622 bp primer )

Conventional polymerase chain reaction and real-time polymerase chain reaction were performed on the food groups contaminated with Staphylococcus aureus using the primer pairs of SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Was performed using an ABI 7700 real-time polymerase chain reaction apparatus, and the results are shown in FIGS. 29 and 30. Each lane of FIG. 29 is as follows.

Lane M: Size marker

N: negative control (does not contain DNA)

1: Escherichia coli O157: H7 105 CFU / mL-Hamburger Patty

2: Escherichia coli O157: H7 105 CFU / mL-ground chicken

3: Staphylococcus aureus KCTC1927 105 CFU / mL-Sundae

4: Staphylococcus aureus KCTC1927 105 CFU / mL-Gimbap

5: Vibrio parahaemolyticus KCCM41654 105 CFU / mL-seawater

6: Vibrio parahaemolyticus KCCM41654 105 CFU / ml-Shrimp

7: Listeria monocytogenes ATCC86152 105 CFU / mL-Salad

8: Listeria monocytogenes ATCC86152 105 CFU / ml-Ice Cream

9: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Frozen Chicken

10: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Salad

11: Bacillus cereus KCTC1661 105 CFU / mL-Miso

12: Bacillus cereus KCTC1661 105 CFU / mL-Gochujang

13: Thermoscene Entericulica KCCM41657 105 CFU / mL-Bottled water

14: Thermocinia Entericulica KCCM41657 105 CFU / mL-Milk

15: Shigella Flexneri ATCC12022 105 CFU / mL-Eating Spring Water

16: Shigella Flexnery ATCC12022 105 CFU / mL-Oyster

P: positive control (Staphylococcus aureus KCTC1927 105 CFU / mL)

As shown in FIG. 29, a band of the correct size was confirmed only in the food inoculated with E. coli O157: H7 and the positive control group, and as shown in FIG. 30, the SYBR green I PCR product using the ABI 7700 device. The Tm value was found to be a Tm value of 80.8 ° C. In more detail, the Tm value of the product was found to be 80.8 ° C in both the primary and secondary foods, and the error range of the SYBR Green I system using the ABI 7700 was found to be ± 0 ° C.

14-3. vibrio Parahemolyticus  (167 bp primer )

Conventional polymerase chain reaction and real-time polymerase chain reaction were performed using the primer pairs of SEQ ID NO: 7 and SEQ ID NO: 8 for the food groups contaminated with Vibrio parahemolyticus, and real-time polymerase chain reaction. Was performed using an ABI 7700 real-time polymerase chain reaction device and TMC-1000 real-time polymerase chain reaction device, and the results are shown in FIGS. 31 to 33. Each lane of FIG. 31 is as follows.

Lane M: Size marker

N: negative control (does not contain DNA)

1: Escherichia coli O157: H7 105 CFU / mL-Hamburger Patty

2: Escherichia coli O157: H7 105 CFU / mL-ground chicken

3: Staphylococcus aureus KCTC1927 105 CFU / mL-Sundae

4: Staphylococcus aureus KCTC1927 105 CFU / mL-Gimbap

5: Vibrio parahaemolyticus KCCM41654 105 CFU / mL-seawater

6: Vibrio parahaemolyticus KCCM41654 105 CFU / ml-Shrimp

7: Listeria monocytogenes ATCC86152 105 CFU / mL-Salad

8: Listeria monocytogenes ATCC86152 105 CFU / ml-Ice Cream

9: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Frozen Chicken

10: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Salad

11: Bacillus cereus KCTC1661 105 CFU / ㎖-Doenjang

12: Bacillus cereus KCTC1661 105 CFU / mL-Gochujang

13: Thermoscene Entericulica KCCM41657 105 CFU / mL-Bottled water

14: Thermocinia Entericulica KCCM41657 105 CFU / mL-Milk

15: Shigella Flexneri ATCC12022 105 CFU / mL-Eating Spring Water

16: Shigella Flexnery ATCC12022 105 CFU / mL-Oyster

P: positive control (Vibrio parahemolyticus KCCM41654 105 CFU / mL)

As shown in FIG. 31, a band of the correct size was identified only in the food inoculated with Vibrio parahaemolyticus and the positive control group, and as shown in FIG. 32, SYBR green I PCR using an ABI 7700 device. The Tm value of the product was found to be a Tm value of 84.9 ° C. More specifically, the Tm value of the Vibrio parahemolyticus PCR product was found to be 84.9 ° C. in both the primary and secondary food assays. From the results, it was confirmed that the error range of the SYBR Green I system using the ABI 7700 is ± 0 ℃, the difference in the Tm value between the primary food and the secondary food ± 0.5 ℃ when using the TMC-1000 Was confirmed.

14-4. Listeria Monocytogenes   (451 bp primer )

Conventional polymerase chain reaction and real-time polymerase chain reaction were performed on the food groups contaminated with Listeria monocytogenes using primer pairs of SEQ ID NO: 9 and SEQ ID NO: 10, respectively. ABI 7700 real-time polymerase chain reaction device and TMC-1000 real-time polymerase chain reaction device were carried out, and the results of each are shown in FIGS. 34 to 36. Each lane of FIG. 34 is as follows.

Lane M: Size marker

N: negative control (does not contain DNA)

1: Escherichia coli O157: H7 105 CFU / mL-Hamburger Patty

2: Escherichia coli O157: H7 105 CFU / mL-ground chicken

3: Staphylococcus aureus KCTC1927 105 CFU / mL-Sundae

4: Staphylococcus aureus KCTC1927 105 CFU / mL-Gimbap

5: Vibrio parahaemolyticus KCCM41654 105 CFU / mL-seawater

6: Vibrio parahaemolyticus KCCM41654 105 CFU / ml-Shrimp

7: Listeria monocytogenes ATCC86152 105 CFU / mL-Salad

8: Listeria monocytogenes ATCC86152 105 CFU / ml-Ice Cream

9: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Frozen Chicken

10: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Salad

11: Bacillus cereus KCTC1661 105 CFU / mL-Miso

12: Bacillus cereus KCTC1661 105 CFU / mL-Gochujang

13: Thermoscene Entericulica KCCM41657 105 CFU / mL-Bottled water

14: Thermocinia Entericulica KCCM41657 105 CFU / mL-Milk

15: Shigella Flexneri ATCC12022 105 CFU / mL-Eating Spring Water

16: Shigella Flexnery ATCC12022 105 CFU / mL-Oyster

P: positive control (Listeria monocytogenes ATCC86152 105 CFU / ml)

As shown in FIG. 34, a band of the correct size was confirmed only in the food inoculated with Listeria monocytogenes and the positive control group, and as shown in FIG. 35, the SYBR green I PCR product using the ABI 7700 device. The Tm value of was found to be a Tm value of 82.3 ° C. More specifically, the Tm value of the Listeria monocytogenes PCR product was found to be 82.3 ° C. in both the primary and secondary food assays. From the results, it was confirmed that the error range of the SYBR Green I system using the ABI 7700 is ± 0 ℃, the difference in the Tm value between the primary food and the secondary food ± 1 ℃ when using the TMC-1000 Was confirmed.

14-5. Salmonella  Strains (678 bp primer )

Food groups contaminated with Salmonella strains were subjected to real-time polymerase chain reaction using conventional polymerase chain reaction and ABI 7700 real-time polymerase chain reaction apparatus using primer pairs of SEQ ID NO: 15 and SEQ ID NO: 16, respectively. Real time polymerase chain reaction was performed, and the results are shown in FIGS. 37 and 38. Upon electrophoresis of the PCR product subjected to the conventional polymerase chain reaction, each lane is as follows.

Lane M: Size marker

N: negative control (does not contain DNA)

1: Escherichia coli O157: H7 105 CFU / mL-Hamburger Patty

2: Escherichia coli O157: H7 105 CFU / mL-ground chicken

3: Staphylococcus aureus KCTC1927 105 CFU / mL-Sundae

4: Staphylococcus aureus KCTC1927 105 CFU / mL-Gimbap

5: Vibrio parahaemolyticus KCCM41654 105 CFU / mL-seawater

6: Vibrio parahaemolyticus KCCM41654 105 CFU / ml-Shrimp

7: Listeria monocytogenes ATCC86152 105 CFU / mL-Salad

8: Listeria monocytogenes ATCC86152 105 CFU / ml-Ice Cream

9: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Frozen Chicken

10: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Salad

11: Bacillus cereus KCTC1661 105 CFU / mL-Miso

12: Bacillus cereus KCTC1661 105 CFU / mL-Gochujang

13: Thermoscene Entericulica KCCM41657 105 CFU / mL-Bottled water

14: Thermocinia Entericulica KCCM41657 105 CFU / mL-Milk

15: Shigella Flexneri ATCC12022 105 CFU / mL-Eating Spring Water

16: Shigella Flexnery ATCC12022 105 CFU / mL-Oyster

P: positive control (Salmonella enteritidis KCCM12021 105 CFU / mL)

As a result of electrophoresis of the result of the Conventional Polymerase Chain Reaction of Salmonella entericdis, only the food inoculated with Salmonella entericdis and the positive control group showed a band of the correct size, as shown in FIG. The Tm value of the SYBR green I PCR product using ABI 7700 instrument was found to be Tm value of 86.7 ° C. More specifically, the Tm value of the PCR product of Salmonella Entityis was found to be 86.7 ° C in both the primary and secondary food analysis results, and the error range of the SYBR Green I system using ABI 7700 was ± 0 ° C. Was confirmed.

14-6. Bacillus Cereus  (640 bp primer )

Food groups contaminated with Bacillus cereus were subjected to real-time polymerase chain reaction using conventional polymerase chain reaction and ABI 7700 real-time polymerase chain reaction apparatus using primer pairs of SEQ ID NO: 5 and SEQ ID NO: 6, respectively. 39 and 40 show the results of the real-time polymerase chain reaction. Upon electrophoresis of the PCR product subjected to the conventional polymerase chain reaction, each lane is as follows.

Lane M: Size marker

N: negative control (does not contain DNA)

1: Escherichia coli O157: H7 105 CFU / mL-Hamburger Patty

2: Escherichia coli O157: H7 105 CFU / mL-ground chicken

3: Staphylococcus aureus KCTC1927 105 CFU / mL-Sundae

4: Staphylococcus aureus KCTC1927 105 CFU / mL-Gimbap

5: Vibrio parahaemolyticus KCCM41654 105 CFU / mL-seawater

6: Vibrio parahaemolyticus KCCM41654 105 CFU / ml-Shrimp

7: Listeria monocytogenes ATCC86152 105 CFU / mL-Salad

8: Listeria monocytogenes ATCC86152 105 CFU / ml-Ice Cream

9: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Frozen Chicken

10: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Salad

11: Bacillus cereus KCTC1661 105 CFU / mL-Miso

12: Bacillus cereus KCTC1661 105 CFU / mL-Gochujang

13: Thermoscene Entericulica KCCM41657 105 CFU / mL-Bottled water

14: Thermocinia Entericulica KCCM41657 105 CFU / mL-Milk

15: Shigella Flexneri ATCC12022 105 CFU / mL-Eating Spring Water

16: Shigella Flexnery ATCC12022 105 CFU / mL-Oyster

P: positive control (Bacillus cereus KCTC1661 105 CFU / mL)

As a result of electrophoresis of the result of the conventional polymerase chain reaction of Bacillus cereus, the band of the correct size was confirmed only in the food inoculated with Bacillus cereus and the positive control group, as shown in FIG. 40, ABI 7700 The Tm value of the SYBR green I PCR product using the device was found to be Tm value of 82.7 ℃. More specifically, the Tm value of the PCR product of Bacillus cereus was found to be 82.7 ° C in both the primary and secondary food analysis results, and the error range of the SYBR Green I system using ABI 7700 was ± 0 ° C. Confirmed.

14-7. Heat sinia Entericulica  (330 bp primer )

Conventional polymerase chain reaction and real-time polymerase chain reaction were carried out using the primer pairs of SEQ ID NO: 11 and SEQ ID NO: 12 for the food groups contaminated with thermocinia entericitis, and real-time polymerase chain reaction. Was performed using an ABI 7700 real-time polymerase chain reaction device and TMC-1000 real-time polymerase chain reaction device, and the results of each are shown in FIGS. 41 to 43. Upon electrophoresis of the PCR product subjected to the conventional polymerase chain reaction, each lane is as follows.

Lane M: Size marker

N: negative control (does not contain DNA)

1: Escherichia coli O157: H7 105 CFU / mL-Hamburger Patty

2: Escherichia coli O157: H7 105 CFU / mL-ground chicken

3: Staphylococcus aureus KCTC1927 105 CFU / mL-Sundae

4: Staphylococcus aureus KCTC1927 105 CFU / mL-Gimbap

5: Vibrio parahaemolyticus KCCM41654 105 CFU / mL-seawater

6: Vibrio parahaemolyticus KCCM41654 105 CFU / ml-Shrimp

7: Listeria monocytogenes ATCC86152 105 CFU / mL-Salad

8: Listeria monocytogenes ATCC86152 105 CFU / ml-Ice Cream

9: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Frozen Chicken

10: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Salad

11: Bacillus cereus KCTC1661 105 CFU / mL-Miso

12: Bacillus cereus KCTC1661 105 CFU / mL-Gochujang

13: Thermoscene Entericulica KCCM41657 105 CFU / mL-Bottled water

14: Thermocinia Entericulica KCCM41657 105 CFU / mL-Milk

15: Shigella Flexneri ATCC12022 105 CFU / mL-Eating Spring Water

16: Shigella Flexnery ATCC12022 105 CFU / mL-Oyster

P: positive control (therniacina entericulica KCCM41657 105 CFU / mL)

As a result of electrophoresis of the result of the conventional polymerase chain reaction of thermocinia entericitis, the band of the correct size was confirmed only in the food inoculated with Bacillus cereus and the positive control group, as shown in FIG. 42. , The Tm value of the SYBR green I PCR product using ABI 7700 instrument was found to be Tm value of 88.1 ℃. More specifically, the Tm value of the Listeria monocytogenes PCR product was found to be 88.1 ° C. in both the primary and secondary food assays. From the results, it was confirmed that the error range of the SYBR Green I system using the ABI 7700 is ± 0 ℃, the difference in the Tm value between the primary food and the secondary food ± 1 ℃ when using the TMC-1000 Was confirmed.

14-8. Shigella strains (445 bp primer )

Using the primer pairs of SEQ ID NO: 17 and SEQ ID NO: 18 for the food groups contaminated with Shigella strains, real-time polymerase chain reaction was performed using a conventional polymerase chain reaction and an ABI 7700 real-time polymerase chain reaction device. 44 and 45 show the results of real-time polymerase chain reaction. Upon electrophoresis of the PCR product subjected to the conventional polymerase chain reaction, each lane is as follows.

Lane M: Size marker

N: negative control (does not contain DNA)

1: Escherichia coli O157: H7 105 CFU / mL-Hamburger Patty

2: Escherichia coli O157: H7 105 CFU / mL-ground chicken

3: Staphylococcus aureus KCTC1927 105 CFU / mL-Sundae

4: Staphylococcus aureus KCTC1927 105 CFU / mL-Gimbap

5: Vibrio parahaemolyticus KCCM41654 105 CFU / mL-seawater

6: Vibrio parahaemolyticus KCCM41654 105 CFU / ml-Shrimp

7: Listeria monocytogenes ATCC86152 105 CFU / mL-Salad

8: Listeria monocytogenes ATCC86152 105 CFU / ml-Ice Cream

9: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Frozen Chicken

10: Salmonella Entrepreneur KCCM12021 105 CFU / mL-Salad

11: Bacillus cereus KCTC1661 105 CFU / mL-Miso

12: Bacillus cereus KCTC1661 105 CFU / mL-Gochujang

13: Thermoscene Entericulica KCCM41657 105 CFU / mL-Bottled water

14: Thermocinia Entericulica KCCM41657 105 CFU / mL-Milk

15: Shigella Flexneri ATCC12022 105 CFU / mL-Eating Spring Water

16: Shigella Flexnery ATCC12022 105 CFU / mL-Oyster

P: positive control (Shigella flexneri ATCC12022 105 CFU / mL)

As a result of electrophoresis of the result of the conventional polymerase chain reaction of Shigella flexneri, the band of the correct size was confirmed only in the food inoculated with Bacillus cereus and the positive control group, as shown in FIG. The Tm value of the SYBR green I PCR product using a 7700 instrument was found to be a Tm value of 80.9 ° C. More specifically, the Tm value of the PCR product of Bacillus cereus was found to be 82.7 ° C in both the primary and secondary food analysis results, and the error range of the SYBR Green I system using ABI 7700 was ± 0 ° C. Confirmed.

<110> SAMSUNG EVERLAND INC. <120> A PRIMER FOR DETECTING FOOD BORNE PATHOGENIC MICROORGANISM AND A METHOD FOR DETECTING FOOD BORNE MICROORGANISM USING THE SAME <130> dpp20070127kr <160> 41 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> E1 <400> 1 gatagacttt tcgacccaac aaag 24 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> E2 <400> 2 ccgatgtggt cccctgag 18 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SA1 <400> 3 aatttaacag ctaaagagtt tggt 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SA4 <400> 4 acgtatcttc catgaatgat ctaa 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B1 <400> 5 gactacattc acgattacgc agaa 24 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> B4 <400> 6 ttcctaaact tgcaccatct cgg 23 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> V1 <400> 7 ctcatttgta ctgttgaacg ccta 24 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> V4 <400> 8 tactactggc gcttctggtt c 21 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> L1 <400> 9 ctggcacaaa attacttaca acga 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> L3 <400> 10 aactactgga gctgcttgtt tttc 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Y1 <400> 11 aataccgcat aacgtcttcg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Y2 <400> 12 cttcttctgc gagtaacgtc 20 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> C1 <400> 13 tgatatgtcc aaaaatgaac caga 24 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C2 <400> 14 ttgtgattcc cctgtgtcag g 21 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SAL1 <400> 15 gaatcctcag tttttcaacg tttc 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SAL4 <400> 16 tagccgtaac aaccaataca aatg 24 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SH1 <400> 17 caggcatgaa attaagcctg 20 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SH4 <400> 18 tgtacatcga taataccaaa c 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CA1 <400> 19 acttctttat tgcttgctgc 20 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CA2 <400> 20 gccacaacaa gttaaagaag c 21 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> E3 <400> 21 ttgctcaata atcagacgaa gatg 24 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> E4 <400> 22 cgttgtcaca ccgggcactg a 21 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> E5 <400> 23 ccccactctg acaccatcct 20 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SA2 <400> 24 ttcccactaa atgtgtttca taa 23 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SA3 <400> 25 ttcattaaag aaaaagtgta cgag 24 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> B2 <400> 26 gtagaccaat tgttgcttgt aatgg 25 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B3 <400> 27 ctatgctgac gagctacatc cata 24 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> V2 <400> 28 aggcgtcatt gttatcagaa gc 22 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> V3 <400> 29 tcatgagcag aggcgtcatt g 21 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> V5 <400> 30 aatagaaggc aaccagttgt tgat 24 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> V6 <400> 31 gactgccatt catttgatga tagc 24 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> L2 <400> 32 tggagtgctt actgctttgt cag 23 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> L4 <400> 33 gttaccaccc catgaataag c 21 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Y3 <400> 34 cggtattcct ccagatctct ac 22 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> C3 <400> 35 tgccattcat atctagctaa tgct 24 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> C4 <400> 36 tttcctttct tctgcaaaag tctc 24 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C5 <400> 37 cctgctgttc ctttttgaga g 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SAL2 <400> 38 ccagacgaaa gagcgtggta a 21 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SAL3 <400> 39 gtatgcccgg taaacagatg agt 23 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SH2 <400> 40 ctgcattctg gcaatctctt caca 24 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SH3 <400> 41 gcatctgaca gcaatagtac a 21

Claims (5)

Vibrio para, which comprises a primer consisting of a nucleotide sequence of SEQ ID NO: 7 and a primer consisting of a nucleotide sequence of SEQ ID NO: 8, and amplifying a 167 bp Vibrio parahemolyticus specific gene product having a Tm value of 84.9 ° C. Primer pairs for detection of hemolyticus; Vibrio para, which comprises a primer consisting of a nucleotide sequence of SEQ ID NO: 7 and a primer consisting of a nucleotide sequence of SEQ ID NO: 28, and amplifying a 91 bp Vibrio parahemoliticus specific gene product having a Tm value of 81.2 ° C. Primer pairs for detection of hemolyticus; Vibrio para, which comprises a primer consisting of a nucleotide sequence of SEQ ID NO: 7 and a primer consisting of a nucleotide sequence of SEQ ID NO: 29, and amplifying a 101 bp Vibrio parahemolyticus specific gene product having a Tm value of 81.7 ° C. Amplifying a 688 bp Vibrio parahemoliticus specific gene product consisting of a primer pair consisting of a primer pair for hemolyticus and a nucleotide sequence of SEQ ID NO: 7 and a nucleotide sequence of SEQ ID NO: 31, and having a Tm value of 85.5 ° C. A primer for detecting Vibrio parahaemolyticus, which comprises one or more selected primer pairs from the group consisting of primer pairs for detecting Vibrio parahemolyticus consisting of primers. Vibrio parahaemolyticus detection method comprising the step of detecting the Vibrio parahemoliticus by using a polymerase chain reaction (PCR) using the primer for detecting vibrio parahemoliticus of claim 1. The method of claim 2, wherein the polymerase chain reaction is a multiplex polymerase chain reaction. The method of claim 3, wherein the polymerase chain reaction is a real-time polymerase chain reaction. Vibrio parahemolyticus, which comprises the primer for detecting Vibrio parahemolyticus according to claim 1, which detects Vibrio parahemolyticus using a polymerase chain reaction (PCR), a reaction buffer, dNTP and Taq DNA polymerase. Detection kit.