JP2007274934A - Primer set and method for detecting food poisoning bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a primer set for measuring food poisoning bacteria by multiplex PCR method and a method for detecting the food poisoning bacteria. <P>SOLUTION: The primer set detects two or more food poisoning bacteria of a group consisting of intestinal tract bleeding coliform bacillus O-157, Salmonella bacteria, Campylobacter jejuni, Listeria monocytogenes and S. aureus by PCR method. The primer set for multiplex PCR comprises two or more specific primer sets which are mixed depending on bacteria to be detected. The method for detecting the food poisoning bacteria uses the primer for multiplex PCR. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a primer set and a method for detecting food poisoning bacteria. More specifically, the present invention relates to a primer set for multiplex PCR used when a plurality of food poisoning bacteria are simultaneously detected by polymerase chain reaction (PCR) and a method for detecting food poisoning bacteria using the primer set.

  In order to prevent the occurrence and infection of food poisoning, food poisoning bacteria are detected and identified from foods, food raw materials, patient vomits and feces, environmentally derived materials, and the like. When performing such detection and identification, confirmation tests such as enrichment culture, direct isolation culture, primary confirmation culture, secondary confirmation culture, and agglutination reaction with antiserum must be performed. The time required for each of these tests is 18 to 24 hours, 3 to 6 days are required as the total time required, and a great amount of time and cost are required. Further, when testing a plurality of food poisoning bacteria, each food poisoning bacteria must be tested. In addition, it is necessary to apply the optimum medium, culture method and determination method for the inspection of each bacterium. Thus, current inspection methods are complicated and lack rapidness and simplicity.

Therefore, recently, it has become possible to detect food poisoning bacteria by gene amplification reaction (PCR) using oligonucleotides as primers. However, it is necessary to prepare each reaction solution for each target gene, and in order to examine a plurality of food poisoning bacteria, it is necessary to prepare a plurality of reaction solutions and there is a problem that the operation is complicated. It was.
As a technique for overcoming the drawbacks of the above PCR, there is a multiplex PCR method in which a plurality of target gene primer sets are mixed in a single reaction solution, and simultaneously a plurality of different genes are specifically detected (non-patent literature). 1 and 2). However, the conventional multiplex PCR method does not always obtain a specific gene amplification product by simply mixing a plurality of primer sets.
In general, when two or more genes are simultaneously amplified by PCR, the frequency of nonspecific amplification of genes other than the target is increased. Therefore, it was necessary to design a primer that not only specifically binds to a target gene but also has low interference between primers (Non-patent Document 3).
In addition, after selecting a primer specific to the species or genus, the presence or absence of competition was examined, and in the case of competition, it was necessary to optimize the concentration of the primer set and the reaction temperature (Patent Document 1). Furthermore, even when primers are designed with sufficient consideration, there are cases where unexpected reduction in reaction efficiency and nonspecific amplification may be observed, and the design of a specific primer set has not been easy.
Jpn. J. Food Microbiol., 19: 47-55. 2002 J. Food Prot., 68: 551-556. 2005 Clin. Microbiol. Rev., 13: 559-570, 2000 JP-A-2005-34121

As described above, in the conventional multiplex PCR method, it is not always possible to obtain specific gene amplification products by simply mixing a plurality of primer sets.
The present invention has been made in order to solve such problems and specifically detect food poisoning bacteria, and is present in foods, food raw materials, patient vomits and feces, environmentally derived materials, etc. For multiplex PCR to simultaneously detect enterohemorrhagic Escherichia coli O157, Salmonella spp., Campylobacter jejuni , Listeria monocytogenes and / or Staphylococcus aureus Provided are a primer set and a method for detecting the food poisoning bacteria using the primer set.

In order to solve the above problems, the present invention provides a PCR method for two or more food poisoning bacteria of the group consisting of enterohemorrhagic Escherichia coli O157, Salmonella spp., Campylobacter jejuni, Listeria monocytogenes and Staphylococcus aureus. It is a primer set for detection, and is a primer set for multiplex PCR formed by mixing two or more primer sets described in (1) to (5) below according to the bacteria to be detected.
(1) For enterohemorrhagic E. coli O157,
A primer consisting of the base sequence shown in SEQ ID NO: 1 and a primer consisting of the base sequence shown in SEQ ID NO: 4 or 5;
A primer consisting of the base sequence shown in SEQ ID NO: 2 and a primer consisting of the base sequence shown in SEQ ID NO: 4 or 5;
A primer consisting of the base sequence shown in SEQ ID NO: 3 and a primer consisting of the base sequence shown in SEQ ID NO: 5;
A primer consisting of the base sequence shown in SEQ ID NO: 6 and a primer consisting of the base sequence shown in SEQ ID NO: 11 or 13;
A primer consisting of the base sequence shown in SEQ ID NO: 7 and a primer consisting of the base sequence shown in SEQ ID NO: 12 or 13;
A primer consisting of the base sequence shown in SEQ ID NO: 8 and a primer consisting of the base sequence shown in SEQ ID NO: 12 or 13;
A primer consisting of the base sequence shown in SEQ ID NO: 9 and a primer consisting of the base sequence shown in SEQ ID NO: 13;
A primer consisting of the base sequence shown in SEQ ID NO: 10 and a primer consisting of the base sequence shown in SEQ ID NO: 13;
One of the combinations or a combination of complementary sequences thereof.
(2) For Salmonella spp.
A primer consisting of the base sequence shown in SEQ ID NO: 14 and a primer consisting of the base sequence shown in SEQ ID NO: 19 or 21;
A primer consisting of the base sequence shown in SEQ ID NO: 15 and a primer consisting of the base sequence shown in SEQ ID NO: 18, 19 or 20;
A primer consisting of the base sequence shown in SEQ ID NO: 16 and a primer consisting of the base sequence shown in SEQ ID NO: 21;
A primer consisting of the base sequence shown in SEQ ID NO: 17 and a primer consisting of the base sequence shown in SEQ ID NO: 22, 23 or 24;
A primer consisting of the base sequence shown in SEQ ID NO: 25 and a primer consisting of the base sequence shown in SEQ ID NO: 27, 29, 30 or 31;
A primer consisting of the base sequence shown in SEQ ID NO: 26 and a primer consisting of the base sequence shown in SEQ ID NO: 27, 28, 29, 30 or 31;
One of the combinations or a combination of complementary sequences thereof.
(3) For Campylobacter jejuni
A primer consisting of the base sequence shown in SEQ ID NO: 32 and a primer consisting of the base sequence shown in SEQ ID NO: 34 or 36;
A primer consisting of the base sequence shown in SEQ ID NO: 33 and a primer consisting of the base sequence shown in SEQ ID NO: 35 or 36;
One of the combinations or a combination of complementary sequences thereof.
(4) For Listeria monocytogenes,
A primer consisting of the base sequence shown in SEQ ID NO: 37 and a primer consisting of the base sequence shown in SEQ ID NO: 41, 42, 43, 44, 45 or 46;
A primer consisting of the base sequence shown in SEQ ID NO: 38 and a primer consisting of the base sequence shown in SEQ ID NO: 41, 42, 43, 44 or 45;
A primer consisting of the base sequence shown in SEQ ID NO: 39 and a primer consisting of the base sequence shown in SEQ ID NO: 43, 44, 45 or 46;
A primer consisting of the base sequence shown in SEQ ID NO: 40 and a primer consisting of the base sequence shown in SEQ ID NO: 44, 45 or 46;
One of the combinations or a combination of complementary sequences thereof.
(5) Against Staphylococcus aureus
A primer consisting of the base sequence shown in SEQ ID NO: 47 and a primer consisting of the base sequence shown in SEQ ID NO: 49 or 50;
A primer consisting of the base sequence shown in SEQ ID NO: 48 and a primer consisting of the base sequence shown in SEQ ID NO: 50;
One of the combinations or a combination of complementary sequences thereof.
In particular, a multiplex PCR primer set including all of the primer sets described in (1) to (5) above is preferably used. Furthermore, it is preferable to select a primer set so that a difference of 10 bases or more in the base length of each PCR amplification product occurs.
Moreover, the oligonucleotide of this invention is an oligonucleotide which consists of a base sequence shown by said sequence number 1-50, or its complementary sequence.
Furthermore, the method for detecting food poisoning bacteria of the present invention comprises (a) a step of extracting DNA from a specimen, (b) PCR using the primer set according to any one of claims 1 to 3 using the DNA as a template. Two or more food poisoning bacteria of the group consisting of enterohemorrhagic Escherichia coli O157, Salmonella, Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus, comprising a step of performing, and (c) detecting the presence or absence of DNA amplification Is a method for simultaneously detecting.

According to the primer set for multiplex PCR of the present invention, the target genes of enterohemorrhagic Escherichia coli O157, Salmonella, Campylobacter jejuni, Listeria monocytogenes and / or Staphylococcus aureus are specified in a single PCR tube. Can be amplified automatically and without causing interference reactions. Therefore, when the amplification product is electrophoresed, enterohemorrhagic Escherichia coli O157, Salmonella, Campylobacter jejuni, Listeria monocytogenes and / or macroscopically distinguishable on a single lane of the electrophoresis gel. Alternatively, a band of PCR amplification product specific for S. aureus can be formed.
Further, the method for detecting food poisoning bacteria of the present invention is a method using the above-mentioned primer set for multiplex PCR, and enterohemorrhagic Escherichia coli O157, Salmonella spp., Listeria monocytogenes, Campylobacter jejuni and Staphylococcus aureus. Two or more kinds of food poisoning bacteria can be detected quickly, easily, specifically and simultaneously.

  In order to detect enterohemorrhagic Escherichia coli O157, Salmonella spp., Campylobacter jejuni, Listeria monocytogenes and / or Staphylococcus aureus that cause food poisoning simultaneously and without interference with the PCR method As a result of studying primer sets for multiplex PCR, intimin gene or O157 antigen synthesis region gene, invA gene of Salmonella invasive factor or Salmonella genus, which is one of the pathogenic factors of enterohemorrhagic Escherichia coli O157 Among primer combinations that specifically detect the oriC gene of fungi, the sodB gene of superoxide dismutase necessary for the survival of Campylobacter jejuni, the hlyA gene of Listeria monocytogenes membrane damage toxin, and the coagulase gene of Staphylococcus aureus , Table 1, Table 2, Table 3, and Table to be described later A group consisting of enterohemorrhagic Escherichia coli O157, Salmonella spp., Campylobacter jejuni, Listeria monocytogenes and Staphylococcus aureus by combining two or more selected from the combinations described in 4 and Table 5 It has been found that two or more food poisoning bacteria can be detected simultaneously and accurately.

The above simultaneous detection means that (1) one primer selected from the combinations shown in Table 1, Table 2, Table 3, Table 4, and Table 5 using two or more primer groups. In the PCR tube, heat denaturation, annealing, and polymerization reaction are amplified under a single PCR condition to prepare a PCR amplification product, and (2) the PCR amplification product is electrophoresed on a single lane. PCR that is specific to the test primer can accurately detect food poisoning bacteria corresponding to two or more selected primers of the group consisting of hemorrhagic Escherichia coli O157, Salmonella spp., Campylobacter jejuni, Listeria monocytogenes and Staphylococcus aureus It means that all of the amplification products form a band that can be visually recognized.
In order to form a visually distinguishable band, the minimum value of the difference in size between adjacent amplification products is preferably 10 bp or more, preferably 20 bp or more.
Moreover, being able to detect correctly means that the appearance frequency of false negative or false positive is less than 10%, respectively.

  The oligonucleotide used as a primer in the present invention is a nucleotide fragment having a length of 10 bases or more, preferably 15 bases or more in consideration of selectivity, detection sensitivity and reproducibility, and may be either chemically synthesized or natural. It does not matter whether or not the primer is labeled. In addition, as long as it functions as a primer, one or more bases may be added to the 5 ′ end side of the primer (oligonucleotide), and one or more bases are substituted or deleted. Also good.

  The base length of the PCR amplification product is defined by the selected primer set, but the amplification region in the nucleotide sequence of the food poisoning bacterium defined by the primer set of the present invention is 50 to 2,000 bases, preferably 100 to 1 bases. Select to be 1,000 bases.

Hereinafter, the primer set of this invention is demonstrated concretely.
Enterohemorrhagic Escherichia coli ( Escherichia
The primer set for detecting E. coli O157) is as follows.
A sequence for detecting the intimin gene of enterohemorrhagic E. coli O157;
(5 ')-CTGAACACAGTACGCAGAAG- (3') ... (Ia: SEQ ID NO: 1)
(5 ')-GCACAAGACTACCAGGCTAT- (3') ... (Ib: SEQ ID NO: 2)
(5 ')-GTCGTGTCTGCTAAAAC- (3') ... (Ic: SEQ ID NO: 3)
(5 ')-GTAAAGTCCGTTACCCCAAC- (3') ... (Id: SEQ ID NO: 4)
(5 ')-ACTGGCTGACCGTTTTTCA- (3') ... (Ie: SEQ ID NO: 5)
Or a sequence for detecting the O157 antigen synthesis region gene of enterohemorrhagic Escherichia coli O157;
(5 ′)-GGCGTATTACATGCGTTC- (3 ′) (IIa: SEQ ID NO: 6)
(5 ′)-TAGTCTCCAGTTGTATGGGG- (3 ′) (IIb: SEQ ID NO: 7)
(5 ′)-GTAGGGGTTGTATGCTCGT- (3 ′) (IIc: SEQ ID NO: 8)
(5 ′)-CTTTCCGACACCAGAGTTAG- (3 ′) (IId: SEQ ID NO: 9)
(5 ′)-GCAATACTTAGTGGCTGGG- (3 ′) (IIe: SEQ ID NO: 10)
(5 ′)-CCCCATACAACTGGAGACTA- (3 ′) (IIf: SEQ ID NO: 11)
(5 ′)-CTAACTCTGGTGTCGGAAAG- (3 ′) (IIg: SEQ ID NO: 12)
(5 ′)-GTAGAGCCGCCATCAACTAT- (3 ′) (IIh: SEQ ID NO: 13)
One of the combinations shown in Table 1 or combinations of complementary sequences thereof.
It is obvious to those skilled in the art that an oligonucleotide set consisting of a complementary sequence of a primer set functions as a primer set.

The primer set for detecting the above enterohemorrhagic Escherichia coli is specifically shown as follows.
A primer consisting of the base sequence shown in SEQ ID NO: 1 and a primer consisting of the base sequence shown in SEQ ID NO: 4 or 5;
A primer consisting of the base sequence shown in SEQ ID NO: 2 and a primer consisting of the base sequence shown in SEQ ID NO: 4 or 5;
A primer consisting of the base sequence shown in SEQ ID NO: 3 and a primer consisting of the base sequence shown in SEQ ID NO: 5;
A primer consisting of the base sequence shown in SEQ ID NO: 6 and a primer consisting of the base sequence shown in SEQ ID NO: 11 or 13;
A primer consisting of the base sequence shown in SEQ ID NO: 7 and a primer consisting of the base sequence shown in SEQ ID NO: 12 or 13;
A primer consisting of the base sequence shown in SEQ ID NO: 8 and a primer consisting of the base sequence shown in SEQ ID NO: 12 or 13;
A primer consisting of the base sequence shown in SEQ ID NO: 9 and a primer consisting of the base sequence shown in SEQ ID NO: 13;
A primer consisting of the base sequence shown in SEQ ID NO: 10 and a primer consisting of the base sequence shown in SEQ ID NO: 13;
One of the combinations or a combination of complementary sequences thereof.

The primer set for detecting Salmonella is as follows.
A sequence for detecting the invA gene of Salmonella;
(5 ')-GCGTCTACGTAGTCAGTTC- (3') (IIIa: SEQ ID NO: 14)
(5 ′)-ATCAGCAAGGTAGCAGTCAGT- (3 ′) (IIIb: SEQ ID NO: 15)
(5 ′)-GGTATCTGCTGAAGTTGAGG- (3 ′) (IIIc: SEQ ID NO: 16)
(5 ′)-GTGCTCGTTTACGACCTG- (3 ′) (IIId: SEQ ID NO: 17)
(5 ')-GATATACGTTGTACCGTGGC- (3') ... (IIIe: SEQ ID NO: 18)
(5 ′)-GCTCCACGAATATGCTCTAC- (3 ′) (IIIf: SEQ ID NO: 19)
(5 ′)-CCTCAACTTCAGCAGATACC- (3 ′) (IIIg: SEQ ID NO: 20)
(5 ′)-GACAGACGTAAGGAGGACAA- (3 ′) (IIIh: SEQ ID NO: 21)
(5 ′)-CCACGGTGACAATAGAGAAG- (3 ′) (IIIi: SEQ ID NO: 22)
(5 ')-GTTCAACAGCTGCGTCAT- (3') ... (IIIj: SEQ ID NO: 23)
(5 ′)-ATCGAGATCGCCAATCAGTC- (3 ′) (IIIk: SEQ ID NO: 24)
Or Salmonella oriC
Sequences for detecting genes;
(5 ′)-CAACTTTAGCCGATGGAG- (3 ′) (IVa: SEQ ID NO: 25)
(5 ′)-GGATAACCCGGATCCTGTA- (3 ′) (IVb: SEQ ID NO: 26)
(5 ′)-CACAGCTAGTGATCCTTTCC- (3 ′) (IVc: SEQ ID NO: 27)
(5 ′)-AGATTCCGGAAGATCCAG- (3 ′) (IVd: SEQ ID NO: 28)
(5 ′)-ACAGGTTGGGGAAACACT- (3 ′) (IVe: SEQ ID NO: 29)
(5 ′)-GTTGGGGAAACACTGAAG- (3 ′) (IVf: SEQ ID NO: 30)
(5 ′)-AGAAGCGGAACTGAAAGG- (3 ′) (IVg: SEQ ID NO: 31)
Is one of the combinations shown in Table 2 or a combination of complementary sequences thereof.

The primer set for detecting the above-mentioned Salmonella is specifically as follows.
A primer consisting of the base sequence shown in SEQ ID NO: 14 and a primer consisting of the base sequence shown in SEQ ID NO: 19 or 21;
A primer consisting of the base sequence shown in SEQ ID NO: 15 and a primer consisting of the base sequence shown in SEQ ID NO: 18, 19 or 20;
A primer consisting of the base sequence shown in SEQ ID NO: 16 and a primer consisting of the base sequence shown in SEQ ID NO: 21;
A primer consisting of the base sequence shown in SEQ ID NO: 17 and a primer consisting of the base sequence shown in SEQ ID NO: 22, 23 or 24;
A primer consisting of the base sequence shown in SEQ ID NO: 25 and a primer consisting of the base sequence shown in SEQ ID NO: 27, 29, 30 or 31;
A primer consisting of the base sequence shown in SEQ ID NO: 26 and a primer consisting of the base sequence shown in SEQ ID NO: 27, 28, 29, 30 or 31;
One of the combinations or a combination of complementary sequences thereof.

Campylobacter
The primer set for detecting jejuni ) is as follows.
A sequence for detecting the Campylobacter jejuni superoxide dismutase (sodB) gene;
(5 ')-CTTCAAATGGGGGCGTAT- (3') ... (Va: SEQ ID NO: 32)
(5 ')-GGCGGTTCATGTCAAAGT- (3') ... (Vb: SEQ ID NO: 33)
(5 ′)-CTAAATAAGCAGGGCGTG- (3 ′) (Vc: SEQ ID NO: 34)
(5 ′)-CTAACTGATCCCATGCCTTC- (3 ′) (Vd: SEQ ID NO: 35)
(5 ′)-AGCTAACTGATCCCATGC- (3 ′) (Ve: SEQ ID NO: 36)
Is one of the combinations shown in Table 3 or combinations of complementary sequences thereof.

The primer set for detecting the above Campylobacter jejuni is specifically shown as follows.
A primer consisting of the base sequence shown in SEQ ID NO: 32 and a primer consisting of the base sequence shown in SEQ ID NO: 34 or 36;
A primer consisting of the base sequence shown in SEQ ID NO: 33 and a primer consisting of the base sequence shown in SEQ ID NO: 35 or 36;
One of the combinations or a combination of complementary sequences thereof.

The primer set for detecting Listeria monocytogenes is as follows.
A sequence for detecting the hlyA gene of Listeria monocytogenes;
(5 ′)-CAAGTCCTAAGACGCCAATC- (3 ′) (VIa: SEQ ID NO: 37)
(5 ')-GTATACCACGGAGATGCAGT- (3') ... (VIb: SEQ ID NO: 38)
(5 ′)-CAAACGTTAACAACGCAGTA- (3 ′) (VIc: SEQ ID NO: 39)
(5 ')-GGATACGTTGCTCAATTC- (3') ... (VId: SEQ ID NO: 40)
(5 ′)-GAGAGCACCTGGATAGGTTA- (3 ′) (VIe: SEQ ID NO: 41)
(5 ')-GTCATACCTGGCAAATCAAT- (3') ... (VIf: SEQ ID NO: 42)
(5 ')-GCAACGTATCCTCCAGAGT- (3') ... (VIg: SEQ ID NO: 43)
(5 ′)-GGACGATGTGAAATGAGC- (3 ′) (VIh: SEQ ID NO: 44)
(5 ′)-AAACCAGTGCATTCTTTAGC- (3 ′) (VIi: SEQ ID NO: 45)
(5 ′)-GCCCCAGATGGAGATATTTCTATT- (3 ′) (VIj: SEQ ID NO: 46)
Is one of the combinations shown in Table 4 or combinations of complementary sequences thereof.

The primer set for detecting the above-mentioned Listeria monocytogenes is specifically shown as follows.
A primer consisting of the base sequence shown in SEQ ID NO: 37 and a primer consisting of the base sequence shown in SEQ ID NO: 41, 42, 43, 44, 45 or 46;
A primer consisting of the base sequence shown in SEQ ID NO: 38 and a primer consisting of the base sequence shown in SEQ ID NO: 41, 42, 43, 44 or 45;
A primer consisting of the base sequence shown in SEQ ID NO: 39 and a primer consisting of the base sequence shown in SEQ ID NO: 43, 44, 45 or 46;
A primer consisting of the base sequence shown in SEQ ID NO: 40 and a primer consisting of the base sequence shown in SEQ ID NO: 44, 45 or 46;
One of the combinations or a combination of complementary sequences thereof.

The primer set for detecting Staphylococcus aureus is as follows.
A sequence for detecting the coagulase gene of S. aureus,
(5 ′)-CGACATATGGGCCTAGAGTA- (3 ′) (VIIa: SEQ ID NO: 47)
(5 ′)-CGTACCGAACTGGAACTTAC- (3 ′) (VIIb: SEQ ID NO: 48)
(5 ')-GTAAGTTCCAGTTCGGTACG- (3') ... (VIIc: SEQ ID NO: 49)
(5 ')-GGTCATCCATTAGGTGCTAC- (3') ... (VIId: SEQ ID NO: 50)
Is one of the combinations shown in Table 5 or combinations of complementary sequences thereof.

A specific primer set for detecting the above S. aureus is as follows.
A primer consisting of the base sequence shown in SEQ ID NO: 47 and a primer consisting of the base sequence shown in SEQ ID NO: 49 or 50;
A primer consisting of the base sequence shown in SEQ ID NO: 48 and a primer consisting of the base sequence shown in SEQ ID NO: 50;
One of the combinations or a combination of complementary sequences thereof.

In the primer set for multiplex PCR of the present invention, a primer set for multiplex PCR is configured and used by appropriately combining the above primer sets according to the food poisoning bacteria species to be detected. Since the type of food poisoning bacteria contained in the specimen is often unknown, it is preferable to mix and use five kinds of primer sets because the food poisoning bacteria contained in the specimen can be reliably detected.
When constructing a multiplex PCR primer set, the PCR amplification products listed in Table 1 to Table 5 are selected so that the base length of each PCR amplification product is 10 bases or more, preferably 15 bases or more. Configure and use plex PCR primer sets. By doing so, it is possible to form a band that can visually distinguish all the PCR amplification products specific to the test primer.

  The method for detecting food poisoning bacteria of the present invention comprises (a) a step of extracting DNA from a specimen, (b) a step of performing PCR using the DNA as a template, and using the above-described multiplex PCR primer set, (c) DNA A method for simultaneously detecting two or more food poisoning bacteria of the group consisting of enterohemorrhagic Escherichia coli O157, Salmonella, Campylobacter jejuni, Listeria monocytogenes and Staphylococcus aureus, comprising a step of detecting the presence or absence of amplification of .

  The specimen is not limited to food and food ingredients, but may be environment-derived materials such as patient vomit, feces, sewage, and swabs. In order to use these materials as PCR samples, an operation for releasing DNA components from the cells present in the materials is required as a pretreatment. However, PCR will proceed if there are several to several tens of centimeters of DNA that can be hybridized with the primer. A sample solution having a DNA amount can be prepared. If necessary, the DNA sample may be prepared after growing the food poisoning bacteria by culturing the test material in an appropriate medium.

Next, PCR is performed using the DNA in the sample prepared above as a template and the multiplex PCR primer set of the present invention.
Such a PCR step can be performed according to a conventional method, and can be performed using a commercially available PCR apparatus or the like.
More specifically, for example, as a reaction solution used for the PCR reaction, a reaction solution attached to a commercially available PCR measurement kit or the like may be used.
Moreover, the thermostable DNA polymerase used in the PCR reaction may be derived from any species as long as the activity is maintained at a temperature of 90 to 95 ° C. Examples of the thermostable DNA polymerase used include Gene Taq, TaKaRa Taq, ExTaq HS and the like.
As PCR conditions, the temperature of heat denaturation is 90 to 95 ° C., the temperature of the annealing operation for hybridizing the primers is 37 to 65 ° C., the polymerization reaction is 50 to 75 ° C., and PCR using this as one cycle is 20 to 42. Amplify by cycling. In particular, the present invention enables simultaneous detection of a plurality of food poisoning bacteria genes in a single PCR reaction, the temperature of heat denaturation is 94 ° C., the Tm value and GC content of all primers are 45 to 55 ° C., respectively. The design was made to be in the range of 50 to 55%.
The amount of the primer set used is not particularly limited, but generally it is preferably used at about 20 μM.

After amplification by PCR is completed, amplified DNA in the reaction solution is detected. DNA may be detected according to a conventional method. For example, the reaction solution after PCR is subjected to conventional gel electrophoresis using agarose or the like, and the DNA fragment after electrophoresis is stained with ethidium bromide, fluorescent reagent The method of detecting by the method etc. is illustrated and the presence and length of the amplified DNA fragment can be confirmed. As shown in Tables 1 to 5 above, since the base length of the amplified DNA is defined by the primer set used, the presence or absence of food poisoning bacteria to be examined can be determined based on the base length of the amplified DNA. it can.
For detection of the amplified nucleotide fragment, electrophoresis method or chromatography using a support other than agarose gel can be applied. The primer set can be fixed to a support such as a membrane, an ELISA plate, a slide glass, etc., and used as a probe for Southern blotting.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to the example which concerns.

Example 1
Sample Preparation Table 6 and Table 7 Enterohemorrhagic Escherichia coli O157 strain, Table 8 and Table 9 Salmonella strain, Table 10 Campylobacter jejuni strain, Listeria strain shown in Table 11 and epidermal grapes shown in Table 12 Each of the staphylococci and Staphylococcus aureus strains was cultured in a commonly used enrichment medium and culture conditions. 1 ml of the culture solution was centrifuged at 6000 rpm for 2 minutes, collected, suspended in 100 μl of sterilized purified water, heated at 96 ° C. for 5 minutes, and then centrifuged at 15000 rpm for 10 minutes, and the supernatant was used as a specimen.

Oligonucleotides having the sequences shown in PCR primer SEQ ID NOs: 1 to 50 were chemically synthesized. Chemical synthesis was performed by the β-cyanoethyl phosphoramidite method. The synthesized oligonucleotide was purified by high performance liquid chromatography using a C18 reverse phase column.

PCR method Using 1 μl of the sample solution, 37.7 μl of sterile distilled water, 10 × reaction buffer solution (500 mM)
KCl, 100 mM Tris-HCl pH 8.3, 17.5 mM magnesium chloride) 5 μl, dNTP solution 4 μl (dATP, dCTP, dGTP, dTTP mixture; final concentration 2.5 mM), one from each of the primer sets shown in Tables 1 to 5 1.0 μl (20 μM) of each selected primer set and 0.3 μl of thermostable DNA polymerase (Taq DNA polymerase (5 unit / μl; manufactured by Roche)) were added to prepare a total reaction volume of 50 μl. Reaction conditions: (thermal denaturation) 94 ° C., 1 minute, (annealing) 55 ° C., 1 minute, (polymerization reaction)
68 ° C, 1 minute. The process from thermal denaturation through annealing to the polymerization reaction was defined as one cycle, and this was performed for 35 cycles (total time required: about 3 hours). These operations were performed by programming the above reaction conditions in a PCR device (Gene Amp ™ PCR System 9700, Applied Biosystems, iCycler ™, Bio-Rad, PTC-200 DNA Engine, MJ Japan).

In order to detect the amplified nucleotide fragments from the detection reaction solution of the amplified DNA , agarose gel electrophoresis was performed as follows. An agarose gel having a gel concentration of 1% (W / V) and containing ethidium bromide (concentration 0.5 μg / ml) was used. The electrophoresis conditions were a constant voltage of 100 V and 25 minutes. The operation method and other conditions were performed by the technique described in Molecular Cloning 3rd edition (2001) by Sambrook et. Al. In addition to the reaction solution, a molecular weight marker was also migrated, and the size of the nucleotide fragment was calculated by comparing the relative mobility.

Results The above results are shown in Tables 6 to 12. As shown in Tables 6 to 12, only the DNA of the gene-positive strain was amplified and did not react at all with the DNA of the gene-negative strain. That is, it was revealed that the primer set (oligonucleotide set) of the present invention correctly amplifies the target gene and selectively amplifies only the bacteria having the target gene. That is, it has been clarified that the primer set indicated by “◯” in Tables 1 to 5 can be applied to the detection of each food poisoning bacterium.

Example 2
Method The same as in Example 1 except that each culture solution described in Example 1 was mixed and subjected to the experiment, and the multiplex PCR primer sets (primer sets 1 to 4) described in Table 13 were used. Operated. In addition, the primer set used here is a primer set set so that five types of food poisoning bacteria can be detected.

Results The above results are shown in FIG. In the figure, (1), (2), (3) and (4) show electrophoresis images of gene amplification products by primer set 1, primer set 2, primer set 3 and primer set 4, respectively. M in the figure represents an electrophoretic image of the molecular weight marker. As shown in FIG. 1, when the multiplex PCR primer set shown in Table 13 is used, five types of food poisoning bacteria of enterohemorrhagic Escherichia coli O157, Salmonella spp., Listeria monocytogenes, Campylobacter jejuni and Staphylococcus aureus Could be detected simultaneously. In addition, when the PCR amplification product was electrophoresed, it was possible to form five independent bands that could be visually distinguished on the same lane.

Comparative example
Method The procedure was performed in the same manner as in Example 2 except that the primer set for multiplex PCR described in Table 14 (primer sets 5 and 6) was used.

Results The above results are shown in FIG. (5) and (6) in the figure show electrophoresis images of gene amplification products by primer set 5 and primer set 6, respectively. As shown in FIG. 2, when the multiplex PCR primer set shown in Table 14 is used, five types of food poisoning bacteria of enterohemorrhagic Escherichia coli O157, Salmonella spp., Listeria monocytogenes, Campylobacter jejuni and Staphylococcus aureus Of these, it was found that in the case of primer set 5, enterohemorrhagic Escherichia coli O157 could not be detected in spite of using the primer set (IIa and IIg) based on the base sequence of the enterohemorrhagic Escherichia coli O157 genome. In addition, it was found that the primer set 6 cannot detect L. monocytogenes despite the use of primer sets (VIb and VIj) based on the base sequence of the L. monocytogenes genome.

It is an electrophoresis image which shows that five types of food poisoning bacteria can be detected using the primer set shown in Table 13. It is an electrophoretic image which shows that only four types of food poisoning bacteria are detected if the primer set shown in Table 14 is used.

Claims (5)

A primer set for detecting two or more food poisoning bacteria of the group consisting of enterohemorrhagic Escherichia coli O157, Salmonella spp., Campylobacter jejuni, Listeria monocytogenes and Staphylococcus aureus by PCR, and the target bacteria A primer set for multiplex PCR obtained by mixing two or more of the primer sets described in (1) to (5) below.
(1) For enterohemorrhagic E. coli O157,
A primer consisting of the base sequence shown in SEQ ID NO: 1 and a primer consisting of the base sequence shown in SEQ ID NO: 4 or 5;
A primer consisting of the base sequence shown in SEQ ID NO: 2 and a primer consisting of the base sequence shown in SEQ ID NO: 4 or 5;
A primer consisting of the base sequence shown in SEQ ID NO: 3 and a primer consisting of the base sequence shown in SEQ ID NO: 5;
A primer consisting of the base sequence shown in SEQ ID NO: 6 and a primer consisting of the base sequence shown in SEQ ID NO: 11 or 13;
A primer consisting of the base sequence shown in SEQ ID NO: 7 and a primer consisting of the base sequence shown in SEQ ID NO: 12 or 13;
A primer consisting of the base sequence shown in SEQ ID NO: 8 and a primer consisting of the base sequence shown in SEQ ID NO: 12 or 13;
A primer consisting of the base sequence shown in SEQ ID NO: 9 and a primer consisting of the base sequence shown in SEQ ID NO: 13;
A primer consisting of the base sequence shown in SEQ ID NO: 10 and a primer consisting of the base sequence shown in SEQ ID NO: 13;
One of the combinations or a combination of complementary sequences thereof.
(2) For Salmonella spp.
A primer consisting of the base sequence shown in SEQ ID NO: 14 and a primer consisting of the base sequence shown in SEQ ID NO: 19 or 21;
A primer consisting of the base sequence shown in SEQ ID NO: 15 and a primer consisting of the base sequence shown in SEQ ID NO: 18, 19 or 20;
A primer consisting of the base sequence shown in SEQ ID NO: 16 and a primer consisting of the base sequence shown in SEQ ID NO: 21;
A primer consisting of the base sequence shown in SEQ ID NO: 17 and a primer consisting of the base sequence shown in SEQ ID NO: 22, 23 or 24;
A primer consisting of the base sequence shown in SEQ ID NO: 25 and a primer consisting of the base sequence shown in SEQ ID NO: 27, 29, 30 or 31;
A primer consisting of the base sequence shown in SEQ ID NO: 26 and a primer consisting of the base sequence shown in SEQ ID NO: 27, 28, 29, 30 or 31;
One of the combinations or a combination of complementary sequences thereof.
(3) For Campylobacter jejuni
A primer consisting of the base sequence shown in SEQ ID NO: 32 and a primer consisting of the base sequence shown in SEQ ID NO: 34 or 36;
A primer consisting of the base sequence shown in SEQ ID NO: 33 and a primer consisting of the base sequence shown in SEQ ID NO: 35 or 36;
One of the combinations or a combination of complementary sequences thereof.
(4) For Listeria monocytogenes,
A primer consisting of the base sequence shown in SEQ ID NO: 37 and a primer consisting of the base sequence shown in SEQ ID NO: 41, 42, 43, 44, 45 or 46;
A primer consisting of the base sequence shown in SEQ ID NO: 38 and a primer consisting of the base sequence shown in SEQ ID NO: 41, 42, 43, 44 or 45;
A primer consisting of the base sequence shown in SEQ ID NO: 39 and a primer consisting of the base sequence shown in SEQ ID NO: 43, 44, 45 or 46;
A primer consisting of the base sequence shown in SEQ ID NO: 40 and a primer consisting of the base sequence shown in SEQ ID NO: 44, 45 or 46;
One of the combinations or a combination of complementary sequences thereof.
(5) Against Staphylococcus aureus
A primer consisting of the base sequence shown in SEQ ID NO: 47 and a primer consisting of the base sequence shown in SEQ ID NO: 49 or 50;
A primer consisting of the base sequence shown in SEQ ID NO: 48 and a primer consisting of the base sequence shown in SEQ ID NO: 50;
One of the combinations or a combination of complementary sequences thereof.   A primer set for multiplex PCR comprising all one of the primer sets described in (1) to (5) of claim 1.   The primer set for multiplex PCR according to claim 1 or 2, wherein each primer set is selected so that a base length of each PCR amplification product is different by 10 bases or more.   An oligonucleotide comprising the base sequence shown in SEQ ID NOs: 1 to 50 or a complementary sequence thereof.   (a) a step of extracting DNA from a specimen, (b) a step of performing PCR using the primer set according to any one of claims 1 to 3 using the DNA as a template, and (c) presence or absence of amplification of DNA. A method for simultaneously detecting two or more food poisoning bacteria of the group consisting of enterohemorrhagic Escherichia coli O157, Salmonella, Campylobacter jejuni, Listeria monocytogenes and Staphylococcus aureus, comprising a step of detecting.

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