JP5867147B2 - Enterobacteriaceae group detection method - Google Patents

Description

  The present invention relates to a primer set for detecting Enterobacteriaceae group, an Enterobacteriaceae group detection kit, and an Enterobacteriaceae group detection method.

  In food microbiological tests, the rapidity and accuracy of test results are extremely important for maintaining the freshness of test objects and ensuring food safety. In a conventional food microbiological test, a predetermined test including a microorganism culturing process has been performed (Non-patent Documents 1 and 2). However, in the inspection method including the culture process, the culture period is rate-determined, and there is a time problem that it takes 3 days to 1 week before the test result is obtained.

  In recent years, detection and quantification of hygienic index bacteria by molecular biological techniques using PCR has been performed. The hygiene indicator bacteria are bacteria that have the same origin as intestinal food poisoning bacteria such as Salmonella and Shigella, or are likely to behave together with them. It is used as an index for estimating the presence or absence of food contamination by food poisoning bacteria.

  By the way, conventionally, in Japan, the hygienic indicator bacteria corresponded to coliforms and fecal coliforms. Therefore, detection and quantification of coliform bacteria using the PCR method is also employed in food microbiological tests (Non-patent Document 3). However, the definition of Escherichia coli group is a lactose-degrading bacterium, and there is a big problem that intestinal food poisoning bacteria such as Salmonella and Shigella that are non-lactose-degrading bacteria are not included in the hygiene indicator bacteria. . Since 2006, there has been a shift from Escherichia coli to the Enterobacteriaceae family that uses glucose degradability as an indicator, mainly in Europe. Therefore, there is a need for a new method for rapidly and specifically detecting Enterobacteriaceae family by molecular biological techniques.

  The Enterobacteriaceae family is characterized by degrading glucose to produce acid and being negative for oxidase reaction, but the bacteria that fall under these are so diverse that the Enterobacteriaceae family There is no specific gene that can only be a target for nucleic acid amplification. Therefore, a method for detecting Enterobacteriaceae has been developed using 16SrRNA, RpoB, gyrB and other genes as housekeeping genes as target genes. However, none of the methods using genes can specifically detect only the Enterobacteriaceae group, and there are problems in terms of detection accuracy such as high false positive rate or high false negative rate. there were. For the above reasons, a nucleic acid amplification method capable of specifically detecting only the Enterobacteriaceae family has not been established so far.

Ministry of Health, Labor and Welfare, Food Sanitation Inspection Guidelines, 2004 Jasson V. , Et al., 2010, Food Microbiology, 27, 710-730 Martin, MC., Et al., 2009, International Journal of Systematic and Evolutionary Microbiology, 59, 234-247.

  An object of the present invention is to develop and provide a primer set that can specifically detect only Enterobacteriaceae group by a nucleic acid amplification method without passing through a culture step.

  Another object of the present invention is to provide an enterobacteriaceae group detection method capable of detecting enterobacteriaceae group rapidly and with high accuracy using the developed primer set. .

  In order to solve the above problems, the present inventors designed a large number of primer sets based on the alignment of various genes in the Enterobacteriaceae family, and used them for PCR. As a result, only when a primer set for a specific region of the ribosomal protein L16 gene (rplP), which has been a housekeeping gene but has not attracted much attention as a marker gene, is used, the group of Enterobacteriaceae is extremely specific. It was found that it could be detected. The present invention has been completed based on the above findings, and specifically provides the following.

(1) The base sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 1 containing a base sequence of 17 or more consecutive bases in which 1 to 3 bases are added, deleted or substituted Consists of a nucleotide comprising a forward primer composed of nucleotides and a base sequence represented by SEQ ID NO: 2 or a base sequence in which 1 to 3 bases are added, deleted or substituted in the base sequence represented by SEQ ID NO: 2 A primer set for detecting Enterobacteriaceae family comprising reverse primers.
(2) The primer set for detecting enterobacteriaceae group according to (1), wherein the reverse primer is composed of a nucleotide comprising the base sequence represented by SEQ ID NO: 3.
(3) The primer set for detecting Enterobacteriaceae group according to (1) or (2), wherein the reverse primer is composed of nucleotides of 50 bases or less.
(4) The reverse primer is composed of a nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence in which 1 to 3 bases are added, deleted or substituted in the nucleotide sequence shown in SEQ ID NO: 2. A primer set for detecting Enterobacteriaceae family according to (1).
(5) The primer set for enterobacteriaceae group detection according to (4), wherein the reverse primer is composed of a nucleotide having a base sequence represented by SEQ ID NO: 3.
(6) The forward primer is composed of a nucleotide sequence comprising the nucleotide sequence represented by SEQ ID NO: 4, or a nucleotide sequence comprising 1 to 3 bases added, deleted or substituted in the nucleotide sequence represented by SEQ ID NO: 4. A primer set for detecting Enterobacteriaceae family according to any one of (1) to (5).
(7) The primer set for enterobacteriaceae group detection according to (6), wherein the forward primer is composed of a nucleotide comprising the base sequence represented by SEQ ID NO: 5.
(8) The primer set for enterobacteriaceae group detection according to (6) or 7), wherein the forward primer is composed of nucleotides of 50 bases or less.
(9) The forward primer has a base sequence represented by SEQ ID NO: 1 or a base sequence represented by SEQ ID NO: 1 having a base sequence of 1 to 3 bases added, deleted or substituted, having 17 or more consecutive bases The primer set for detecting Enterobacteriaceae group according to any one of (1) to (5), comprising nucleotides comprising a base sequence.
(10) The forward primer is composed of a nucleotide sequence consisting of the base sequence represented by SEQ ID NO: 4, or a base sequence comprising 1 to 3 bases added, deleted or substituted in the base sequence represented by SEQ ID NO: 4. (9) A primer set for detecting Enterobacteriaceae family.
(11) The primer set for enterobacteriaceae group detection according to (10), wherein the forward primer is composed of a nucleotide having the base sequence represented by SEQ ID NO: 5.
(12) Enterobacteriaceae according to any one of (1) to (11), wherein the Enterobacteriaceae family is Citrobacter, Edwardsiella, Erwinia, Escherichia, Proteus, Salmonella, or Yersinia Primer set for detecting bacteria group.
(13) An enterobacteriaceae group detection kit comprising the primer set for detecting enterobacteriaceae group according to any one of (1) to (12).
(14) An enterobacteriaceae group detection method, wherein an extraction step of extracting a nucleic acid from a sample, and an intestine according to any one of (1) to (12) using the nucleic acid extracted in the nucleic acid extraction step as a template An amplification step of performing a nucleic acid amplification reaction using a primer set for detecting the bacteriophage group, and a determination step of determining whether the enterobacteriaceae group is positive or negative in the sample based on the amount of amplification product after the amplification step Said method.
(15) The enterobacteriaceae group detection method according to (14), wherein a quantitative nucleic acid amplification method is used in the amplification step.
(16) In the determination step, when the amount of the amplified product after the amplification step is statistically significantly higher than the amount of the amplified product of the control sample not including the Enterobacteriaceae family, The method for detecting Enterobacteriaceae family according to (14) or (15), wherein the family bacteria family is determined to be positive.
(17) The enterobacteriaceae group detection method according to any one of (14) to (16), wherein the sample is food.
(18) An enterobacteriaceae group quantification method, wherein an extraction step of extracting a nucleic acid from a sample, and an intestine according to any one of (1) to (12) using the nucleic acid extracted in the nucleic acid extraction step as a template Quantitative amplification process that performs quantitative nucleic acid amplification reaction using primer set for bacteriophage group detection, Ct value obtained in the quantitative amplification process is substituted into calibration formula, and enterobacteriaceae group in sample is quantitative And the calibration formula is a primer set for detecting Enterobacteriaceae group according to any one of (1) to (12) using a nucleic acid extracted from a serially diluted sample of a known number of bacteria as a template. The method, which is a formula derived based on the amount of amplification product of each serially diluted sample by performing a quantitative nucleic acid amplification reaction using
(19) The enterobacteriaceae group quantification method according to (18), wherein the calibration formula is Ct value = -3 (logCFU / g) +37).
(20) The enterobacteriaceae group quantification method according to (18) or (19), wherein the sample is food.

  According to the primer set for detecting Enterobacteriaceae group of the present invention, the Enterobacteriaceae group can be specifically detected by a nucleic acid amplification method.

  According to the enterobacteriaceae group detection kit of the present invention, reagents and the like necessary for simple detection of enterobacteriaceae group can be supplied.

  According to the method for detecting Enterobacteriaceae group of the present invention, the Enterobacteriaceae group can be detected quickly and accurately without going through a bacterial culture step.

  According to the enterobacteriaceae group quantification method of the present invention, the enterobacteriaceae group can be quantified quickly and accurately without going through a bacterial culture step.

The result of having electrophoresed the amplified fragment of the rplP gene by agarose gel when PCR was performed using the primer set for detecting Enterobacteriaceae family of the present invention is shown. The arrow indicates the amplified fragment of the rplP gene to be detected. Each lane number corresponds to the strain indicated as “FIG. 1 number” in Table 1. The result of having electrophoresed the amplified fragment of the rplP gene on an agarose gel when PCR was performed with various DNA polymerases using the Enterobacteriaceae family primer of the present invention is shown. The arrow indicates the amplified fragment of the rplP gene to be detected. A used TaKaRa Taq (TaKaRa Bio), B used TaKaRa ExTaq (TaKaRa Bio), and C used Taq DNA polymerase Native (Invitrogen). Each lane number corresponds to the strain shown in FIG. The result of having electrophoresed the amplified fragment of the rplP gene on an agarose gel when PCR was performed with various DNA polymerases using the Enterobacteriaceae family primer of the present invention is shown. The arrow indicates the amplified fragment of the rplP gene to be detected. D used Platium PCR Super Mix (Invitrogen), and E used MightyAmp DNA Polymerase Ver. 2 (Takara Bio). Each lane number corresponds to the strain shown in FIG. The result of the PCR growth curve in each dilution step of each bacterial species obtained by real-time PCR is shown. A is Citrobacter freudii, B is Enterobacter aerogenes, and C is Escherichia coli. The result of the PCR growth curve in each dilution step of each bacterial species obtained by real-time PCR is shown. D is Klebsiella pneumoniae and E is Proteus mirabillis. 3 shows a calibration curve obtained based on the growth curve of FIG. 3-1. The calibration curve obtained based on the growth curve of FIG. 3-2 is shown. The result of Table 2 is made into a graph. Using the calibration curves obtained in FIGS. 4-1 and 4-2, the number of Enterobacteriaceae groups in food was quantified by real-time PCR and the number of Enterobacteriaceae groups determined by the culture method A comparison of quantitative results is shown. The horizontal axis shows the results by real-time PCR, and the vertical axis shows the results by the culture method.

1. 1. Primer set for detecting Enterobacteriaceae group 1-1. Configuration The first aspect of the present invention is a primer set for detecting Enterobacteriaceae family. The primer set for detecting Enterobacteriaceae family of this embodiment is a forward primer composed of nucleotides having a base sequence of a specific region in the ribosomal protein L16 gene and complementary to the base sequence of the specific region of the gene It consists of the reverse primer comprised by the nucleotide which has a base sequence.

In this specification, the “Enterobacteriaceae group” (Enterobacteriaceae group) forms a characteristic colony of pink, red, purple, etc. on a violet red bile glucose (VRBG) agar medium. , Refers to a group of Gram-negative bacilli that are negative for oxidase reactions that ferment glucose. However, in this specification, not only the case of being composed of multiple types of Enterobacteriaceae but also the case of being composed of only one type of Enterobacteriaceae, it shall be included in the Enterobacteriaceae group And Enterobacteriaceae include, for example, the genus Alishewanella, Alterococcus, Aquamonas, Aranicola, Arsenophonus, Azotivirga, Blochmannia, Brenneria, Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Genus, Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella, Grimontella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella, Moellerella, Morganella, Obesumbacterium, Pantobacterium, Pectobacterium Phlomobacter, Photorhabdus, Plesiomonas, Pragiomonas, Progius, Proteus, Providencia, Rahnella, Raoultella, Salmonella, Samsonia, Serratia, Shigella, Sodalis, Tatumella, Trabulsiella, X , Species belonging to the genus Yersinia and Yokenella.
In the present specification, a group of bacteria other than the group of Enterobacteriaceae is referred to as a “non-Enterobacteriaceae group”.

  In the present specification, the “primer set for detecting Enterobacteriaceae family” is a set of primer pairs for nucleic acid amplification reaction consisting of a forward primer and a reverse primer. The primer set for detecting Enterobacteriaceae group is composed of nucleotides having a base sequence that can specifically amplify a specific region of the ribosomal protein L16 gene in microorganisms only in the Enterobacteriaceae group. When subjected to a nucleic acid amplification reaction, in principle, any primer is composed of a single-stranded nucleic acid molecule.

  In the present specification, “ribosomal protein L16” (hereinafter referred to as “rplP”) is one of the constituent molecules of the ribosomal protein 50S subunit (large subunit). In E. coli, it is known to bind directly to 23S rRNA and localize to the A site of peptidyltransferase. In the present specification, the “rplp gene” refers to a gene encoding rplp.

  In this embodiment, the nucleotide constituting the primer is an oligonucleotide or a polynucleotide, and includes a natural nucleotide, a non-natural nucleotide or a combination thereof.

  “Natural nucleotide” refers to DNA and RNA in which only nucleotides existing in nature are linked. In addition, “non-natural nucleotide” refers to an artificially constructed polymer compound having a structure and / or similar properties similar to those of a natural nucleotide, or a nucleotide that does not exist in nature that has been artificially chemically modified. Say. For example, cross-linked nucleic acid (BNA / LNA: Bridged Nucleic Acid / Locked Nucleic Acid; LNA is a registered trademark), peptide nucleic acid (PNA: Peptide Nucleic Acid), peptide nucleic acid having a phosphate group (PHONA), morpholino nucleic acid (morpholino) Including oligo), methylphosphonate DNA / RNA, phosphorothioate DNA / RNA, phosphoramidate DNA / RNA, 2′-O-methyl DNA / RNA, and the like. The nucleotide used for the primer of the present invention is preferably a nucleotide consisting only of DNA, a nucleotide consisting only of a cross-linked nucleic acid, a nucleotide consisting of DNA and RNA, or a DNA and LNA. Each primer may be composed of different nucleotides. For example, the forward primer may be a nucleotide consisting only of DNA, and the reverse primer may be a nucleotide consisting only of a crosslinked nucleic acid.

The primer in this embodiment may be labeled with a phosphate group, a sugar and / or a base, if necessary. As the label, a labeler known in the art can be used. For example, radioisotopes (eg, ³² P, ³ H, ¹⁴ C), DIG, biotin, fluorescent dyes (eg, FITC, Texas, cy3, cy5, cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493 , NBD, TAMRA), or a luminescent substance (for example, acridinium ester). Further, if necessary, a nucleotide having a base sequence including a restriction enzyme site may be linked to the 5 ′ end side of the primer.

  Hereinafter, the forward primer and the reverse primer constituting the primer set for detecting Enterobacteriaceae family in this embodiment will be specifically described. The primer set for detecting Enterobacteriaceae in this embodiment can be arbitrarily combined with various forward primers and reverse primers described below.

(1) Forward primer The forward primer in the primer set for detecting Enterobacteriaceae family of this embodiment consists of a nucleotide containing a part of the base sequence on the sense strand side including the start codon of the rplp gene. Specifically, it is a nucleotide comprising a nucleotide sequence of 17 bases or more consecutive in the nucleotide sequence represented by SEQ ID NO: 1 (5′-ATGTTACAACCWAAGCGTACAAAATTCCGTAA-3 ′. In the nucleotide sequence represented by SEQ ID NO: 1, W is A (adenine ) Or T (thymine), or a nucleotide comprising a nucleotide sequence of 17 or more consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 1, wherein 1 to 3 nucleotides are added, deleted or substituted Even when 1 to 3 bases are added, deleted or substituted in a nucleotide of 17 bases or more containing a predetermined base sequence, annealing to the target nucleic acid is possible, and primer However, it is desirable that the addition, deletion, or substitution of the base is at a position other than the 3 ′ end in the extension direction of the primer. The addition, deletion, or substitution is performed for the same reason.Preferable nucleotides are 1 to 3 in the nucleotide sequence represented by SEQ ID NO: 4 (5'-ATGTTACAACCWAAGCGTACA-3 ') or the nucleotide sequence represented by SEQ ID NO: 4. A nucleotide containing a base sequence in which one base is added, deleted or substituted, more preferably a nucleotide containing the base sequence shown in SEQ ID NO: 5 (5′-ATGTTACAACCAAAGCGTACA-3 ′), more preferably a sequence. It is a nucleotide sequence consisting of a base sequence represented by No. 4 or a base sequence having 1 to 3 bases added, deleted or substituted in the base sequence represented by SEQ ID No. 4. Particularly preferred is the base represented by SEQ ID No. 5. A nucleotide consisting of a sequence.

  The base length of the forward primer is not particularly limited as long as the target nucleic acid fragment, that is, a partial fragment of the rplp gene can be amplified by the primer set of this embodiment. Usually, it is 100 bases or less. Preferably it is 50 bases or less, More preferably, it is 40 bases or less, More preferably, it is 30 bases or less, Most preferably, it is 17-25 bases.

(2) Reverse primer The reverse primer in the primer set for detecting Enterobacteriaceae family of this embodiment consists of a nucleotide containing a part of the base sequence on the antisense strand side near the 3 ′ end in the rplp gene. Specifically, a base sequence represented by SEQ ID NO: 2 (5'-TTACCYTGACGCTTAAYYGC-3 '), or a base sequence in which 1 to 3 bases are added, deleted or substituted in the base sequence represented by SEQ ID NO: 2, Is a nucleotide. In the base sequence represented by SEQ ID NO: 2, Y represents pyrimidine, that is, C (cytosine), T (thymine), or U (uracil). Preferably, it is a nucleotide containing the base sequence (5′-TTACCYTGACGCTTAACTGC-3 ′) represented by SEQ ID NO: 3. More preferably, it is a nucleotide consisting of the base sequence shown by SEQ ID NO: 2 or the base sequence shown by the base sequence shown by adding, deleting or replacing 1 to 3 bases in the base sequence shown by SEQ ID NO: 2. More preferably, it is a nucleotide consisting of the base sequence represented by SEQ ID NO: 3.

1-2. Effect The primer set for detecting Enterobacteriaceae family of this embodiment can be used as a primer pair for nucleic acid amplification reaction to specifically amplify a specific region of rplP gene only by Enterobacteriaceae family . Thereby, the Enterobacteriaceae family can be detected with high accuracy.

2. Enterobacteriaceae group detection kit 2-1. Configuration The second aspect of the present invention is an enterobacteriaceae group detection kit. The enterobacteriaceae group detection kit of this aspect includes the primer set for detecting enterobacteriaceae group of the first aspect as an essential component. Two or more primer pairs may be included. For example, in the case of performing nucleic acid amplification reaction in two steps and detecting Enterobacteriaceae family, as in nested PCR, the first step forward primer is the nucleotide of the nucleotide sequence shown in SEQ ID NO: 5, When the two-step forward primer is a nucleotide having a nucleotide sequence located 3 'further than the nucleotide sequence shown in SEQ ID NO: 5 in the nucleotide sequence shown in SEQ ID NO: 1, and includes two types of forward primers Is applicable.

The enterobacteriaceae group detection kit of this embodiment can include various reagents necessary for nucleic acid amplification reaction, if necessary, in addition to the above-mentioned enterobacteriaceae group detection primer set. For example, four types of deoxyribonucleotides (dATP, dGTP, dCTP, dTTP; hereinafter, these are collectively referred to as “dNTP”), a nucleic acid amplification reaction buffer, a salt such as MgCl ₂ , and / or a nucleic acid polymerase (DNA polymerase, In particular, it may contain a thermostable DNA polymerase). Moreover, when performing a quantitative nucleic acid amplification method, the probe which has a fluorescence reporter and a quencher can also be included. Furthermore, it can also include a manual describing a protocol for detecting Enterobacteriaceae family.

2-2. Effect According to the enterobacteriaceae group detection kit of this embodiment, a primer set for detecting the enterobacteriaceae group necessary for rapidly detecting the enterobacteriaceae group can be provided.

3. 3. Enterobacteriaceae group detection method 3-1. Configuration The third aspect of the present invention is a method for detecting Enterobacteriaceae family. The enterobacteriaceae group detection method of this aspect includes an extraction step, an amplification step, and a determination step. Hereinafter, each step will be specifically described.

(1) Extraction Step The “extraction step” is a step of extracting nucleic acid from a sample.
In this embodiment, “sample” refers to a test substance to be used in the method for detecting Enterobacteriaceae family of this embodiment. Preferred are foods, tableware (plates, bowls, chopsticks, spoons, forks, knives, etc.) or cooking utensils (knives, cutting boards, cloths, balls, pans, etc.). More preferably, non-heat-treated foods such as raw meat (including internal organs such as minced meat, liver, heart, intestine, etc.), processed meat (hamburg, sausage, bacon, ham, etc.) before heating, fresh seafood (fish fillet or stripped) , And sashimi), vegetables (including root vegetables and potatoes), or fruits. In particular, raw edible vegetables, fruits, raw edible meat, and raw edible seafood are suitable as samples in the method for detecting enterobacteriaceae in this embodiment. The sample may be thawed after freezing.

  “Nucleic acid” in this embodiment refers to nucleic acid contained in a sample or nucleic acid contained in a cell attached to the sample, and refers to DNA and / or RNA. Preferred is DNA, particularly genomic DNA of a microorganism containing the rplP gene or a fragment thereof.

When nucleic acid is extracted from a sample, when the sample is a food, an appropriate amount (usually 0.5 g to 50 g is sufficient, preferably 25 g) of the food is weighed, homogenized as necessary, and suspended. To prepare. When the sample is a tableware or cooking utensil, the solution after washing or immersing them with water, physiological saline, or a buffer such as Tris / EDTA (pH 8.0) buffer, Homogenize if necessary to prepare a suspension. Nucleic acids can be extracted directly from the above suspension, but if necessary, a medium such as BPW (Buffered Pepton Water) medium (1% peptone, 0.5% NaCl, 0.35% Na ₂ HPO ₄ , 0.15% KH ₂ PO ₄ : pH 7.2 ± 0.2) may be added and cultured at 37 ° C. for 5-24 hours.

  The nucleic acid can be extracted from the suspension by a method known in the art, for example, an alkali method or a boiling method. These specific extraction methods are described with reference to, for example, the method described in Sambrook, J. et.al., 2001, Molecular Cloning: A Laboratory Manual Third Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. do it. In addition, various nucleic acid extraction kits commercially available from various manufacturers such as NucleoSpin Tissue (MACHEREY-NAGEL) can also be used.

(2) Amplification step The “amplification step” is a step of performing a nucleic acid amplification reaction using the nucleic acid extracted in the nucleic acid extraction step as a template and using the enterobacteriaceae group detection primer set according to the first aspect. .

  As used herein, “nucleic acid amplification reaction” refers to a reaction in which a specific region sandwiched between two primers is amplified by an enzymatic reaction such as polymerase using a nucleic acid such as DNA as a template and dNTP as a substrate.

  When the nucleic acid extracted in the nucleic acid extraction step is DNA, using DNA (including genomic DNA and cDNA) as a template, using a primer set and an enzyme such as DNA polymerase, the specific region in the rplP gene of the template DNA is determined. Duplicate and amplify (DNA amplification step). As a DNA amplification method used in this step, any method known in the art may be used. For example, PCR (polymerase chain reaction) method, ICAN (isothermal gene amplification) method and the like can be mentioned. The PCR method is preferred. (PCR: Clinical Diagnostics and Research, A. Rolfs, I. Schuller, U. Finckh, I. Weber-Rolfs, 1992, Springer-verlag Berlin Hidelberg.)

  The DNA polymerase used in the reaction is appropriately determined depending on the nucleic acid amplification method to be used. Usually, a heat resistant DNA polymerase (for example, Taq DNA polymerase, Pfu DNA polymerase, etc.) is used. Such thermostable nucleic acid polymerases include TaKaRa Bio (for example, TaKaRa Taq, Takara ExTaq, Takara KOD, MightyAmp DNA Polymerase Ver.2, etc.), Life Technology (for example, Taq DNA polymerase, Native, Platium PCR Super Mix, etc.) ), New England Biolab, Roche, Promega, etc., various types of products are commercially available and can be used.

  The reaction conditions for the nucleic acid amplification reaction may be determined in consideration of the length of the nucleotide to be amplified, the amount of template DNA, the Tm value of the primer, the optimum reaction temperature and the optimum pH of the DNA polymerase used. The length of the nucleotide to be amplified by the enterobacteriaceae group detection method of this embodiment is in the range of 170 to 350 nucleotides, preferably 170 to 250 nucleotides, more preferably 170 to 200 nucleotides. The amount of template DNA per reaction is 50 fg to 50 ng. If a commercially available enzyme is used, the optimum reaction temperature and optimum pH of the DNA polymerase to be used may be adjusted to the optimum reaction temperature and optimum pH described in the attached manual.

  For PCR conditions, when the reaction is performed in three steps of denaturation, annealing, and extension, for example, initial denaturation is performed at a temperature of 92 ° C or higher for 1 to 5 minutes, and then a heat denaturation step is performed at 90 ° C to 98 ° C for 30 seconds. The annealing step may be performed at 50 ° C to 60 ° C for 30 seconds to 1 minute, and the extension step at 70 ° C to 75 ° C for 40 seconds to 2 minutes. When the reaction is performed in two steps, denaturation and annealing extension, for example, after initial denaturation at a temperature of 92 ° C or higher for 1 to 5 minutes, the thermal denaturation step is performed at 90 ° C to 98 ° C for 30 seconds to 1 minute. The step may be performed at 50 ° C. to 60 ° C. for about 20 seconds to 1 minute. Further, the number of cycles is usually 10 to 50 cycles. Preferably it is 15 to 45 cycles, more preferably 20 to 40 cycles.

  The amplified DNA obtained after this step can be purified as necessary. The purification method may be any method known in the art. For example, an ethanol precipitation method or a purification method using a spin gel filtration column can be used.

  When the nucleic acid extracted in the nucleic acid extraction step is RNA, a reverse transcription step is required prior to the DNA amplification step performed when the nucleic acid is DNA. The reverse transcription step is a step of generating cDNA by performing a reverse transcription reaction using dNTP as a substrate and a primer and an enzyme such as reverse transcription polymerase. As a method used in the reverse transcription reaction, a method known in the art can be used. For example, the reverse transcription method described in Sambrook, J. et. Al., 2001, Molecular Cloning: A Laboratory Manual Third Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York may be used. The cDNA obtained in the reverse transcription step is subjected to the DNA amplification step.

  In this step, if the primer is labeled with the label, the double-stranded nucleic acid as the amplification product is selectively separated and purified from the reaction solution after the amplification reaction based on the label, The other single-stranded nucleic acid complementary to the target single-stranded nucleic acid among the double-stranded nucleic acids can be conveniently separated and removed based on the label.

  The nucleic acid amplification reaction in this step may use a quantitative nucleic acid amplification method. The “quantitative nucleic acid amplification method” referred to here is, for example, a method for detecting and quantifying an amplification product in real time in a nucleic acid amplification reaction, and includes, for example, a real-time PCR method. The detection method of the amplification product in the quantitative nucleic acid amplification method is not particularly limited as long as it can detect an increase in fluorescence intensity reflecting the increase in the amplification product over time. For example, a nucleic acid probe labeled with a fluorescent reporter and a quencher at the 5 ′ and 3 ′ ends, respectively, is degraded by a DNA polymerase having 5 ′ → 3 ′ exonuclease activity, resulting in the release of a nucleic acid fragment containing the 5 ′ end. In the presence of RNaseH, a nucleic acid amplification reaction is performed using a nucleic acid probe containing RNA with the same label, and only the RNA portion of the diffusion probe is detected. Fluorescence emitted by the degradation of selenium is detected in real time (cycling probe method), or a nucleic acid stain such as SYBER Green I is added to the reaction solution for gene amplification and stained with the gene amplification reaction, and excitation light And a method for measuring the intensity of fluorescence emitted from the nucleic acid stain that intercalates the increase in amplification products while irradiating . Kits using quantitative nucleic acid amplification methods are commercially available from various manufacturers (for example, SYBR Premix Extaq (TaKaRa Bio), etc.), and they may be used. The detection and quantification of the amplification product may be performed using a device commercially available from each manufacturer, for example, ABI 7900HT real-time PCR system, ABI7000 real-time RT-PCR (both from Applied Biosystems). A specific method may be performed according to the method described in the instruction manual attached to the apparatus.

(3) Determination Step The “determination step” is a step of determining whether the Enterobacteriaceae family is positive or negative in the sample based on the amount of amplification product after the amplification step. If it is positive, it means that the sample is contaminated with Enterobacteriaceae, and if it is negative, it means that the sample is not contaminated with Enterobacteriaceae.

  The determination method is performed based on the amount of amplification product after the amplification step. As the simplest method, when an amplification product of the rplp gene is detected after the amplification step, that is, when the amount of amplification product is not 0, it is determined as positive, and when it is not detected, that is, the amount of amplification product is When it is 0, the method of determining as negative is mentioned. These can be determined by, for example, whether or not a band having an expected size can be confirmed by gel electrophoresis of the amplification product. If the band is confirmed, it can be determined as positive. In addition, when a quantitative nucleic acid amplification method is used in the amplification step, the determination can also be made by an increase in the fluorescence value accompanying the generation of the amplification product. For example, in real-time PCR, if the fluorescence value rises 32 to 38 cycles, preferably 33 to 37 cycles, more preferably 34 to 36 cycles, and most preferably 35 cycles before, it may be determined as positive.

  In addition, the amount of amplification product after the amplification step is compared with the amount of amplification product of a control sample that does not include the Enterobacteriaceae family, and when the amount of amplification product is statistically significantly higher than the control sample, A method of determining that the sample is positive for Enterobacteriaceae family may be used.

  The term “control sample” as used herein refers to a sample that serves as a control for the sample, and is a sample that is the same or approximately the same as the sample and that is the same or approximately the same amount and does not include at least the Enterobacteriaceae family. Say. For example, when 25 g of raw beef meat is used as a sample, it becomes clear that the control sample is 25 g of raw beef and does not contain the Enterobacteriaceae family. As used herein, “statistically significant” means that there is a significant difference between the sample and the control sample when the quantitative difference in the amplification product is statistically processed. Specifically, for example, the risk rate (significance level) is less than 5%, 1%, or 0.1%. The test method for statistical processing is not particularly limited as long as a known test method capable of determining the presence or absence of significance is appropriately used. For example, Student's t test or multiple comparison test can be used.

3-2. Effect According to the method for detecting Enterobacteriaceae group of this embodiment, it is possible to specifically detect only the Enterobacteriaceae group from a sample without passing through a culture step. Therefore, it is possible to provide a food microorganism testing method that can be detected in a short time.

4). Enterobacteriaceae group quantification method 4-1. Configuration A fourth aspect of the present invention is a method for quantifying Enterobacteriaceae family. The enterobacteriaceae group quantification method of this aspect includes an extraction step, a quantitative amplification step, and a measurement step. Hereinafter, each step will be specifically described. Among these, the extraction process is the same as that in the third aspect, and therefore the description thereof is omitted. Here, the quantitative amplification process and the measurement process characteristic of this aspect will be described.

(1) Quantitative amplification process The "quantitative amplification process" refers to a quantitative nucleic acid using the nucleic acid extracted in the nucleic acid extraction process as a template and any of the Enterobacteriaceae family detection primer sets described in the first aspect. This is a step of performing an amplification reaction. This step is substantially the same as the quantitative nucleic acid amplification method described in the amplification step in the enterobacteriaceae family detection method of the third aspect. Therefore, it may be performed according to the quantitative nucleic acid amplification method described in the amplification step.

(2) Measurement step The “measurement step” is a step of quantitatively measuring the Enterobacteriaceae group in the sample by substituting the Ct value obtained in the quantitative amplification step into a calibration formula. The “Ct value (Threshold Cycle value)” here refers to the number of cycles when the amplification product reaches a certain amount, and the “calibration formula” here refers to a serially diluted sample with a known number of bacteria. A quantitative nucleic acid amplification reaction is performed based on the amount of amplification product of each step-diluted sample by performing a quantitative nucleic acid amplification reaction using the extracted nucleic acid as a template and any of the Enterobacteriaceae family detection primer sets described in the first embodiment. That is, the calibration formula performs the extraction step and the quantitative amplification step in the enterobacteriaceae family quantification method of this embodiment on a serially diluted sample obtained by serially diluting a bacterial solution whose number of bacteria has been measured in advance. After creating a calibration curve based on the amplification curve showing the amount of amplification product at each dilution step obtained as a result of quantitative nuclear amplification amplification reaction, it is only necessary to derive from the calibration curve.The Ct value obtained in the quantitative amplification step is calibrated. By assigning to the formula, The number of bacteria in 1 g of the sample can be calculated, and an example of a calibration formula is Ct value = −3 (logCFU / g) +37). Of course, the calibration formula is not limited to this, and a calibration formula may be newly derived from a serially diluted sample of another known number of bacteria.

4-2. Effect According to the enterobacteriaceae group quantification method of this embodiment, only the enterobacteriaceae group can be rapidly quantified from the sample without passing through the culture step.

<Example 1: Specific detection of Enterobacteriaceae group by primer set for detecting Enterobacteriaceae group of the present invention>
(the purpose)
It is verified that the enterobacteriaceae group primer set designed for the rplP gene of the present invention can specifically detect the enterobacteriaceae group.

(Method)
1. Bacterial Cell Culture The bacterial species listed in Table 1 were used for the Enterobacteriaceae family and the Non-Enterobacteriaceae family. These are standard strains derived from strain distribution agencies and obtained from ATCC, JCM, or IAM.

Add 10 mL of TSB medium (Bacto-trypticase soy broth; 1.7% Pancreatic Digest of Casein, 0.3% Papaic Digest Soybean, 0.5% NaCl, 0.25% K ₂ HPO ₄ , 0.25% Dextrose)) to each of the bacteria listed in Table 1. And cultured at 30 ° C. for 16 hours.

2. DNA preparation After culturing, 1 mL of each culture solution was centrifuged at 15,000 G for 5 minutes at 4 ° C. to collect the cells. DNA extraction from the cells was prepared using NucleoSpin Tissue (MACHEREY-NAGEL) according to the attached protocol. The prepared DNA was dissolved in 50 μL of Elution Buffer (5 mM Tris / HCl, pH 8.5 composition is described) (about 50 to 300 ng / μL). The extracted DNA was prepared to 5 ng / μL and used as template DNA.

3. PCR and Gel Electrophoresis Subsequently, a nucleic acid amplification reaction was performed by PCR using the prepared DNA as a template. The nucleotide of the base sequence shown in SEQ ID NO: 5 was used as the forward primer, and the nucleotide of the base sequence shown in SEQ ID NO: 3 was used as the reverse primer. When these primer sets are used, a DNA fragment having a length of 185 bases is amplified from the Enterobacteriaceae family.

TaKaRa Taq (Takara Bio) was used as the DNA polymerase. Reagents attached to TaKaRa Taq were used as PCR reagents such as a reaction buffer. As the composition of the reaction solution, the reaction composition recommended in the protocol attached to TaKaRa Taq was used. Specifically, 5 μL of template DNA (corresponding to 250 ng), 5 μL of 10 × Taq buffer containing 15 mM MgCl ₂ , 4 μL of 2.5 mM dNTP, 4.5 μL each of 10 pmol / μL forward primer and reverse primer, and Taq polymerase 0.25 μL was mixed with sterilized water to make a total of 50 μL. As PCR reaction conditions, initial denaturation was performed at 95 ° C for 5 minutes, and then 30 cycles of 2 steps of 95 ° C for 15 seconds and 60 ° C for 31 seconds were performed. 5 μL of the reaction solution after the reaction was taken and subjected to 3.0% agarose gel electrophoresis (NuSieve 3: 1 agarose gel, FMC Bioproducts) to confirm the presence or absence of amplification products in each bacterium.

(result)
The results are shown in FIG. The arrow indicates the amplified fragment of the rplP gene amplified by the PCR. As shown in this figure, when PCR was performed using the primer set of the present invention, the amplified fragment of the rplP gene was detected only in the Enterobacteriaceae group and not in the non-Enterobacteriaceae group. It was. From this result, it was proved that the primer set of the present invention was a primer set capable of specifically detecting only the Enterobacteriaceae group.

<Example 2: Adaptability of the primer set for detecting Enterobacteriaceae family of the present invention to various DNA polymerases>
(the purpose)
It is confirmed whether the Enterobacteriaceae group primer of the present invention can specifically detect Enterobacteriaceae group with respect to various DNA polymerases other than TaKaRa Taq.

(Method)
As the DNA polymerase, TaKaRa Taq (Takara Bio), TaKaRa ExTaq (Takara Bio), Taq DNA polymerase, Native (Invitrogen), Platium PCR Super Mix (Invitrogen), and Mighty Amp DNA Polymerase Ver. 2 (Takara Bio) were used. The primer set used is the same as in Example 1. PCR reagents such as reaction buffers used the reagents attached to each DNA polymerase in the same manner as TaKaRa Taq, and the composition of the reaction solution conformed to the recommended composition in the attached protocol. Specifically, for TaKaRa Taq (Takara Bio), 5 μL of template DNA (corresponding to 250 ng), 5 μL of 10 × Taq buffer containing 15 mM MgCl ₂ , 4 μL of 2.5 mM dNTP, 10 pmol / μL of forward primer and reverse primer each 4.5 μL and 0.25 μL of Taq polymerase and sterilized water were mixed to make a total of 50 μL. In TaKaRa ExTaq (Takara Bio), 5 μL of template DNA (corresponding to 250 ng), 5 μL of 10 mM ExTaq buffer containing 20 mM MgCl ₂ , 4 μL of 2.5 mM dNTP, 4.5 μL each of 10 pmol / μL forward primer and reverse primer, and Ex Taq polymerase was mixed with 0.25 μL of sterile water to make a total of 50 μL. For Taq DNA polymerase, Native (Invitrogen), 5 μL of 10 × PCR buffer, 4 μL of 2.5 mM dNTP Mix, 1.5 μL of 50 mM MgCl ₂ , 4.5 μL each of 10 pmol / μL forward primer and reverse primer, and 0.25 Taq DNA polymerase μL and sterilized water were mixed to make a total of 50 μL. In Platium PCR Super Mix (Invitrogen), 5 μL of template DNA (corresponding to 250 ng), 1.65 mM MgCl ₂ , 220 μM dNTP, 45 U of Platium PCR Super Mix containing 22 U / mL Taq DNA polymerase, 10 pmol / μL forward primer and reverse Primers were mixed with 1 μL of sterilized water for a total of 50 μL. In MightyAmp DNA Polymerase Ver.2 (Takara Bio), sterilize 4 mM MgCl ₂ , 800 μM dNTP-containing 2 × MightyAmp Taq PCR Buffer 25 μL, 10 pmol / μL forward primer and reverse primer 4.5 μL each, and MightyAmp Taq DNA polymerase 1 μL Mix in water to make a total of 50 μL. PCR reaction conditions and amplification products were confirmed according to the method described in Example 1.

(result)
The results are shown in FIGS. 2-1 and 2-2. As in Example 1, the arrow indicates an amplified fragment of the rplP gene amplified by the PCR. As shown in these figures, when a nucleic acid amplification reaction is performed under common PCR conditions using the reagents attached to each DNA polymerase, any DNA polymerase can specifically detect Enterobacteriaceae. Became clear. That is, the use of the Enterobacteriaceae tuberculosis detection primer set of the present invention, regardless of the type of DNA polymerase, also, and various dNTP concentrations, primer concentrations, only Enterobacteriaceae bacterial group also in MgCl ₂ concentration It was proved that can be specifically detected.

<Example 3: Confirmation of quantification of primer set for detecting Enterobacteriaceae group of the present invention>
(the purpose)
It is confirmed that the primer set for detecting Enterobacteriaceae family of the present invention can be used for quantification of amplification products.

(Method)
1. Bacterial cell culture For the Enterobacteriaceae family, five species (Citrobacter freundii, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) which are representative species of the group were used. Each strain was obtained from a standard strain-derived standard strain (JCM, IAM). Each strain is 10 mL TSB ((Bacto-trypticase soy broth; 1.7% Pancreatic Digest of Casein, 0.3% Papaic Digest Soybean, 0.5% NaCl, 0.25% K ₂ HPO ₄ , 0.25% Dextrose)) at 37 ° C after 16 h incubation, 10-fold in saline 9 mL, 10 ² times, was serially diluted 10 ³ times. After taking 1 mL from each diluted solution and collecting the cells by centrifugation at 15,000 g for 5 minutes at 4 ° C., DNA was extracted from the cells using NucleoSpin Tissue (MACHEREY-NAGEL) according to the attached protocol. The prepared DNA was dissolved in 50 μL of Elution Buffer (5 mM Tris / HCl, pH 8.5).

2. Real-time PCR
Whether or not the primer set for detecting Enterobacteriaceae family of the present invention can be quantitatively used was confirmed by real-time PCR. DNA prepared from each bacterial species was subjected to real-time PCR using SYBR Premix Ex Taq (Takara Bio). The primer set used is the same as in Example 1. The reaction solution composition was 5 μL of template DNA, 25 μL of 2 × SYBR Premix Extaq (Takara Bio), 4.5 μL each of 10 pmol / μL forward primer and reverse primer, and 11 μL of sterilized water, for a total of 50 μL. The reaction conditions for real-time PCR were in accordance with the PCR reaction conditions described in Example 1 (40 cycles of 2 steps of 95 ° C. for 15 seconds and 60 ° C. for 31 seconds after initial denaturation at 95 ° C. for 5 minutes). For measurement of real-time PCR, ABI 7900HT real-time PCR system (Applied Biosystems) was used, and the specific operation was in accordance with the attached instruction manual.

  In parallel with the PCR, TSA (Bacto-trypticase soy agar; 0.5% Papaic Digest of Casein, 0.5% Papaic Digest Soybean, 0.5% NaCl, 0.25%, 1.5% Agar) Smeared on a flat plate. Counting was performed by counting the number of colonies formed by incubating the smeared plate overnight at 37 ° C.

(result)
Fig. 3-1 and Fig. 3-2 show the results of real-time PCR at each dilution stage of each bacterial species. Fig. 3-1 shows the growth curves for each dilution stage, A for Citrobacter freundii, B for Enterobacter aerogenes, C for Escherichia coli, D for Klebsiella pneumoniae, and E for Proteus mirabilis. Show. From this result, it was revealed that the primer set for detecting Enterobacteriaceae family of the present invention can also be used in quantitative nucleic acid amplification reactions.

  Subsequently, a calibration curve was prepared based on the obtained growth curve and the number of viable bacteria obtained by plate culture. Specifically, a baseline was set between 3 and 12 cycles in which no increase in fluorescence was confirmed, and the point at which the threshold and amplification curve crossed was taken as the Threshold cycle (Ct value) on the vertical axis and obtained by plating. A calibration curve was created with the number of viable bacteria as the horizontal axis. FIGS. 4-1 and 4-2 show calibration curves for each bacterial species obtained from the growth curves of FIGS. 3-1 and 3-2, respectively. As FIG. 4-1 and FIG. 4-2 show, the calibration curve was very close between the bacterial species. Therefore, as a result of averaging these five calibration curves, Ct value = −3 (logCFU / g) +37 could be obtained. In Example 4 below, this formula was used as a calibration formula for quantification of the Enterobacteriaceae family.

<Example 4: Bacterial count comparison evaluation with culture method>
(the purpose)
The number of bacteria (colony number) in the Enterobacteriaceae group calculated from the calibration formula (Ct value = -3 (logCFU / g) +37) obtained in Example 3 should match the number of bacteria by the culture method. Is confirmed using commercially available food.

(Method)
1. Materials Food samples were purchased at a retail store in Tokyo on the day of the experiment. A total of 53 samples were used: 16 samples of salads, 11 samples of vegetables, 16 samples of meat, and 10 samples of fresh seafood (see Table 2).

  First, 25 g from each food sample was aseptically weighed and homogenized at 230 rpm for 60 seconds in a 225 mL BPW medium using Stomacker 400-T (Seward). Thereafter, the mixture was appropriately diluted with 9 mL BPW medium according to the food.

2. Enterobacteriaceae group quantification method by culture method Detection and quantification of Enterobacteriaceae group by culture method were performed based on ISO21528-2 which is the official method of Enterobacteriaceae group detection method.

  Take 1 mL of the homogenized sample diluted suspension in a sterile petri dish, and cool it to 47 ° C. VRBG medium (0.3% Yeast extract, 0.7% Pepton, 0.5% NaCl, 0.15) About 10 mL of Bile salts No. 3, 1.0% Glucose, 0.003% Neutral red, 0.0002% Crystal violet, 1.2% Agar) was added, and the mixture was mixed. After solidifying the VRBG medium, about 15 mL of new VRBG medium was further layered and solidified. After culturing at 37 ° C. for 24 hours, the number of colonies was counted using dark red and purple colonies as typical colonies of the Enterobacteriaceae family.

3. Real-time PCR method (a) DNA extraction by enterobacteriaceae group detection method of the present invention 1 mL of the homogenized sample diluted suspension was centrifuged at 15,000 G for 5 minutes at 4 ° C. to collect the cells. DNA extraction from the cells was performed using NucleoSpin Tissue (MACHEREY-NAGEL) according to the attached protocol. The prepared DNA was dissolved in 50 μL of Elution Buffer.

(B) Detection by real-time PCR The reaction solution composition and reaction conditions for real-time PCR were performed according to the method described in Example 3. The baseline was set between 3 and 12 cycles when no increase in fluorescence was observed. The point where the threshold and the amplification curve intersect is the threshold cycle (Ct value), and using the calibration formula (Ct value = -3 (logCFU / g) +37) obtained in Example 3, enterobacteriaceae in the food The number was estimated.

(result)
The results are shown in Table 2 and FIG. FIG. 5 is a graph of Table 2. The vertical axis represents the results of the culture method, and the horizontal axis represents the results of the real-time PCR method using the enterobacteriaceae family quantification method of the present invention.

  As shown in Table 2, Enterobacteriaceae obtained by each of the enterobacteriaceae group detection method (A: realtime PCR count) and culture method (B: VRBG Count) of the present invention using real-time PCR. From the results of the number of fungal groups, the number of bacteria by both methods was a difference of about ± 1 order (AB in the table). Also from FIG. 5, the plot of the number of bacteria obtained by the culture method and the number of bacteria obtained by the real-time PCR method is almost located on a diagonal line (shown by a broken line) indicating that the number of both bacteria is equal. I understand that. As a result, the number of bacteria by the culture method and the real-time PCR method are almost the same, and the real-time PCR method by the enterobacteriaceae group quantification method of the present invention can be used for quantification of enterobacteriaceae group in foods. Proven.

  Although there were few bacteria detected by the culture method, there were spots (indicated by arrows) that were slightly off the diagonal line due to the detection by the real-time PCR method. The real-time PCR method of the present invention is an error due to the fact that it can be detected if it is a bacterium that is shortly after death. As described above, in the quantification method by the real-time PCR method, new dead bacteria can be added to the quantification result, but as described above, the error range is extremely small and does not greatly affect the result of the quantification method of the present invention. .

Claims (13)

Consists of nucleotides comprising a base sequence of 17 or more bases in the base sequence shown in SEQ ID NO: 1 or a base sequence in which 1 to 3 bases are added, deleted or substituted in the base sequence shown in SEQ ID NO: 1. And a reverse primer comprising a nucleotide sequence comprising a forward primer and a nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence having 1 to 3 bases added, deleted or substituted in the nucleotide sequence represented by SEQ ID NO: 2 Primer set for detecting Enterobacteriaceae family consisting of primers.   The primer set for detecting Enterobacteriaceae family according to claim 1, wherein the reverse primer comprises a nucleotide comprising the base sequence represented by SEQ ID NO: 3.   The forward primer is composed of a nucleotide sequence comprising the nucleotide sequence represented by SEQ ID NO: 4, or a nucleotide sequence comprising 1 to 3 bases added, deleted or substituted in the nucleotide sequence represented by SEQ ID NO: 4. A primer set for detecting Enterobacteriaceae family according to 1 or 2.   The primer set for detecting Enterobacteriaceae family according to claim 3, wherein the forward primer is composed of a nucleotide comprising the base sequence represented by SEQ ID NO: 5.   The primer set for enterobacteriaceae group detection according to any one of claims 1 to 4, wherein the forward primer and the reverse primer are composed of nucleotides of 50 bases or less.   The enterobacteriaceae group detection kit provided with the primer set for enterobacteriaceae group detection as described in any one of Claims 1-5. An enterobacteriaceae group detection method comprising:
An extraction process for extracting nucleic acids from the sample;
An amplification step of performing a nucleic acid amplification reaction using the nucleic acid extracted in the nucleic acid extraction step as a template using the enterobacteriaceae family primer set for detection according to any one of claims 1 to 5,
The said method including the determination process of determining the positive or negative of the enterobacteriaceae group in the said sample based on the amount of amplification products after the said amplification process.   The method for detecting Enterobacteriaceae group according to claim 7, wherein a quantitative nucleic acid amplification method is used in the amplification step.   In the determination step, when the amount of the amplification product after the amplification step is statistically significantly larger than the amount of the amplification product of the control sample not including the Enterobacteriaceae group, the sample is in the Enterobacteriaceae group. The method for detecting Enterobacteriaceae group according to claim 7 or 8, wherein the method is determined to be positive.   The method for detecting Enterobacteriaceae family according to any one of claims 7 to 9, wherein the sample is food. Enterobacteriaceae group quantification method,
An extraction process for extracting nucleic acids from the sample;
A quantitative amplification step of performing a quantitative nucleic acid amplification reaction using the nucleic acid extracted in the nucleic acid extraction step as a template using the enterobacteriaceae family primer set for detection according to any one of claims 1 to 5,

Including the measurement step of quantitatively measuring the Enterobacteriaceae group in the sample by substituting the Ct value obtained in the quantitative amplification step into a calibration formula,
The calibration formula is a quantitative nucleic acid amplification reaction using a primer set for detecting Enterobacteriaceae family according to any one of claims 1 to 5 using a nucleic acid extracted from a serially diluted sample of a known number of bacteria as a template. The method as described above, which is a formula derived on the basis of the amount of amplification product of each serially diluted sample.   The enterobacteriaceae group quantification method according to claim 11, wherein the calibration formula is Ct value = -3 (logCFU / g) +37).   The method for quantifying Enterobacteriaceae family according to claim 11 or 12, wherein the sample is food.

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