WO2015190107A1 - Method for detecting vibrio parahaemolyticus, and carrier for detecting vibrio parahaemolyticus - Google Patents

Method for detecting vibrio parahaemolyticus, and carrier for detecting vibrio parahaemolyticus Download PDF

Info

Publication number
WO2015190107A1
WO2015190107A1 PCT/JP2015/002932 JP2015002932W WO2015190107A1 WO 2015190107 A1 WO2015190107 A1 WO 2015190107A1 JP 2015002932 W JP2015002932 W JP 2015002932W WO 2015190107 A1 WO2015190107 A1 WO 2015190107A1
Authority
WO
WIPO (PCT)
Prior art keywords
vibrio parahemolyticus
seq
probe
gene
vibrio
Prior art date
Application number
PCT/JP2015/002932
Other languages
French (fr)
Japanese (ja)
Inventor
和輝 中島
隆明 山崎
聡史 古川
Original Assignee
東洋製罐グループホールディングス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 東洋製罐グループホールディングス株式会社 filed Critical 東洋製罐グループホールディングス株式会社
Publication of WO2015190107A1 publication Critical patent/WO2015190107A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a technique for detecting microorganisms such as food poisoning bacteria, and more particularly to a method for detecting Vibrio parahemolyticus and a carrier for detecting Vibrio parahemolyticus.
  • amplification target regions amplification target gene region, target
  • PCR polymerase chain reaction
  • a DNA fragment in the gene region is amplified and the size of the amplified product is analyzed by electrophoresis.
  • a DNA containing the gene by PCR using a primer capable of amplifying a heat-resistant hemolytic toxin gene (tdh) possessed by pathogenic Vibrio parahemolyticus.
  • Pathogenic Vibrio parahemolyticus is detected using a DNA chip on which a fragment is amplified and a probe that binds complementarily to the amplified product is immobilized.
  • toxin genes of pathogenic Vibrio parahemolyticus there are various types of toxin genes of pathogenic Vibrio parahemolyticus, and it is preferable to identify them and make them detectable.
  • the present invention has been made in view of the above circumstances, and simultaneously and highly specifically detects Vibrio parahemolyticus including those having no pathogenicity and pathogenic Vibrio parahemolyticus causing food poisoning. It is an object of the present invention to provide a method for detecting Vibrio parahemolyticus that can be performed by sex, and a carrier for detecting Vibrio parahemolyticus.
  • the method for detecting Vibrio parahemolyticus comprises vibrio parahaemolyticus and vibrio parahemolyticus (including those that are pathogenic) simultaneously.
  • a method for detecting parahemolyticus wherein a genomic DNA extracted from a sample is used as a template and a DNA fragment containing a vibrio parahemolyticus virulence expression regulatory gene (toxR) and a thermostable hemolysin gene (tdh ) And a DNA fragment containing a thermostable hemolysin-like toxin gene (trh) are simultaneously amplified by multiplex PCR, and the presence or absence of each amplification product is detected, thereby causing pathogenic Vibrio in the sample.
  • ToxR vibrio parahemolyticus virulence expression regulatory gene
  • tdh thermostable hemolysin gene
  • trh thermostable hemolysin-like toxin gene
  • the carrier for detecting Vibrio parahemolyticus of the present invention is Vibrio parahemolyticus for simultaneously detecting pathogenic Vibrio parahemolyticus and Vibrio parahemolyticus (including pathogenic ones).
  • a scab detection carrier comprising at least one of the base sequences shown in SEQ ID NOs: 7 to 10 selected from a pathogenic expression regulatory gene (toxR) of Vibrio parahemolyticus, At least one probe consisting of the base sequences shown in SEQ ID NOs: 11 to 15 selected from the heat-resistant hemolytic toxin gene (tdh) of the residue, and the heat-resistant hemolytic toxin-like toxin type 1 gene (trh1) of Vibrio parahemolyticus At least one probe consisting of the nucleotide sequence shown in SEQ ID NOs: 16 to 19 and Vibrio parahaemo At least one probe consisting Tikasu heat hemolysin similar toxin type 2 gene nucleotide sequence shown in SEQ ID NO: 20 or
  • ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to detect simultaneously Vibrio parahemolyticus including what does not have pathogenicity, and pathogenic Vibrio parahemolyticus causing food poisoning with high specificity. .
  • thermostable hemolysin toxin-like toxin gene (trh) region in the method for detecting Vibrio parahemolyticus according to an embodiment of the present invention, and a vibrio according to an embodiment of the present invention
  • a probe selected from the thermostable hemolysin-like toxin type 1 gene (trh1) and a probe selected from the thermostable hemolysin-like toxin type 2 gene (trh2) in the carrier for detection of parahemolyticus (trh2) It is a figure which shows the detection result (fluorescence intensity) of Vibrio parahemolyticus using a probe.
  • the method for detecting Vibrio parahemolyticus according to the present embodiment is a method of vibrio parahemolyticus that simultaneously detects pathogenic Vibrio parahemolyticus and Vibrio parahemolyticus (including pathogenic ones).
  • a detection method using a genomic DNA extracted from a sample as a template, a DNA fragment containing a vibrio parahemolyticus pathogenic expression regulatory gene (toxR), a DNA fragment containing a thermostable hemolysin gene (tdh), , A DNA fragment containing a thermostable hemolysin-like toxin gene (trh), and simultaneously amplified by multiplex PCR, and detecting the presence or absence of each amplified product, thereby detecting pathogenic Vibrio parahemolyticus in the sample. The presence or absence and the presence or absence of Vibrio parahemolyticus are detected simultaneously.
  • toxR vibrio parahemolyticus pathogenic expression regulatory gene
  • tdh DNA fragment containing a thermostable hemolysin gene
  • trh thermostable hemolysin-like toxin gene
  • Vibrio parahaemolyticus is a Gram-negative and halophilic gonococci.
  • pathogenic Vibrio parahemolyticus Vibrio parahaemolyticus
  • non-pathogenic Vibrio parahemolyticus exists.
  • a pathogenic expression regulatory gene (toxR) commonly held in the genomic DNA of Vibrio parahemolyticus is amplified by PCR using the PCR method.
  • ToxR pathogenic expression regulatory gene
  • pathogenic Vibrio parahemolyticus includes a gene having a heat-resistant hemolytic toxin gene (tdh) and / or a heat-resistant hemolytic toxin-like toxin gene (trh) as a gene that produces a toxin.
  • tdh heat-resistant hemolytic toxin gene
  • trh heat-resistant hemolytic toxin-like toxin gene
  • FIG. 1 there are strains in which the state of possession of these toxin genes is known.
  • the heat-resistant hemolytic toxin-like toxin gene (trh) is classified into a heat-resistant hemolytic toxin-like toxin type 1 gene (trh1) and a heat-resistant hemolytic toxin-like toxin type 2 gene (trh2).
  • these strains are used to simultaneously detect three genes, toxR, tdh, and trh, and to further distinguish between trh1 and trh2.
  • the pathogenic expression regulation gene toxR
  • the heat-resistant hemolytic toxin gene tdh
  • the heat-resistant hemolytic toxin-like toxin gene trh
  • Vibrio parahemolyticus for Vibrio parahemolyticus, two types of indicators, hygiene indicator bacteria and food poisoning harmful bacteria, which could not be achieved by the conventional method, are obtained. It is possible to identify at the same time.
  • a primer set for amplifying the pathogenic expression regulatory gene (toxR) region of Vibrio parahemolyticus by PCR It is preferable to use a primer consisting of a base sequence shown in SEQ ID NO: 1 (F: forward primer) and a primer consisting of a base sequence shown in SEQ ID NO: 2 (R: reverse primer).
  • a primer set for amplifying the heat-resistant hemolytic venom gene (tdh) region of pathogenic Vibrio parahemolyticus by PCR
  • a primer (F: It is preferable to use a primer composed of a forward primer) and a primer (R: reverse primer) comprising the base sequence shown in SEQ ID NO: 4.
  • a primer set for amplifying the heat-resistant hemolytic toxin-like toxin gene (trh) region of pathogenic Vibrio parahaemolyticus by PCR
  • a primer comprising the nucleotide sequence shown in SEQ ID NO: 5 (trh primer set) F: a forward primer) and a primer composed of the base sequence shown in SEQ ID NO: 6 (R: reverse primer) are preferably used.
  • the PCR reaction solution contains the above-described toxR primer set, tdh primer set, and trh primer set, and other components are general ones. Can be used. Specifically, a buffer solution, a nucleic acid synthesis substrate, a nucleic acid synthesizing enzyme such as Ex Taq, a labeling component such as Cy5, a sample DNA, and water containing water can be used. Then, a part of the genomic DNA in the sample is amplified by a nucleic acid amplification device such as a thermal cycler using such a PCR reaction solution. That is, when genomic DNA having a region to be amplified by the primer set is present in the sample, the region to be amplified is amplified.
  • Primers used in the method for detecting Vibrio parahemolyticus are not limited to the above base sequences, and one or several bases are deleted, substituted, or added in each base sequence. Things can be used. Moreover, what consists of a nucleic acid fragment which can be hybridized on stringent conditions with respect to the nucleic acid fragment which consists of a base sequence complementary to each base sequence can also be used.
  • Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed.
  • a DNA having high homology (with a homology of 90% or more, preferably 95% or more) with respect to the DNA comprising the sequences represented by SEQ ID NOs: 1 to 6 is determined from the sequences represented by SEQ ID NOs: 1 to 6.
  • the conditions for hybridizing with DNA consisting of a base sequence complementary to the DNA to be obtained can be mentioned. Usually, it means a case where hybridization occurs at a temperature about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the melting temperature (Tm) of the complete hybrid.
  • Tm melting temperature
  • Electrophoresis can be performed by a general method such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, or microchip electrophoresis.
  • the PCR amplification product is dropped on the Vibrio parahemolyticus detection carrier (DNA chip) according to this embodiment, and the label of the amplification product hybridized with the probe immobilized on the carrier is detected. It is also preferable to detect the presence or absence of Vibrio parahemolyticus and pathogenic Vibrio parahemolyticus in the sample.
  • the detection of the label can be performed by using a general label detection device such as a fluorescence scanning device, for example, by measuring the fluorescence intensity of the amplification product using BIOSHOT (R) of Toyo Kohan Co., Ltd. Can do.
  • BIOSHOT BIOSHOT
  • the label is not limited to fluorescence, and other labels may be used.
  • the carrier for detecting Vibrio parahemolyticus is represented by SEQ ID NOs: 7 to 10 as probes (toxR probes) selected from the pathogenic expression regulatory gene (toxR) region of Vibrio parahemolyticus. It is preferable that at least one probe consisting of a base sequence is immobilized.
  • the Vibrio parahemolyticus detection carrier according to this embodiment is SEQ ID NO: 11 as a probe (tdh probe) selected from the heat-resistant hemolytic toxin gene (tdh) region of pathogenic Vibrio parahemolyticus. It is preferable that at least one of the probes having the base sequences shown in .about.15 is immobilized.
  • the carrier for detecting Vibrio parahemolyticus is selected from the thermostable hemolysin-like toxin type 1 gene in the thermostable hemolysin-like toxin gene (trh) region of pathogenic Vibrio parahemolyticus It is preferable that at least one of the base sequences shown in SEQ ID NOs: 16 to 19 is immobilized as the probe (trh1 probe), and the heat-resistant hemolytic toxin-like toxin 2 in the same gene (trh) region As a probe selected from a type gene (trh2 probe), it is preferable to fix at least one of the probes consisting of the nucleotide sequence shown in SEQ ID NO: 20 or 21.
  • Vibrio parahemolyticus detection carrier By configuring the Vibrio parahemolyticus detection carrier according to the present embodiment in such a configuration, together with Vibrio parahemolyticus (including pathogenic Vibrio parahemolyticus), pathogenic Vibrio parahaemolyticus It becomes possible to specifically detect lyticus simultaneously. It is also possible to identify and detect whether or not a pathogenic Vibrio parahaemolyticus has a heat-resistant hemolytic toxin gene (tdh) and a heat-resistant hemolytic toxin-like toxin gene (trh).
  • tdh heat-resistant hemolytic toxin gene
  • trh heat-resistant hemolytic toxin-like toxin gene
  • thermostable hemolysin-like toxin type 1 gene and the thermostable hemolysin-like toxin type 2 gene are not distinguished from each other by the size of the amplification product using a dedicated primer, but using the same primer. It is possible to reduce the number of primers in multiplex PCR by making each identifiable using a dedicated probe with high specificity after obtaining amplification products, which can improve accuracy and reduce costs. It has become.
  • the probe used in the Vibrio parahemolyticus detection carrier according to the present embodiment is not limited to the above base sequence, and one or several bases are deleted, substituted or added in each base sequence. Things can be used. Moreover, what consists of a nucleic acid fragment which can be hybridized on stringent conditions with respect to the nucleic acid fragment which consists of a base sequence complementary to each base sequence can also be used. Furthermore, probes having a base sequence complementary to these probes can also be used.
  • Vibrio parahemolyticus detection carrier specifically, a predetermined buffer solution is mixed with the PCR amplification product and dropped onto the carrier. Next, the carrier is allowed to stand at 45 ° C. for 1 hour, and then PCR amplification products and the like that have not been hybridized with a predetermined buffer are washed away from the carrier. Then, by detecting the label by applying the carrier to a label detection apparatus, pathogenic Vibrio parahemolyticus and Vibrio parahemolyticus (including pathogenic Vibrio parahemolyticus) exist in the sample. It can be detected at the same time.
  • the vibrio parahemolyticus including those having no pathogenicity It is possible to detect pathogenic Vibrio parahaemolyticus that causes food poisoning at the same time and with high specificity.
  • ⁇ Test 1 Verification of simultaneous amplification using three types of primer sets> DNA was extracted from these Vibrio parahemolyticus by a conventional method using seven types of Vibrio parahemolyticus strains known in FIG. These are all derived from the Osaka University Institute for Microbial Diseases. Next, using the toxR primer set, tdh primer set, and trh primer set shown in FIG. 2, for each strain, the respective amplification target regions can be simultaneously amplified by multiplex PCR, and whether or not the obtained amplification product can be detected. I verified. Specifically, it was performed as follows.
  • Vibrio parahemolyticus is abbreviated as Vibrio.
  • a thermal cycler ep gradient (Eppendorf Co., Ltd.) was used for gene amplification by PCR.
  • the reaction conditions are as follows. (1) 95 ° C for 2 minutes (2) 95 ° C for 10 seconds (DNA strand dissociation step) (3) 68 ° C. 30 seconds (annealing process) (4) 72 ° C. for 30 seconds (DNA synthesis step) (5) 40 cycles of 72 ° C 2 minutes (2) to (4)
  • the PCR reaction solution containing the PCR amplification product thus obtained was subjected to a microchip electrophoresis apparatus MultiNA (Shimadzu Corporation), and size analysis of the PCR amplification product was performed.
  • the result is shown in FIG.
  • amplification products (estimated size 319 bp) by the toxR primer set were detected for all seven types of strains.
  • the amplification product (estimated size 195 bp) by the tdh primer set is detected only for the strains (4), (5) and (6) having the tdh gene, and the amplification product (estimated size 274 bp) by the trh primer set is trh Only the strains (1), (2), (3) and (6) having the gene were detected.
  • each of these regions could be specifically and simultaneously detected by the toxR primer set, tdh primer set, and trh primer set in the method for detecting Vibrio parahemolyticus according to the present embodiment.
  • the Vibrio parahemolyticus detection method according to this embodiment it was confirmed that Vibrio parahemolyticus and pathogenic Vibrio parahemolyticus can be detected simultaneously.
  • ⁇ Test 2 Verification of discrimination between heat-resistant hemolytic toxin-like toxin type 1 and type 2 genes (trh1, trh2)>
  • the trh gene region was amplified by PCR for each strain using 10 ng / ⁇ l of each DNA extract of strains (1), (2), and (3) prepared in Test 1 and the trh primer set.
  • a PCR reaction solution was prepared with the following composition, and the PCR reaction solution containing the PCR amplification product was obtained in the same manner as in Test 1 for other points.
  • a heat-resistant hemolytic toxin-like toxin type 1 gene detection probe consisting of the nucleotide sequences of SEQ ID NOs: 16 to 19 shown in FIG. 5 and heat-resistant hemolysis consisting of the nucleotide sequences of SEQ ID NOs: 20 to 21 are shown in advance.
  • a DNA chip on which a probe for detecting a toxin-like toxin type 2 gene (trh2 probe) was immobilized was prepared.
  • a mixture of 4 ⁇ L of the PCR reaction solution and 2 ⁇ L of the hybridization buffer solution (3 ⁇ SSC / 0.3% SDS citrate-saline-sodium dodecyl sulfate) was dropped onto the DNA chip. And reacted at 45 ° C. for 1 hour.
  • the DNA chip is immersed in a washing solution (2 ⁇ SSC / 0.2% SDS solution, 2 ⁇ SSC solution in this order) at room temperature for washing, and a cover glass is placed on the fluorescence detector Bioshot (manufactured by Toyo Kohan Co., Ltd.) ) To detect the fluorescence in the spot area of each probe.
  • the label component (Cy5) of the amplification product hybridized with the probe was excited by laser light to emit light, and the amount of light was detected by a CCD camera mounted in the detector.
  • the light intensity was replaced with an electric signal and digitized to obtain fluorescence intensity.
  • This fluorescence intensity is an intensity index in the apparatus, and has a unit, and was calculated by correcting so that the background value becomes zero. The result is shown in FIG.
  • the strains (1) and (2) As shown in the figure, for the strains (1) and (2), a high fluorescence intensity was obtained with the trh1 detection probe, and a low fluorescence intensity was obtained with the trh2 detection probe.
  • the strain (1) and the strain (2) are trh1 gene-bearing bacteria among the pathogenic Vibrio parahemolyticus trh gene-bearing bacteria.
  • the strain (3) a high fluorescence intensity was obtained with the trh2 detection probe, and a relatively low fluorescence intensity was obtained with the trh1 detection probe. From this, it can be seen that the strain (3) is a trh2 gene-bearing bacterium among the pathogenic Vibrio parahemolyticus trh gene-bearing bacterium.
  • the Vibrio parahemolyticus detection carrier according to the present embodiment, by using the trh1 detection probe and the trh2 detection probe in combination, the trh gene possessed by the pathogenic Vibrio parahemolyticus It was found that when bacteria are included in the sample to be detected, trh1 gene-bearing bacteria and trh2 gene-bearing bacteria can be discriminated, and false negative determination for the trh gene can be avoided.
  • ⁇ Test 3 Verification of simultaneous detection by DNA chip> ToxR, tdh, trh in Vibrio parahemolyticus is detected by the carrier for detecting Vibrio parahemolyticus according to this embodiment in which a probe that can bind to a DNA fragment containing toxR, tdh, tr1, and trh2 genes is immobilized. It was verified whether the three types of genes could be specifically detected simultaneously. Specifically, a DNA chip on which each probe consisting of the nucleotide sequences of SEQ ID NOs: 7 to 21 shown in FIG. 5 was immobilized was prepared. Then, using 7 types of Vibrio parahemolyticus strains shown in FIG.
  • the toxR detection probes of SEQ ID NOs: 7 to 10 showed high fluorescence intensity for all seven types of Vibrio parahemolyticus strains.
  • the tdh detection probes of SEQ ID NOs: 11 to 15 show high fluorescence intensity only for the strains (4), (5) and (6) having the tdh gene, and the strains having no tdh gene (1 ), (2), (3), (7) did not show high fluorescence intensity.
  • the trh1 detection probes of SEQ ID NOs: 16 to 19 show high fluorescence intensity only for the strains (1), (2), and (6) that possess trh1, and the strains (3) and (4) that do not possess trh1 , (5) and (7) do not show high fluorescence intensity
  • the trh2 detection probes of SEQ ID NOs: 20 to 21 show high fluorescence intensity only for the strain (3) having trh2, and possess trh2.
  • the strains (1), (2), (4), (5), (6) and (7) which did not show no high fluorescence intensity.
  • the three types of gene regions of toxR, tdh, and trh in Vibrio parahemolyticus can be specifically and simultaneously detected. all right.
  • trh if only one of trh1 and trh2 is present in the sample, it is clear that it can be identified and that false negative determination can be prevented. became.
  • the present invention is not limited to the above embodiments and examples, and various modifications can be made within the scope of the present invention.
  • the PCR reaction solution may contain other components, or the Vibrio parahemolyticus detection carrier according to the present embodiment may have other than the above. It is possible to make appropriate changes such as adding additional probes and fixing them.
  • the present invention can be suitably used for detecting Vibrio parahemolyticus in food inspection, epidemiological environmental inspection, environmental inspection, clinical test, livestock hygiene, and the like.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

To enable simultaneous and highly specific detection of Vibrio parahaemolyticus (hereinafter Vibrio) including non-virulent strains and virulent Vibrio parahaemolyticus (hereinafter virulent Vibrio) causing food poisoning. A method for simultaneously detecting virulent Vibrio and Vibrio (including virulent strains), said method comprising: using as a template a genomic DNA extracted from a sample; simultaneously amplifying a DNA fragment containing Vibrio virulence expression regulatory gene (toxR), a DNA fragment containing thermostable direct hemolysin gene (tdh) and a DNA fragment containing thermostable direct hemolysin-related hemolysin gene (trh) by multiplex PCR; and detecting the presence or absence of the respective amplification products to thereby simultaneously detect the presence or absence of virulent Vibrio and the presence or absence of Vibrio in the sample.

Description

ビブリオ・パラヘモリティカスの検出方法、及びビブリオ・パラヘモリティカス検出用担体Vibrio parahemolyticus detection method and vibrio parahemolyticus detection carrier
　本発明は、食中毒菌などの微生物を検出するための技術に関し、特にビブリオ・パラヘモリティカスの検出方法、及びビブリオ・パラヘモリティカス検出用担体に関する。 The present invention relates to a technique for detecting microorganisms such as food poisoning bacteria, and more particularly to a method for detecting Vibrio parahemolyticus and a carrier for detecting Vibrio parahemolyticus.
　近年、食品や環境などの公衆衛生の分野における衛生水準の向上により、食中毒の発生には減少傾向が見られるものの、現在でもなお日本国内で毎年２万人以上が食中毒に罹患している。この中には、病原性ビブリオ・パラヘモリティカス（腸炎ビブリオ）を原因とするものも少なからず含まれており、その発生を防止するために、食中毒菌等の検査において、食品や環境中の病原性ビブリオ・パラヘモリティカスを精度高く検出することが重要になっている。 In recent years, due to the improvement of the level of hygiene in the field of public health such as food and the environment, the incidence of food poisoning has been decreasing, but even now in Japan, more than 20,000 people suffer from food poisoning every year. This includes not only a few caused by pathogenic Vibrio parahaemolyticus (Vibrio parahaemolyticus), but in order to prevent its occurrence, in the inspection of food poisoning bacteria, etc. It is important to detect pathogenic Vibrio parahaemolyticus with high accuracy.
　病原性ビブリオ・パラヘモリティカスを検出する方法としては、例えば特許文献１に記載のように、所定のプライマーを用いて、ＰＣＲ（ポリメラーゼ連鎖反応）法により増幅対象領域（増幅対象遺伝子領域、標的遺伝子領域）のＤＮＡ断片を増幅し、その増幅産物のサイズを電気泳動法で分析することによって行う方法がある。
　また、本出願人による特許文献２に記載の発明では、病原性ビブリオ・パラヘモリティカスが有する耐熱性溶血毒遺伝子（tdh）を増幅可能なプライマーを用いて、ＰＣＲ法により当該遺伝子を含むＤＮＡ断片を増幅し、その増幅産物と相補的に結合するプローブを固定化したＤＮＡチップを用いて、病原性ビブリオ・パラヘモリティカスの検出を行っている。 As a method for detecting pathogenic Vibrio parahemolyticus, for example, as described in Patent Document 1, amplification target regions (amplification target gene region, target) using a predetermined primer and PCR (polymerase chain reaction) method There is a method in which a DNA fragment in the gene region) is amplified and the size of the amplified product is analyzed by electrophoresis.
In the invention described in Patent Document 2 by the present applicant, a DNA containing the gene by PCR using a primer capable of amplifying a heat-resistant hemolytic toxin gene (tdh) possessed by pathogenic Vibrio parahemolyticus. Pathogenic Vibrio parahemolyticus is detected using a DNA chip on which a fragment is amplified and a probe that binds complementarily to the amplified product is immobilized.
特表２００８－５３８０７５号公報Special table 2008-538075 gazette 国際公開第２０１１／１４２１１９号公報International Publication No. 2011/142119
　ところで、公衆衛生の分野においては、食中毒の原因となる病原性ビブリオ・パラヘモリティカスの有無の検査のみならず、衛生学的指標として、病原性に拘わらず、ビブリオ・パラヘモリティカスの有無を同時に検査できることが望ましい。
　しかしながら、上述した従来の技術では、病原性を持たないものを含むビブリオ・パラヘモリティカスを、病原性ビブリオ・パラヘモリティカスと同時に検出することはできなかった。
　また、ビブリオ・パラヘモリティカスの検査を行う実際の現場においては、検査試料に食物の遺伝子など様々なＤＮＡが混入することから、特異性に優れた検出精度が求められていた。
　さらに、病原性ビブリオ・パラヘモリティカスの毒素遺伝子には種々のものが存在しているところ、これらを識別して検出可能にすることが好ましい。
　本発明は、上記事情に鑑みなされたものであり、病原性を持たないものを含むビブリオ・パラヘモリティカスと、食中毒の原因となる病原性ビブリオ・パラヘモリティカスの検出を同時にかつ高い特異性で行うことが可能なビブリオ・パラヘモリティカスの検出方法、及びビブリオ・パラヘモリティカス検出用担体の提供を目的とする。 By the way, in the field of public health, not only the presence or absence of pathogenic Vibrio parahemolyticus causing food poisoning, but also the presence or absence of Vibrio parahemolyticus as a hygienic indicator regardless of pathogenicity It is desirable to be able to inspect simultaneously.
However, with the above-described conventional technology, it has not been possible to detect Vibrio parahemolyticus including those having no pathogenicity simultaneously with the pathogenic Vibrio parahemolyticus.
In actual sites where Vibrio parahemolyticus is tested, various DNAs such as food genes are mixed in the test sample, so that detection accuracy with excellent specificity is required.
Furthermore, there are various types of toxin genes of pathogenic Vibrio parahemolyticus, and it is preferable to identify them and make them detectable.
The present invention has been made in view of the above circumstances, and simultaneously and highly specifically detects Vibrio parahemolyticus including those having no pathogenicity and pathogenic Vibrio parahemolyticus causing food poisoning. It is an object of the present invention to provide a method for detecting Vibrio parahemolyticus that can be performed by sex, and a carrier for detecting Vibrio parahemolyticus.
　上記目的を達成するために、本発明のビブリオ・パラヘモリティカスの検出方法は、病原性ビブリオ・パラヘモリティカス及びビブリオ・パラヘモリティカス（病原性のものを含む）を同時に検出するビブリオ・パラヘモリティカスの検出方法であって、試料から抽出したゲノムＤＮＡをテンプレートとして、ビブリオ・パラヘモリティカスの病原性発現調節遺伝子（toxR）を含むＤＮＡ断片と、耐熱性溶血毒遺伝子（tdh）を含むＤＮＡ断片と、耐熱性溶血毒類似毒素遺伝子（trh）を含むＤＮＡ断片と、をマルチプレックスＰＣＲにより同時に増幅させ、それぞれの増幅産物の有無を検出することによって、試料中における病原性ビブリオ・パラヘモリティカスの有無及びビブリオ・パラヘモリティカスの有無を同時に検出する方法としてある。 In order to achieve the above object, the method for detecting Vibrio parahemolyticus according to the present invention comprises vibrio parahaemolyticus and vibrio parahemolyticus (including those that are pathogenic) simultaneously. A method for detecting parahemolyticus, wherein a genomic DNA extracted from a sample is used as a template and a DNA fragment containing a vibrio parahemolyticus virulence expression regulatory gene (toxR) and a thermostable hemolysin gene (tdh ) And a DNA fragment containing a thermostable hemolysin-like toxin gene (trh) are simultaneously amplified by multiplex PCR, and the presence or absence of each amplification product is detected, thereby causing pathogenic Vibrio in the sample.・ As a method to detect the presence or absence of parahemolyticus and the presence or absence of vibrio or parahemolyticus at the same time. The
　また、本発明のビブリオ・パラヘモリティカス検出用担体は、病原性ビブリオ・パラヘモリティカス及びビブリオ・パラヘモリティカス（病原性のものを含む）を同時に検出するためのビブリオ・パラヘモリティカス検出用担体であって、ビブリオ・パラヘモリティカスの病原性発現調節遺伝子（toxR）から選択された配列番号７～１０に示す塩基配列からなる少なくともいずれかのプローブと、ビブリオ・パラヘモリティカスの耐熱性溶血毒遺伝子（tdh）から選択された配列番号１１～１５に示す塩基配列からなる少なくともいずれかのプローブと、ビブリオ・パラヘモリティカスの耐熱性溶血毒類似毒素1型遺伝子（trh1）から選択された配列番号１６～１９に示す塩基配列からなる少なくともいずれかのプローブと、ビブリオ・パラヘモリティカスの耐熱性溶血毒類似毒素２型遺伝子（trh2）から選択された配列番号２０又は２１に示す塩基配列からなる少なくともいずれかのプローブと、を固定化した構成としてある。 Further, the carrier for detecting Vibrio parahemolyticus of the present invention is Vibrio parahemolyticus for simultaneously detecting pathogenic Vibrio parahemolyticus and Vibrio parahemolyticus (including pathogenic ones). A scab detection carrier comprising at least one of the base sequences shown in SEQ ID NOs: 7 to 10 selected from a pathogenic expression regulatory gene (toxR) of Vibrio parahemolyticus, At least one probe consisting of the base sequences shown in SEQ ID NOs: 11 to 15 selected from the heat-resistant hemolytic toxin gene (tdh) of the residue, and the heat-resistant hemolytic toxin-like toxin type 1 gene (trh1) of Vibrio parahemolyticus At least one probe consisting of the nucleotide sequence shown in SEQ ID NOs: 16 to 19 and Vibrio parahaemo At least one probe consisting Tikasu heat hemolysin similar toxin type 2 gene nucleotide sequence shown in SEQ ID NO: 20 or 21 selected from (trh2), it is constituted with immobilized.
　本発明によれば、病原性を持たないものを含むビブリオ・パラヘモリティカスと、食中毒の原因となる病原性ビブリオ・パラヘモリティカスの検出を同時にかつ高い特異性で行うことが可能となる。 ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to detect simultaneously Vibrio parahemolyticus including what does not have pathogenicity, and pathogenic Vibrio parahemolyticus causing food poisoning with high specificity. .
本発明の実施形態に係るビブリオ・パラヘモリティカスの検出方法及びビブリオ・パラヘモリティカス検出用担体の試験１～３で用いた菌株の毒素遺伝子保有状況を示す図である。It is a figure which shows the detection method of the vibrio parahemolyticus which concerns on embodiment of this invention, and the toxin gene possession situation of the strain used by the tests 1-3 of the support | carrier for Vibrio parahemolyticus detection. 本発明の実施形態に係るビブリオ・パラヘモリティカスの検出方法において用いられる３種類のプライマーセットの塩基配列（プライマー配列）を示す図である。It is a figure which shows the base sequence (primer sequence) of three types of primer sets used in the detection method of Vibrio parahemolyticus which concerns on embodiment of this invention. 本発明の実施形態に係るビブリオ・パラヘモリティカスの検出方法におけるマルチプレックスＰＣＲによる増幅産物の電気泳動の結果を示す図である。It is a figure which shows the result of the electrophoresis of the amplification product by multiplex PCR in the detection method of Vibrio parahemolyticus which concerns on embodiment of this invention. 本発明の実施形態に係るビブリオ・パラヘモリティカスの検出方法における耐熱性溶血毒類似毒素遺伝子（trh）領域を増幅するためのプライマーセット（trhプライマーセット）と、本発明の実施形態に係るビブリオ・パラヘモリティカス検出用担体における耐熱性溶血毒類似毒素１型遺伝子（trh1）から選択されたプローブ（trh1プローブ）及び耐熱性溶血毒類似毒素２型遺伝子（trh2）から選択されたプローブ（trh2プローブ）を用いたビブリオ・パラヘモリティカスの検出結果（蛍光強度）を示す図である。A primer set (trh primer set) for amplifying a thermostable hemolysin toxin-like toxin gene (trh) region in the method for detecting Vibrio parahemolyticus according to an embodiment of the present invention, and a vibrio according to an embodiment of the present invention A probe selected from the thermostable hemolysin-like toxin type 1 gene (trh1) and a probe selected from the thermostable hemolysin-like toxin type 2 gene (trh2) in the carrier for detection of parahemolyticus (trh2) It is a figure which shows the detection result (fluorescence intensity) of Vibrio parahemolyticus using a probe. 本発明の実施形態に係るビブリオ・パラヘモリティカス検出用担体におけるプローブの塩基配列（プローブ配列）を示す図である。It is a figure which shows the base sequence (probe sequence) of the probe in the support | carrier for Vibrio parahemolyticus detection which concerns on embodiment of this invention. 本発明の実施形態に係るビブリオ・パラヘモリティカスの検出方法及びビブリオ・パラヘモリティカス検出用担体における各プローブによるビブリオ・パラヘモリティカスの検出結果（蛍光強度）を示す図である。It is a figure which shows the detection result (fluorescence intensity) of Vibrio parahemolyticus by each probe in the detection method of Vibrio parahemolyticus which concerns on embodiment of this invention, and the support | carrier for Vibrio parahemolyticus detection.
　以下、本発明の実施形態に係るビブリオ・パラヘモリティカスの検出方法、及びビブリオ・パラヘモリティカス検出用担体について、詳細に説明する。
　本実施形態に係るビブリオ・パラヘモリティカスの検出方法は、病原性ビブリオ・パラヘモリティカス及びビブリオ・パラヘモリティカス（病原性のものを含む）を同時に検出するビブリオ・パラヘモリティカスの検出方法であって、試料から抽出したゲノムＤＮＡをテンプレートとして、ビブリオ・パラヘモリティカスの病原性発現調節遺伝子（toxR）を含むＤＮＡ断片と、耐熱性溶血毒遺伝子（tdh）を含むＤＮＡ断片と、耐熱性溶血毒類似毒素遺伝子（trh）を含むＤＮＡ断片と、をマルチプレックスＰＣＲにより同時に増幅させ、それぞれの増幅産物の有無を検出することによって、試料中における病原性ビブリオ・パラヘモリティカスの有無及びビブリオ・パラヘモリティカスの有無を同時に検出することを特徴とする。 Hereinafter, a method for detecting Vibrio parahemolyticus and a carrier for detecting Vibrio parahemolyticus according to an embodiment of the present invention will be described in detail.
The method for detecting Vibrio parahemolyticus according to the present embodiment is a method of vibrio parahemolyticus that simultaneously detects pathogenic Vibrio parahemolyticus and Vibrio parahemolyticus (including pathogenic ones). A detection method, using a genomic DNA extracted from a sample as a template, a DNA fragment containing a vibrio parahemolyticus pathogenic expression regulatory gene (toxR), a DNA fragment containing a thermostable hemolysin gene (tdh), , A DNA fragment containing a thermostable hemolysin-like toxin gene (trh), and simultaneously amplified by multiplex PCR, and detecting the presence or absence of each amplified product, thereby detecting pathogenic Vibrio parahemolyticus in the sample. The presence or absence and the presence or absence of Vibrio parahemolyticus are detected simultaneously.
　ビブリオ・パラヘモリティカス（Vibrio　parahaemolyticus）は、グラム陰性で好塩性の桿菌である。主として海水中に生息し、ヒトに感染すると、食中毒を発症させる病原性ビブリオ・パラヘモリティカス（腸炎ビブリオ）が存在する。一方、非病原性ビブリオ・パラヘモリティカスも存在している。しかしながら、公衆衛生の分野においては、衛生学的指標として、このような病原性の有無に拘わらず、ビブリオ・パラヘモリティカスを幅広く検出可能にすることが好ましい。
　そこで、本実施形態に係るビブリオ・パラヘモリティカスの検出方法では、ビブリオ・パラヘモリティカスのゲノムＤＮＡに共通して保有される病原性発現調節遺伝子（toxR）を増幅対象領域としてＰＣＲ法により増幅し、その増幅産物を検出することによって、試料中における、病原性のあるものとないものとを含むビブリオ・パラヘモリティカスの有無を検出可能にしている。 Vibrio parahaemolyticus is a Gram-negative and halophilic gonococci. There are pathogenic Vibrio parahemolyticus (Vibrio parahaemolyticus) that inhabits mainly in seawater and causes food poisoning when it infects humans. On the other hand, non-pathogenic Vibrio parahemolyticus exists. However, in the field of public health, it is preferable to make Vibrio parahemolyticus widely detectable as a hygienic index regardless of the presence or absence of such pathogenicity.
Therefore, in the method for detecting Vibrio parahemolyticus according to the present embodiment, a pathogenic expression regulatory gene (toxR) commonly held in the genomic DNA of Vibrio parahemolyticus is amplified by PCR using the PCR method. By amplifying and detecting the amplified product, it is possible to detect the presence or absence of Vibrio parahemolyticus in the sample including those that are pathogenic and those that are not pathogenic.
　また、病原性ビブリオ・パラヘモリティカスには、毒素を産生する遺伝子として、耐熱性溶血毒遺伝子（tdh）及び／又は耐熱性溶血毒類似毒素遺伝子（trh）を有するものが存在する。
　具体的には、例えば図１に示すように、これらの毒素遺伝子の保有状況が分っている菌株が存在している。さらに、耐熱性溶血毒類似毒素遺伝子（trh）は、耐熱性溶血毒類似毒素１型遺伝子（trh1）と耐熱性溶血毒類似毒素２型遺伝子（trh2）に区別される。後述する実施例では、これらの菌株を使用して、toxR、tdh、及びtrhの３つの遺伝子を同時に検出すると共に、さらにtrh1とtrh2とを識別可能にしている。 In addition, pathogenic Vibrio parahemolyticus includes a gene having a heat-resistant hemolytic toxin gene (tdh) and / or a heat-resistant hemolytic toxin-like toxin gene (trh) as a gene that produces a toxin.
Specifically, for example, as shown in FIG. 1, there are strains in which the state of possession of these toxin genes is known. Furthermore, the heat-resistant hemolytic toxin-like toxin gene (trh) is classified into a heat-resistant hemolytic toxin-like toxin type 1 gene (trh1) and a heat-resistant hemolytic toxin-like toxin type 2 gene (trh2). In the examples described later, these strains are used to simultaneously detect three genes, toxR, tdh, and trh, and to further distinguish between trh1 and trh2.
　本実施形態に係るビブリオ・パラヘモリティカスの検出方法では、上記の病原性発現調節遺伝子（toxR）と、耐熱性溶血毒遺伝子（tdh）と、耐熱性溶血毒類似毒素遺伝子（trh）とを増幅対象領域としてＰＣＲ法により同時に増幅し、その増幅産物を検出することによって、試料中における、病原性ビブリオ・パラヘモリティカスを含むビブリオ・パラヘモリティカスの有無と、病原性ビブリオ・パラヘモリティカスの有無とを同時に検出可能にしている。
　これによって、本実施形態に係るビブリオ・パラヘモリティカスの検出方法によれば、ビブリオ・パラヘモリティカスについて、従来の方法ではなし得なかった衛生指標菌及び食中毒危害菌の２種類の指標を同時に識別することが可能となっている。 In the detection method of Vibrio parahemolyticus according to the present embodiment, the pathogenic expression regulation gene (toxR), the heat-resistant hemolytic toxin gene (tdh), and the heat-resistant hemolytic toxin-like toxin gene (trh) By simultaneously amplifying by PCR method as a region to be amplified and detecting the amplified product, the presence or absence of Vibrio parahemolyticus including pathogenic Vibrio parahemolyticus in the sample and pathogenic Vibrio parahaemolyticus are detected. It is possible to detect the presence or absence of lyticus at the same time.
Thus, according to the method for detecting Vibrio parahemolyticus according to the present embodiment, for Vibrio parahemolyticus, two types of indicators, hygiene indicator bacteria and food poisoning harmful bacteria, which could not be achieved by the conventional method, are obtained. It is possible to identify at the same time.
　本実施形態に係るビブリオ・パラヘモリティカスの検出方法において、図２に示すように、ビブリオ・パラヘモリティカスの病原性発現調節遺伝子（toxR）領域をＰＣＲ法により増幅するためのプライマーセット（toxRプライマーセット）として、配列番号１に示す塩基配列からなるプライマー（Ｆ：フォワードプライマー）と、配列番号２に示す塩基配列からなるプライマー（Ｒ：リバースプライマー）とからなるものを用いることが好ましい。
　また、病原性ビブリオ・パラヘモリティカスの耐熱性溶血毒遺伝子（tdh）領域をＰＣＲ法により増幅するためのプライマーセット（tdhプライマーセット）として、配列番号３に示す塩基配列からなるプライマー（Ｆ：フォワードプライマー）と、配列番号４に示す塩基配列からなるプライマー（Ｒ：リバースプライマー）とからなるものを用いることが好ましい。
　また、病原性ビブリオ・パラヘモリティカスの耐熱性溶血毒類似毒素遺伝子（trh）領域をＰＣＲ法により増幅するためのプライマーセット（trhプライマーセット）として、配列番号５に示す塩基配列からなるプライマー（Ｆ：フォワードプライマー）と、配列番号６に示す塩基配列からなるプライマー（Ｒ：リバースプライマー）とからなるものを用いることが好ましい。 In the method for detecting Vibrio parahemolyticus according to the present embodiment, as shown in FIG. 2, a primer set for amplifying the pathogenic expression regulatory gene (toxR) region of Vibrio parahemolyticus by PCR ( It is preferable to use a primer consisting of a base sequence shown in SEQ ID NO: 1 (F: forward primer) and a primer consisting of a base sequence shown in SEQ ID NO: 2 (R: reverse primer).
In addition, as a primer set (tdh primer set) for amplifying the heat-resistant hemolytic venom gene (tdh) region of pathogenic Vibrio parahemolyticus by PCR, a primer (F: It is preferable to use a primer composed of a forward primer) and a primer (R: reverse primer) comprising the base sequence shown in SEQ ID NO: 4.
In addition, as a primer set (trh primer set) for amplifying the heat-resistant hemolytic toxin-like toxin gene (trh) region of pathogenic Vibrio parahaemolyticus by PCR, a primer comprising the nucleotide sequence shown in SEQ ID NO: 5 (trh primer set) F: a forward primer) and a primer composed of the base sequence shown in SEQ ID NO: 6 (R: reverse primer) are preferably used.
　本実施形態に係るビブリオ・パラヘモリティカスの検出方法において、ＰＣＲ反応液は、上記のtoxRプライマーセット、tdhプライマーセット、及びtrhプライマーセットを含有し、それ以外の成分には、一般的なものを用いることができる。具体的には、緩衝液、核酸合成基質、Ex　Taq等の核酸合成酵素、Cy5等の標識成分、試料のＤＮＡ、及び水を含むものなどを用いることができる。
　そして、このようなＰＣＲ反応液を用いてサーマルサイクラーなどの核酸増幅装置により、試料中のゲノムＤＮＡの一部が増幅される。すなわち、上記プライマーセットによる増幅対象領域を有するゲノムＤＮＡが試料中に存在している場合、その対象領域が増幅される。 In the method for detecting Vibrio parahemolyticus according to the present embodiment, the PCR reaction solution contains the above-described toxR primer set, tdh primer set, and trh primer set, and other components are general ones. Can be used. Specifically, a buffer solution, a nucleic acid synthesis substrate, a nucleic acid synthesizing enzyme such as Ex Taq, a labeling component such as Cy5, a sample DNA, and water containing water can be used.
Then, a part of the genomic DNA in the sample is amplified by a nucleic acid amplification device such as a thermal cycler using such a PCR reaction solution. That is, when genomic DNA having a region to be amplified by the primer set is present in the sample, the region to be amplified is amplified.
　本実施形態に係るビブリオ・パラヘモリティカスの検出方法において用いるプライマーは、上記の塩基配列に限定されるものではなく、それぞれの塩基配列において１又は数個の塩基が欠損、置換又は付加されたものを用いることができる。また、それぞれの塩基配列に対して相補的な塩基配列からなる核酸断片に対してストリンジェントな条件下でハイブリダイズできる核酸断片からなるものを用いることもできる。 Primers used in the method for detecting Vibrio parahemolyticus according to this embodiment are not limited to the above base sequences, and one or several bases are deleted, substituted, or added in each base sequence. Things can be used. Moreover, what consists of a nucleic acid fragment which can be hybridized on stringent conditions with respect to the nucleic acid fragment which consists of a base sequence complementary to each base sequence can also be used.
　ストリンジェントな条件とは、特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。例えば、配列番号１～６で表される配列からなるＤＮＡに対し高い相同性（相同性が９０％以上、好ましくは９５％以上）を有するＤＮＡが、配列番号１～６で表される配列からなるＤＮＡと相補的な塩基配列からなるＤＮＡと、ハイブリダイズする条件が挙げられる。通常、完全ハイブリッドの溶解温度（Ｔｍ）より約５℃～約３０℃、好ましくは約１０℃～約２５℃低い温度でハイブリダイゼーションが起こる場合をいう。ストリンジェントな条件については、Ｊ．Ｓａｍｂｒｏｏｋら，Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）、特に11.45節「Ｃｏｎｄｉｔｉｏｎｓ　ｆｏｒ　Ｈｙｂｒｉｄｉｚａｔｉｏｎ　ｏｆ　Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ　Ｐｒｏｂｅｓ」に記載されている条件等を使用することができる。 Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed. For example, a DNA having high homology (with a homology of 90% or more, preferably 95% or more) with respect to the DNA comprising the sequences represented by SEQ ID NOs: 1 to 6 is determined from the sequences represented by SEQ ID NOs: 1 to 6. The conditions for hybridizing with DNA consisting of a base sequence complementary to the DNA to be obtained can be mentioned. Usually, it means a case where hybridization occurs at a temperature about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the melting temperature (Tm) of the complete hybrid. For stringent conditions, see J.M. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), etc. can be used, especially in Section 11.45 “Conditions for HybridOboliprozation”.
　次に、ＰＣＲ法により得られた増幅産物（ＰＣＲ増幅産物）を用いて、例えば電気泳動を行うことにより、本実施形態における上記各プライマーセットによる増幅産物が得られているか否かを確認して、試料中におけるビブリオ・パラヘモリティカス及び病原性ビブリオ・パラヘモリティカスの有無を検出することが好ましい。電気泳動は、アガロースゲル電気泳動やポリアクリルアミドゲル電気泳動、マイクロチップ電気泳動など一般的な方法により行うことができる。 Next, using the amplification product (PCR amplification product) obtained by the PCR method, for example, by performing electrophoresis, it is confirmed whether or not the amplification product by each of the primer sets in the present embodiment is obtained. It is preferable to detect the presence or absence of Vibrio parahemolyticus and pathogenic Vibrio parahemolyticus in the sample. Electrophoresis can be performed by a general method such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, or microchip electrophoresis.
　また、ＰＣＲ増幅産物を本実施形態に係るビブリオ・パラヘモリティカス検出用担体上（ＤＮＡチップ）に滴下して、当該担体に固定化されたプローブにハイブリダイズした増幅産物の標識を検出することにより、試料中におけるビブリオ・パラヘモリティカス及び病原性ビブリオ・パラヘモリティカスの有無を検出することも好ましい。
　標識の検出は、蛍光スキャニング装置など一般的な標識検出装置を用いて行うことができ、例えば東洋鋼鈑株式会社のＢＩＯＳＨＯＴ（Ｒ）を用いて、増幅産物の蛍光強度を測定することにより行うことができる。なお、標識は、蛍光に限定されず、その他のものを用いてもよい。 Moreover, the PCR amplification product is dropped on the Vibrio parahemolyticus detection carrier (DNA chip) according to this embodiment, and the label of the amplification product hybridized with the probe immobilized on the carrier is detected. It is also preferable to detect the presence or absence of Vibrio parahemolyticus and pathogenic Vibrio parahemolyticus in the sample.
The detection of the label can be performed by using a general label detection device such as a fluorescence scanning device, for example, by measuring the fluorescence intensity of the amplification product using BIOSHOT (R) of Toyo Kohan Co., Ltd. Can do. The label is not limited to fluorescence, and other labels may be used.
　本実施形態に係るビブリオ・パラヘモリティカス検出用担体は、ビブリオ・パラヘモリティカスの病原性発現調節遺伝子（toxR）領域から選択されたプローブ（toxRプローブ）として、配列番号７～１０に示す塩基配列からなる少なくともいずれかのプローブを固定化したものとすることが好ましい。
　また、本実施形態に係るビブリオ・パラヘモリティカス検出用担体は、病原性ビブリオ・パラヘモリティカスの耐熱性溶血毒遺伝子（tdh）領域から選択されたプローブ（tdhプローブ）として、配列番号１１～１５に示す塩基配列からなる少なくともいずれかのプローブを固定化したものとすることが好ましい。
　さらに、本実施形態に係るビブリオ・パラヘモリティカス検出用担体は、病原性ビブリオ・パラヘモリティカスの耐熱性溶血毒類似毒素遺伝子（trh）領域における耐熱性溶血毒類似毒素１型遺伝子から選択されたプローブ（trh1プローブ）として、配列番号１６～１９に示す塩基配列からなる少なくともいずれかのプローブを固定化したものとすることが好ましく、同遺伝子（trh）領域における耐熱性溶血毒類似毒素２型遺伝子から選択されたプローブ（trh2プローブ）として、配列番号２０又は２１に示す塩基配列からなる少なくともいずれかのプローブを固定化したものとすることが好ましい。 The carrier for detecting Vibrio parahemolyticus according to this embodiment is represented by SEQ ID NOs: 7 to 10 as probes (toxR probes) selected from the pathogenic expression regulatory gene (toxR) region of Vibrio parahemolyticus. It is preferable that at least one probe consisting of a base sequence is immobilized.
Further, the Vibrio parahemolyticus detection carrier according to this embodiment is SEQ ID NO: 11 as a probe (tdh probe) selected from the heat-resistant hemolytic toxin gene (tdh) region of pathogenic Vibrio parahemolyticus. It is preferable that at least one of the probes having the base sequences shown in .about.15 is immobilized.
Furthermore, the carrier for detecting Vibrio parahemolyticus according to the present embodiment is selected from the thermostable hemolysin-like toxin type 1 gene in the thermostable hemolysin-like toxin gene (trh) region of pathogenic Vibrio parahemolyticus It is preferable that at least one of the base sequences shown in SEQ ID NOs: 16 to 19 is immobilized as the probe (trh1 probe), and the heat-resistant hemolytic toxin-like toxin 2 in the same gene (trh) region As a probe selected from a type gene (trh2 probe), it is preferable to fix at least one of the probes consisting of the nucleotide sequence shown in SEQ ID NO: 20 or 21.
　本実施形態に係るビブリオ・パラヘモリティカス検出用担体をこのような構成にすることによって、ビブリオ・パラヘモリティカス（病原性ビブリオ・パラヘモリティカスを含む）と共に、病原性ビブリオ・パラヘモリティカスを同時に特異的に検出することが可能になる。また、病原性ビブリオ・パラヘモリティカスとして、耐熱性溶血毒遺伝子（tdh）と耐熱性溶血毒類似毒素遺伝子（trh）を有するか否かを識別して検出することも可能となる。さらに、耐熱性溶血毒類似毒素遺伝子（trh）として、耐熱性溶血毒類似毒素１型遺伝子と耐熱性溶血毒類似毒素２型遺伝子を識別して検出することも可能となる。
　このように、耐熱性溶血毒類似毒素１型遺伝子と耐熱性溶血毒類似毒素２型遺伝子とを、それぞれ専用のプライマーを用いて増幅産物のサイズで識別するのではなく、同一のプライマーを用いて増幅産物を得た後に、それぞれ専用の特異性の高いプローブを用いて識別可能にすることによって、マルチプレックスＰＣＲにおけるプライマー数を減らすことができ、精度の向上とコストの低減を実現することが可能になっている。 By configuring the Vibrio parahemolyticus detection carrier according to the present embodiment in such a configuration, together with Vibrio parahemolyticus (including pathogenic Vibrio parahemolyticus), pathogenic Vibrio parahaemolyticus It becomes possible to specifically detect lyticus simultaneously. It is also possible to identify and detect whether or not a pathogenic Vibrio parahaemolyticus has a heat-resistant hemolytic toxin gene (tdh) and a heat-resistant hemolytic toxin-like toxin gene (trh). Furthermore, as a heat-resistant hemolytic toxin-like toxin gene (trh), a heat-resistant hemolytic toxin-like toxin type 1 gene and a heat-resistant hemolytic toxin-like toxin type 2 gene can be identified and detected.
In this way, the thermostable hemolysin-like toxin type 1 gene and the thermostable hemolysin-like toxin type 2 gene are not distinguished from each other by the size of the amplification product using a dedicated primer, but using the same primer. It is possible to reduce the number of primers in multiplex PCR by making each identifiable using a dedicated probe with high specificity after obtaining amplification products, which can improve accuracy and reduce costs. It has become.
　本実施形態に係るビブリオ・パラヘモリティカス検出用担体において用いるプローブも、上記の塩基配列に限定されるものではなく、それぞれの塩基配列において１又は数個の塩基が欠損、置換又は付加されたものを用いることができる。また、それぞれの塩基配列に対して相補的な塩基配列からなる核酸断片に対してストリンジェントな条件下でハイブリダイズできる核酸断片からなるものを用いることもできる。さらに、これらのようなプローブに対して相補的な塩基配列を有するプローブを用いることもできる。 The probe used in the Vibrio parahemolyticus detection carrier according to the present embodiment is not limited to the above base sequence, and one or several bases are deleted, substituted or added in each base sequence. Things can be used. Moreover, what consists of a nucleic acid fragment which can be hybridized on stringent conditions with respect to the nucleic acid fragment which consists of a base sequence complementary to each base sequence can also be used. Furthermore, probes having a base sequence complementary to these probes can also be used.
　このようなビブリオ・パラヘモリティカス検出用担体を用いて、具体的には、ＰＣＲ増幅産物に所定の緩衝液を混合し、当該担体に滴下する。次に、当該担体を４５℃で１時間静置し、その後、所定の緩衝液によりハイブリダイズしなかったＰＣＲ増幅産物等を当該担体から洗い流す。そして、当該担体を標識検出装置にかけて標識の検出を行うことにより、試料中に病原性ビブリオ・パラヘモリティカス及びビブリオ・パラヘモリティカス（病原性ビブリオ・パラヘモリティカスを含む）が存在するか否かを同時に検出することができる。 Using such a Vibrio parahemolyticus detection carrier, specifically, a predetermined buffer solution is mixed with the PCR amplification product and dropped onto the carrier. Next, the carrier is allowed to stand at 45 ° C. for 1 hour, and then PCR amplification products and the like that have not been hybridized with a predetermined buffer are washed away from the carrier. Then, by detecting the label by applying the carrier to a label detection apparatus, pathogenic Vibrio parahemolyticus and Vibrio parahemolyticus (including pathogenic Vibrio parahemolyticus) exist in the sample. It can be detected at the same time.
　以上説明したように、本実施形態に係るビブリオ・パラヘモリティカスの検出方法、及びビブリオ・パラヘモリティカス検出用担体によれば、病原性を持たないものを含むビブリオ・パラヘモリティカスと、食中毒の原因となる病原性ビブリオ・パラヘモリティカスの検出を同時にかつ高い特異性で行うことが可能である。 As described above, according to the method for detecting Vibrio parahemolyticus and the carrier for detecting Vibrio parahemolyticus according to the present embodiment, the vibrio parahemolyticus including those having no pathogenicity It is possible to detect pathogenic Vibrio parahaemolyticus that causes food poisoning at the same time and with high specificity.
＜試験１：３種類のプライマーセットを用いた同時増幅の検証＞
　図１に示す毒素遺伝子の保有状況が既知の７種類のビブリオ・パラヘモリティカスの菌株を用いて、これらのビブリオ・パラヘモリティカスから常法によりＤＮＡを抽出した。これらは、いずれも大阪大学微生物病研究所からの分譲由来のものである。
　次いで、図２に示すtoxRプライマーセット、tdhプライマーセット、及びtrhプライマーセットを用いて、菌株毎に、それぞれの増幅対象領域をマルチプレックスＰＣＲにより同時に増幅し、得られた増幅産物を検出できるか否かを検証した。具体的には、以下のように行った。 <Test 1: Verification of simultaneous amplification using three types of primer sets>
DNA was extracted from these Vibrio parahemolyticus by a conventional method using seven types of Vibrio parahemolyticus strains known in FIG. These are all derived from the Osaka University Institute for Microbial Diseases.
Next, using the toxR primer set, tdh primer set, and trh primer set shown in FIG. 2, for each strain, the respective amplification target regions can be simultaneously amplified by multiplex PCR, and whether or not the obtained amplification product can be detected. I verified. Specifically, it was performed as follows.
　上記７種類のビブリオ・パラヘモリティカスの菌株を、それぞれ１％塩化ナトリウム添加トリプティックソイブロス（日本ＢＤ製）に接種して、３７℃で一晩培養を行ったのち、培養液をそれぞれ１ｍＬずつ回収し、５０００×ｇで、１０分間の遠心分離を行った。
　次に、上清を廃棄し、得られた沈殿に、２０ｍｇ／ｍＬ濃度のリゾチーム溶液（２０ｍＭ　Ｔｒｉｓ－ＨＣｌ，ｐＨ８．０／２ｍＭ　ＥＤＴＡ，１．２％ＴｒｉｔｏｎＸ－１００）を加えて、３７℃で３０分間溶菌処理を行った。さらに、ＤＮｅａｓｙ　Ｂｌｏｏｄ＆Ｔｉｓｓｕｅ　Ｋｉｔ（株式会社キアゲン製）を用いて、カラム精製を行うことにより、ＤＮＡ抽出液を得た。このＤＮＡ抽出液を１０ｐｇ／μＬ濃度に調製したものをＰＣＲにおいて使用する試料とした。 After inoculating each of the above 7 types of Vibrio parahemolyticus strains into tryptic soy broth supplemented with 1% sodium chloride (manufactured by Japan BD) and culturing overnight at 37 ° C., 1 mL each of the culture solution Collected and centrifuged at 5000 × g for 10 minutes.
Next, the supernatant is discarded, and a 20 mg / mL lysozyme solution (20 mM Tris-HCl, pH 8.0 / 2 mM EDTA, 1.2% Triton X-100) is added to the resulting precipitate at 37 ° C. Lysis treatment was performed for 30 minutes. Furthermore, a DNA extract was obtained by performing column purification using DNeasy Blood & Tissue Kit (manufactured by Qiagen). This DNA extract prepared at a concentration of 10 pg / μL was used as a sample for PCR.
　次に、ＰＣＲ反応液を以下の組成で調製した。プライマーはシグマアルドリッチジャパン合同会社に合成委託し、それ以外の試薬はタカラバイオ株式会社製のものを使用した。以下の組成において、ビブリオ・パラヘモリティカスをビブリオと略称している。
・緩衝液（10×Ex　Taq　buffer）　　　　　　　　　　　　　(2.0μl）
・核酸合成基質（dNTP　Mixture）　　　　　　　　　　　　 （1.6μl）
・ビブリオtoxR増幅用Ｆプライマー　　　　　　　　　　　　(0.2μl）
・ビブリオtoxR増幅用Ｒプライマー（5’末端Cy5修飾）　　（0.2μl）
・ビブリオtdh増幅用Ｆプライマー　　　　　　　　　　　　 (0.2μl）
・ビブリオtdh増幅用Ｒプライマー（5’末端Cy5修飾）　　　(0.2μl）
・ビブリオtrh増幅用Ｆプライマー　　　　　　　　　　　　 (0.2μl）
・ビブリオtrh増幅用Ｒプライマー（5’末端Cy5修飾）　　　(0.2μl）
・TaKaRa　Ex　Taq　Hot　Start　Version　　　　　　　　　（0.2μl）
・試料のＤＮＡ　　　　　　　　　　　　　　　　　　　　　（1.0μl）
・滅菌水　　　　　　　　　　　　　　　　　　　　　　　　（14.0μl）
（全量　20μl） Next, a PCR reaction solution was prepared with the following composition. The primers were outsourced to Sigma Aldrich Japan GK, and the other reagents were from Takara Bio Inc. In the following composition, Vibrio parahemolyticus is abbreviated as Vibrio.
・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture) (1.6μl)
・ F primer for amplification of Vibrio toxR (0.2μl)
・ R primer for Vibrio toxR amplification (5 'end Cy5 modification) (0.2μl)
・ F primer for amplification of Vibrio tdh (0.2μl)
・ R primer for Vibrio tdh amplification (5 'end Cy5 modification) (0.2μl)
・ F primer for amplification of vibrio trh (0.2μl)
・ R primer for amplification of vibrio trh (5 'end Cy5 modification) (0.2μl)
・ TaKaRa Ex Taq Hot Start Version (0.2μl)
・ Sample DNA (1.0μl)
・ Sterile water (14.0μl)
(Total 20 μl)
　ＰＣＲによる遺伝子の増幅には、サーマルサイクラーｅｐグラジエント（エッペンドルフ株式会社）を使用した。反応条件は、以下の通りである。
（１）９５℃　２分
（２）９５℃　１０秒（ＤＮＡ鎖の乖離工程）
（３）６８℃　３０秒（アニーリング工程）
（４）７２℃　３０秒（ＤＮＡ合成工程）
（５）７２℃　２分
（２）～（４）を４０サイクル A thermal cycler ep gradient (Eppendorf Co., Ltd.) was used for gene amplification by PCR. The reaction conditions are as follows.
(1) 95 ° C for 2 minutes (2) 95 ° C for 10 seconds (DNA strand dissociation step)
(3) 68 ° C. 30 seconds (annealing process)
(4) 72 ° C. for 30 seconds (DNA synthesis step)
(5) 40 cycles of 72 ° C 2 minutes (2) to (4)
　次に、このようにして得られたＰＣＲ増幅産物を含むＰＣＲ反応液をマイクロチップ電気泳動装置ＭｕｌｔｉＮＡ（株式会社島津製作所）に供し、ＰＣＲ増幅産物のサイズ分析を行った。その結果を図３に示す。
　同図に示すように、電気泳動の結果、toxRプライマーセットによる増幅産物（推定サイズ３１９ｂｐ）は、７種類の菌株の全てについて検出された。
　一方、tdhプライマーセットによる増幅産物（推定サイズ１９５ｂｐ）は、tdh遺伝子を有する菌株(4)、(5)、(6)についてのみ検出され、trhプライマーセットによる増幅産物（推定サイズ２７４ｂｐ）は、trh遺伝子を有する菌株(1)、(2)、(3)、(6)についてのみ検出された。 Next, the PCR reaction solution containing the PCR amplification product thus obtained was subjected to a microchip electrophoresis apparatus MultiNA (Shimadzu Corporation), and size analysis of the PCR amplification product was performed. The result is shown in FIG.
As shown in the figure, as a result of electrophoresis, amplification products (estimated size 319 bp) by the toxR primer set were detected for all seven types of strains.
On the other hand, the amplification product (estimated size 195 bp) by the tdh primer set is detected only for the strains (4), (5) and (6) having the tdh gene, and the amplification product (estimated size 274 bp) by the trh primer set is trh Only the strains (1), (2), (3) and (6) having the gene were detected.
　以上のことから、本実施形態に係るビブリオ・パラヘモリティカスの検出方法におけるtoxRプライマーセット、tdhプライマーセット、及びtrhプライマーセットによって、これらの領域をそれぞれ特異的に同時に検出できたことがわかる。このように、本実施形態に係るビブリオ・パラヘモリティカスの検出方法によれば、ビブリオ・パラヘモリティカスと病原性ビブリオ・パラヘモリティカスの検出を同時に行えることが確認された。 From the above, it can be seen that each of these regions could be specifically and simultaneously detected by the toxR primer set, tdh primer set, and trh primer set in the method for detecting Vibrio parahemolyticus according to the present embodiment. Thus, according to the Vibrio parahemolyticus detection method according to this embodiment, it was confirmed that Vibrio parahemolyticus and pathogenic Vibrio parahemolyticus can be detected simultaneously.
＜試験２：耐熱性溶血毒類似毒素１型及び２型遺伝子（trh1,trh2）の識別の検証＞
　試験１で調製した菌株(1)、(2)、(3)の各ＤＮＡ抽出液１０ｎｇ／μｌとtrhプライマーセットを用いて、菌株毎に、ＰＣＲによりtrh遺伝子領域の増幅を行った。ＰＣＲ反応液は以下の組成で調製し、その他の点については試験１と同様にして、ＰＣＲ増幅産物を含むＰＣＲ反応液を得た。 <Test 2: Verification of discrimination between heat-resistant hemolytic toxin-like toxin type 1 and type 2 genes (trh1, trh2)>
The trh gene region was amplified by PCR for each strain using 10 ng / μl of each DNA extract of strains (1), (2), and (3) prepared in Test 1 and the trh primer set. A PCR reaction solution was prepared with the following composition, and the PCR reaction solution containing the PCR amplification product was obtained in the same manner as in Test 1 for other points.
・緩衝液（10×Ex　Taq　buffer）　　　　　　　　　　　　(2.0μl）
・核酸合成基質（dNTP　Mixture）　　　　　　　　　　　 （1.6μl）
・ビブリオtrh増幅用Ｆプライマー　　　　　　　　　　　 (0.2μl）
・ビブリオtrh増幅用Ｒプライマー（5’末端Cy5修飾）　 （0.2μl）
・TaKaRa　Ex　Taq　Hot　Start　Version　　　　　　　　（0.2μl）
・試料のＤＮＡ　　　　　　　　　　　　　　　　　　　　（1.0μl）
・滅菌水　　　　　　　　　　　　　　　　　　　　　　　（14.8μl）
（全量　20μl） ・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture) (1.6μl)
・ F primer for amplification of vibrio trh (0.2μl)
・ R primer for amplification of vibrio trh (5 'end Cy5 modification) (0.2μl)
・ TaKaRa Ex Taq Hot Start Version (0.2μl)
・ Sample DNA (1.0μl)
・ Sterile water (14.8μl)
(Total 20 μl)
　また、予め、図５に示す配列番号１６～１９の塩基配列からなる耐熱性溶血毒類似毒素１型遺伝子検出用のプローブ（trh1プローブ）と、配列番号２０～２１の塩基配列からなる耐熱性溶血毒類似毒素２型遺伝子検出用のプローブ（trh2プローブ）を固定化したＤＮＡチップを作製した。そして、菌株毎に、ＰＣＲ反応液４μＬとハイブリダイゼーション用の緩衝液２μＬ（3×SSC／0.3%SDS　クエン酸－生理食塩水－ドデシル硫酸ナトリウム）を混合したものを、上記ＤＮＡチップに滴下して、４５℃で１時間反応させた。
　反応後、ＤＮＡチップを室温下で洗浄液（2×SSC／0.2%SDS溶液、2×SSC溶液の順に）に浸して洗浄を行い、カバーガラスを載せて蛍光検出器Ｂｉｏｓｈｏｔ(東洋鋼鈑株式会社製)により各プローブのスポット領域の蛍光を検出した。 In addition, a heat-resistant hemolytic toxin-like toxin type 1 gene detection probe (trh1 probe) consisting of the nucleotide sequences of SEQ ID NOs: 16 to 19 shown in FIG. 5 and heat-resistant hemolysis consisting of the nucleotide sequences of SEQ ID NOs: 20 to 21 are shown in advance. A DNA chip on which a probe for detecting a toxin-like toxin type 2 gene (trh2 probe) was immobilized was prepared. For each strain, a mixture of 4 μL of the PCR reaction solution and 2 μL of the hybridization buffer solution (3 × SSC / 0.3% SDS citrate-saline-sodium dodecyl sulfate) was dropped onto the DNA chip. And reacted at 45 ° C. for 1 hour.
After the reaction, the DNA chip is immersed in a washing solution (2 × SSC / 0.2% SDS solution, 2 × SSC solution in this order) at room temperature for washing, and a cover glass is placed on the fluorescence detector Bioshot (manufactured by Toyo Kohan Co., Ltd.) ) To detect the fluorescence in the spot area of each probe.
　具体的には、プローブにハイブリダイズした増幅産物の標識成分（Cy5）をレーザー光により励起して発光させ、その光量を検出器内に取り付けたＣＣＤカメラにより検出した。また、光量を電気信号に置換して数値化し、蛍光強度を得た。この蛍光強度は、当該装置での強度指標であり、単位はなく、バックグラウンドの数値が０になるように補正して算出した。その結果を図４に示す。 Specifically, the label component (Cy5) of the amplification product hybridized with the probe was excited by laser light to emit light, and the amount of light was detected by a CCD camera mounted in the detector. In addition, the light intensity was replaced with an electric signal and digitized to obtain fluorescence intensity. This fluorescence intensity is an intensity index in the apparatus, and has a unit, and was calculated by correcting so that the background value becomes zero. The result is shown in FIG.
　同図に示すように、菌株(1)、菌株(2)については、trh1検出用プローブでは高い蛍光強度が得られ、trh2検出用プローブでは低い蛍光強度しか得られなかった。このことから、菌株(1)、菌株(2)は、病原性ビブリオ・パラヘモリティカスのtrh遺伝子保有菌のうち、trh1遺伝子保有菌であることがわかる。
　一方、菌株(3)については、trh2検出用プローブでは高い蛍光強度が得られ、trh1検出用プローブでは比較的低い蛍光強度しか得られなかった。このことから、菌株(3)は、病原性ビブリオ・パラヘモリティカスのtrh遺伝子保有菌のうち、trh2遺伝子保有菌であることがわかる。 As shown in the figure, for the strains (1) and (2), a high fluorescence intensity was obtained with the trh1 detection probe, and a low fluorescence intensity was obtained with the trh2 detection probe. This shows that the strain (1) and the strain (2) are trh1 gene-bearing bacteria among the pathogenic Vibrio parahemolyticus trh gene-bearing bacteria.
On the other hand, for the strain (3), a high fluorescence intensity was obtained with the trh2 detection probe, and a relatively low fluorescence intensity was obtained with the trh1 detection probe. From this, it can be seen that the strain (3) is a trh2 gene-bearing bacterium among the pathogenic Vibrio parahemolyticus trh gene-bearing bacterium.
　以上のことから、本実施形態に係るビブリオ・パラヘモリティカス検出用担体によれば、trh1検出用プローブとtrh2検出用プローブを併用することで、病原性ビブリオ・パラヘモリティカスのtrh遺伝子保有菌が検出対象の試料に含まれている場合に、trh1遺伝子保有菌とtrh2遺伝子保有菌を識別できると共に、trh遺伝子についての偽陰性の判定を回避できることがわかった。 From the above, according to the Vibrio parahemolyticus detection carrier according to the present embodiment, by using the trh1 detection probe and the trh2 detection probe in combination, the trh gene possessed by the pathogenic Vibrio parahemolyticus It was found that when bacteria are included in the sample to be detected, trh1 gene-bearing bacteria and trh2 gene-bearing bacteria can be discriminated, and false negative determination for the trh gene can be avoided.
＜試験３：ＤＮＡチップによる同時検出の検証＞
　toxR、tdh、tr1、及びtrh2遺伝子を含むＤＮＡ断片と結合可能なプローブを固定化した本実施形態に係るビブリオ・パラヘモリティカス検出用担体によって、ビブリオ・パラヘモリティカスにおけるtoxR、tdh、trhの３種類の遺伝子を、同時に特異的に検出できるか否かを検証した。
　具体的には、図５に示す配列番号７～２１の塩基配列からなる各プローブを固定化したＤＮＡチップを作製した。
　そして、図１に示す７種類のビブリオ・パラヘモリティカスの菌株を用いて、菌株毎に、試験１と同様にして得られたＰＣＲ反応液４μＬと、ハイブリダイゼーション用の緩衝液２μＬ（3×SSC／0.3%SDS　クエン酸－生理食塩水－ドデシル硫酸ナトリウム）を混合したものを、このＤＮＡチップに滴下して、４５℃で１時間反応させた。
　反応後、試験２と同様に、ＤＮＡチップを室温下で洗浄液（2×SSC／0.2%SDS溶液、2×SSC溶液の順に）に浸して洗浄を行い、カバーガラスを載せて蛍光検出器Ｂｉｏｓｈｏｔ(東洋鋼鈑株式会社製)により各プローブのスポット領域の蛍光を検出した。その結果を図６に示す。 <Test 3: Verification of simultaneous detection by DNA chip>
ToxR, tdh, trh in Vibrio parahemolyticus is detected by the carrier for detecting Vibrio parahemolyticus according to this embodiment in which a probe that can bind to a DNA fragment containing toxR, tdh, tr1, and trh2 genes is immobilized. It was verified whether the three types of genes could be specifically detected simultaneously.
Specifically, a DNA chip on which each probe consisting of the nucleotide sequences of SEQ ID NOs: 7 to 21 shown in FIG. 5 was immobilized was prepared.
Then, using 7 types of Vibrio parahemolyticus strains shown in FIG. 1, 4 μL of PCR reaction solution obtained in the same manner as in Test 1 and 2 μL of hybridization buffer solution (3 ×) A mixture of SSC / 0.3% SDS (citric acid-saline-sodium dodecyl sulfate) was dropped onto the DNA chip and reacted at 45 ° C. for 1 hour.
After the reaction, as in Test 2, the DNA chip was washed by immersing it in a washing solution (2 × SSC / 0.2% SDS solution, 2 × SSC solution in this order) at room temperature, and a cover glass was placed on the fluorescence detector Bioshot ( The fluorescence in the spot area of each probe was detected by Toyo Kohan Co., Ltd. The result is shown in FIG.
　同図に示すように、配列番号７～１０のtoxR検出用プローブでは、７種類全てのビブリオ・パラヘモリティカスの菌株について、高い蛍光強度を示した。
　これに対して、配列番号１１～１５のtdh検出用プローブでは、tdh遺伝子を保有する菌株(4)、(5)、(6)のみについて高い蛍光強度を示し、tdh遺伝子を保有しない菌株(1)、(2)、(3)、(7)については、高い蛍光強度を示さなかった。
　また、配列番号１６～１９のtrh1検出用プローブでは、trh1を保有する菌株(1)、(2)、(6)のみについて高い蛍光強度を示し、trh1を保有しない菌株(3)、(4)、(5)、(7)については、高い蛍光強度を示さず、さらに配列番号２０～２１のtrh2検出用プローブでは、trh2を保有する菌株(3)のみについて高い蛍光強度を示し、trh2を保有しない菌株(1)、(2)、(4)、(5)、(6)、(7)については、高い蛍光強度を示さなかった。 As shown in the figure, the toxR detection probes of SEQ ID NOs: 7 to 10 showed high fluorescence intensity for all seven types of Vibrio parahemolyticus strains.
In contrast, the tdh detection probes of SEQ ID NOs: 11 to 15 show high fluorescence intensity only for the strains (4), (5) and (6) having the tdh gene, and the strains having no tdh gene (1 ), (2), (3), (7) did not show high fluorescence intensity.
In addition, the trh1 detection probes of SEQ ID NOs: 16 to 19 show high fluorescence intensity only for the strains (1), (2), and (6) that possess trh1, and the strains (3) and (4) that do not possess trh1 , (5) and (7) do not show high fluorescence intensity, and the trh2 detection probes of SEQ ID NOs: 20 to 21 show high fluorescence intensity only for the strain (3) having trh2, and possess trh2. The strains (1), (2), (4), (5), (6) and (7) which did not show no high fluorescence intensity.
　以上のことから、本実施形態に係るビブリオ・パラヘモリティカス検出用担体によれば、ビブリオ・パラヘモリティカスにおけるtoxR、tdh、trhの３種類の遺伝子領域を、同時に特異的に検出できることがわかった。特に、trhについては、trh1とtrh2のいずれか一方のタイプのもののみが試料中に存在している場合には、そのいずれであるかを識別できると共に、偽陰性の判定を防止できることが明らかとなった。 From the above, according to the Vibrio parahemolyticus detection carrier according to the present embodiment, the three types of gene regions of toxR, tdh, and trh in Vibrio parahemolyticus can be specifically and simultaneously detected. all right. In particular, for trh, if only one of trh1 and trh2 is present in the sample, it is clear that it can be identified and that false negative determination can be prevented. became.
　本発明は、以上の実施形態や実施例に限定されるものではなく、本発明の範囲内において、種々の変更実施が可能である。例えば、本実施形態に係るビブリオ・パラヘモリティカスの検出方法において、ＰＣＲ反応液にその他の成分を含有させたり、あるいは本実施形態に係るビブリオ・パラヘモリティカス検出用担体に、上記以外のプローブをさらに追加して固定化したりするなど適宜変更することが可能である。 The present invention is not limited to the above embodiments and examples, and various modifications can be made within the scope of the present invention. For example, in the method for detecting Vibrio parahemolyticus according to the present embodiment, the PCR reaction solution may contain other components, or the Vibrio parahemolyticus detection carrier according to the present embodiment may have other than the above. It is possible to make appropriate changes such as adding additional probes and fixing them.
　本発明は、食品検査、疫学的環境検査、環境検査、臨床試験、及び家畜衛生等において、ビブリオ・パラヘモリティカスを検出する場合に好適に利用することが可能である。 The present invention can be suitably used for detecting Vibrio parahemolyticus in food inspection, epidemiological environmental inspection, environmental inspection, clinical test, livestock hygiene, and the like.

Claims (6)

  1. 　病原性ビブリオ・パラヘモリティカス及びビブリオ・パラヘモリティカス（病原性のものを含む）を同時に検出するビブリオ・パラヘモリティカスの検出方法であって、
    　試料から抽出したゲノムＤＮＡをテンプレートとして、ビブリオ・パラヘモリティカスの病原性発現調節遺伝子（toxR）を含むＤＮＡ断片と、耐熱性溶血毒遺伝子（tdh）を含むＤＮＡ断片と、耐熱性溶血毒類似毒素遺伝子（trh）を含むＤＮＡ断片と、をマルチプレックスＰＣＲにより同時に増幅させ、それぞれの増幅産物の有無を検出することによって、試料中における病原性ビブリオ・パラヘモリティカスの有無及びビブリオ・パラヘモリティカスの有無を同時に検出するビブリオ・パラヘモリティカスの検出方法。     A method for detecting Vibrio parahemolyticus that simultaneously detects pathogenic Vibrio parahemolyticus and Vibrio parahemolyticus (including pathogenic ones),
    Using DNA extracted from the sample as a template, DNA fragment containing Vibrio parahemolyticus virulence expression regulatory gene (toxR), DNA fragment containing thermostable hemolysin gene (tdh), and thermostable hemolysin A DNA fragment containing a toxin gene (trh) is simultaneously amplified by multiplex PCR, and the presence or absence of each amplification product is detected to detect the presence or absence of pathogenic Vibrio parahemolyticus in the sample and Vibrio parahemo A method for detecting Vibrio parahemolyticus that simultaneously detects the presence or absence of riticas.
  2. 　前記toxRを含むＤＮＡ断片を増幅して得られた増幅産物と相補的に結合するプローブ、前記tdhを含むＤＮＡ断片を増幅して得られた増幅産物と相補的に結合するプローブ、及び前記trhを含むＤＮＡ断片を増幅して得られた増幅産物と相補的に結合するプローブを固定化したビブリオ・パラヘモリティカス検出用担体を用いて、前記それぞれの増幅産物を同時に検出する
    　ことを特徴とする請求項１記載のビブリオ・パラヘモリティカスの検出方法。     A probe that complementarily binds to the amplification product obtained by amplifying the DNA fragment containing toxR, a probe that complementarily binds to the amplification product obtained by amplifying the DNA fragment containing tdh, and trh The amplified products are simultaneously detected using a Vibrio parahemolyticus detection carrier on which a probe that complementarily binds to the amplified product obtained by amplifying the contained DNA fragment is immobilized. The method for detecting Vibrio parahemolyticus according to claim 1.
  3. 　前記toxRを含むＤＮＡ断片を増幅するための配列番号１に示す塩基配列からなるプライマーと配列番号２に示す塩基配列からなるプライマーとからなるプライマーセットと、
    　前記tdhを含むＤＮＡ断片を増幅するための配列番号３に示す塩基配列からなるプライマーと配列番号４に示す塩基配列からなるプライマーとからなるプライマーセットと、
    　前記trhを含むＤＮＡ断片を増幅するための配列番号５に示す塩基配列からなるプライマーと配列番号６に示す塩基配列からなるプライマーとからなるプライマーセットと、を含むＰＣＲ反応液を用いて、前記マルチプレックスＰＣＲを行い、
    　前記toxRから選択された配列番号７～１０に示す塩基配列からなる少なくともいずれかのプローブと、
    　前記tdhから選択された配列番号１１～１５に示す塩基配列からなる少なくともいずれかのプローブと、
    　前記trhにおける耐熱性溶血毒類似毒素1型遺伝子（trh1）から選択された配列番号１６～１９に示す塩基配列からなる少なくともいずれかのプローブと、
    　前記trhにおける耐熱性溶血毒類似毒素２型遺伝子（trh2）から選択された配列番号２０又は２１に示す塩基配列からなる少なくともいずれかのプローブと、を固定化したビブリオ・パラヘモリティカス検出用担体を用いて、前記それぞれの増幅産物の有無の検出を行う
    　ことを特徴とする請求項１又は２記載のビブリオ・パラヘモリティカスの検出方法。     A primer set consisting of 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: 2 for amplifying the DNA fragment containing the toxR;
    A primer set consisting of 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: 4 for amplifying the DNA fragment containing tdh;
    Using a PCR reaction solution containing a primer set consisting of a primer consisting of the base sequence shown in SEQ ID NO: 5 and a primer consisting of the base sequence shown in SEQ ID NO: 6 for amplifying the DNA fragment containing trh, Plex PCR,
    At least one probe consisting of the base sequence shown in SEQ ID NOs: 7 to 10 selected from the toxR;
    At least one of the probes consisting of the base sequences shown in SEQ ID NOs: 11 to 15 selected from the tdh;
    At least one probe consisting of the base sequences shown in SEQ ID NOs: 16 to 19 selected from the heat-resistant hemolytic toxin-like toxin type 1 gene (trh1) in trh;
    A carrier for detecting Vibrio parahemolyticus having immobilized thereon at least one probe consisting of the base sequence represented by SEQ ID NO: 20 or 21 selected from the heat-resistant hemolysin-like toxin type 2 gene (trh2) in trh The method for detecting Vibrio parahemolyticus according to claim 1 or 2, wherein the presence or absence of each of the amplification products is detected using.
  4. 　前記各遺伝子から選択された少なくともいずれかのプローブが、以下の（１）～（３）のいずれかであることを特徴とする請求項３記載のビブリオ・パラヘモリティカスの検出方法。
    （１）配列番号に示す塩基配列において、１又は数個の塩基が欠損、置換又は付加されたプローブ。
    （２）配列番号に示す塩基配列に対して相補的な塩基配列からなる核酸断片に対してストリンジェントな条件下でハイブリダイズできるプローブ。
    （３）（１）又は（２）のプローブに対して相補的な塩基配列を有するプローブ。     4. The method for detecting Vibrio parahemolyticus according to claim 3, wherein at least one of the probes selected from each gene is any one of the following (1) to (3).
    (1) A probe in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO.
    (2) A probe capable of hybridizing under stringent conditions to a nucleic acid fragment consisting of a base sequence complementary to the base sequence shown in SEQ ID NO.
    (3) A probe having a base sequence complementary to the probe of (1) or (2).
  5. 　病原性ビブリオ・パラヘモリティカス及びビブリオ・パラヘモリティカス（病原性のものを含む）を同時に検出するためのビブリオ・パラヘモリティカス検出用担体であって、
    　ビブリオ・パラヘモリティカスの病原性発現調節遺伝子（toxR）から選択された配列番号７～１０に示す塩基配列からなる少なくともいずれかのプローブと、
    　ビブリオ・パラヘモリティカスの耐熱性溶血毒遺伝子（tdh）から選択された配列番号１１～１５に示す塩基配列からなる少なくともいずれかのプローブと、
    　ビブリオ・パラヘモリティカスの耐熱性溶血毒類似毒素1型遺伝子（trh1）から選択された配列番号１６～１９に示す塩基配列からなる少なくともいずれかのプローブと、
    　ビブリオ・パラヘモリティカスの耐熱性溶血毒類似毒素２型遺伝子（trh2）から選択された配列番号２０又は２１に示す塩基配列からなる少なくともいずれかのプローブと、を固定化したことを特徴とするビブリオ・パラヘモリティカス検出用担体。     A vibrio parahemolyticus detection carrier for simultaneously detecting pathogenic vibrio parahemolyticus and vibrio parahemolyticus (including pathogenic ones),
    At least one probe consisting of a base sequence shown in SEQ ID NOs: 7 to 10 selected from a pathogenic expression regulatory gene (toxR) of Vibrio parahemolyticus;
    At least one probe consisting of the nucleotide sequences shown in SEQ ID NOs: 11 to 15 selected from the thermostable hemolysin gene (tdh) of Vibrio parahemolyticus;
    At least any one of the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 16 to 19 selected from the thermostable hemolysin-like toxin type 1 gene (trh1) of Vibrio parahemolyticus;
    It is characterized by immobilizing at least one probe consisting of the nucleotide sequence shown in SEQ ID NO: 20 or 21 selected from heat-resistant hemolytic toxin-like toxin type 2 gene (trh2) of Vibrio parahemolyticus Vibrio parahemolyticus detection carrier.
  6. 　前記各遺伝子から選択された少なくともいずれかのプローブが、以下の（１）～（３）のいずれかであることを特徴とする請求項５記載のビブリオ・パラヘモリティカス検出用担体。
    （１）配列番号に示す塩基配列において、１又は数個の塩基が欠損、置換又は付加されたプローブ。
    （２）配列番号に示す塩基配列に対して相補的な塩基配列からなる核酸断片に対してストリンジェントな条件下でハイブリダイズできるプローブ。
    （３）（１）又は（２）のプローブに対して相補的な塩基配列を有するプローブ。     6. The Vibrio parahemolyticus detection carrier according to claim 5, wherein at least one of the probes selected from each gene is any one of the following (1) to (3).
    (1) A probe in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO.
    (2) A probe capable of hybridizing under stringent conditions to a nucleic acid fragment consisting of a base sequence complementary to the base sequence shown in SEQ ID NO.
    (3) A probe having a base sequence complementary to the probe of (1) or (2).
PCT/JP2015/002932 2014-06-11 2015-06-11 Method for detecting vibrio parahaemolyticus, and carrier for detecting vibrio parahaemolyticus WO2015190107A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2014120917A JP2016000014A (en) 2014-06-11 2014-06-11 Methods for detecting vibrio parahaemolyticus and carriers for detecting v. parahaemolyticus
JP2014-120917 2014-06-11

Publications (1)

Publication Number Publication Date
WO2015190107A1 true WO2015190107A1 (en) 2015-12-17

Family

ID=54833218

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/002932 WO2015190107A1 (en) 2014-06-11 2015-06-11 Method for detecting vibrio parahaemolyticus, and carrier for detecting vibrio parahaemolyticus

Country Status (2)

Country Link
JP (1) JP2016000014A (en)
WO (1) WO2015190107A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107988342A (en) * 2018-01-03 2018-05-04 北京毅新博创生物科技有限公司 The method and product of Mass Spectrometric Identification vibrio parahemolyticus parting
CN108103171A (en) * 2018-01-03 2018-06-01 北京毅新博创生物科技有限公司 The preparation method of vibrio parahemolyticus fingerprint databases
RU2668805C1 (en) * 2017-11-27 2018-10-02 Федеральное казенное учреждение здравоохранения "Ростовский-на-Дону ордена Трудового Красного Знамени научно-исследовательский противочумный институт" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека Strain vibrio parahaemolyticus, used as a producer of direct thermostable hemolysin (tdh)
CN111876499A (en) * 2020-06-24 2020-11-03 河北农业大学 Primer, kit and detection method for detecting vibrio parahaemolyticus
CN112239789A (en) * 2020-11-13 2021-01-19 宁波大学 Primer and kit capable of simultaneously detecting 4 virulence genes of vibrio parahaemolyticus
WO2022095922A1 (en) * 2020-11-05 2022-05-12 Becton, Dickinson And Company Rapid identification and typing of vibrio parahaemolyticus

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011142119A1 (en) * 2010-05-12 2011-11-17 東洋製罐株式会社 Food-poisoning bacteria detection carrier, and method for detecting food-poisoning bacteria
CN102816842A (en) * 2012-07-25 2012-12-12 广东出入境检验检疫局检验检疫技术中心 Multiplex PCR-DHPLC detection primers, kit and detection method for Vibrio parahemolyticus
CN103773858A (en) * 2014-01-09 2014-05-07 嘉兴市疾病预防控制中心 Triple real-time fluorescence PCR (Polymerase Chain Reaction) detection kit of vibrio parahaemolyticus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011142119A1 (en) * 2010-05-12 2011-11-17 東洋製罐株式会社 Food-poisoning bacteria detection carrier, and method for detecting food-poisoning bacteria
CN102816842A (en) * 2012-07-25 2012-12-12 广东出入境检验检疫局检验检疫技术中心 Multiplex PCR-DHPLC detection primers, kit and detection method for Vibrio parahemolyticus
CN103773858A (en) * 2014-01-09 2014-05-07 嘉兴市疾病预防控制中心 Triple real-time fluorescence PCR (Polymerase Chain Reaction) detection kit of vibrio parahaemolyticus

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CARIANI, A. ET AL.: "Detection and characterization of pathogenic vibrios in shellfish by a Ligation Detection Reaction- Universal Array approach", INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, vol. 153, no. 3, 20 November 2011 (2011-11-20), pages 474 - 482, XP028885237 *
HE, P. ET AL.: "Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains", MOLECULAR AND CELLULAR PROBES, vol. 28, no. 5-6, 9 June 2014 (2014-06-09), pages 246 - 250, XP055242130 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2668805C1 (en) * 2017-11-27 2018-10-02 Федеральное казенное учреждение здравоохранения "Ростовский-на-Дону ордена Трудового Красного Знамени научно-исследовательский противочумный институт" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека Strain vibrio parahaemolyticus, used as a producer of direct thermostable hemolysin (tdh)
CN107988342A (en) * 2018-01-03 2018-05-04 北京毅新博创生物科技有限公司 The method and product of Mass Spectrometric Identification vibrio parahemolyticus parting
CN108103171A (en) * 2018-01-03 2018-06-01 北京毅新博创生物科技有限公司 The preparation method of vibrio parahemolyticus fingerprint databases
CN107988342B (en) * 2018-01-03 2021-04-27 北京毅新博创生物科技有限公司 Method for identifying type of vibrio parahaemolyticus by mass spectrum and product
CN108103171B (en) * 2018-01-03 2021-05-21 北京毅新博创生物科技有限公司 Preparation method of vibrio parahaemolyticus fingerprint atlas database
CN111876499A (en) * 2020-06-24 2020-11-03 河北农业大学 Primer, kit and detection method for detecting vibrio parahaemolyticus
WO2022095922A1 (en) * 2020-11-05 2022-05-12 Becton, Dickinson And Company Rapid identification and typing of vibrio parahaemolyticus
CN112239789A (en) * 2020-11-13 2021-01-19 宁波大学 Primer and kit capable of simultaneously detecting 4 virulence genes of vibrio parahaemolyticus

Also Published As

Publication number Publication date
JP2016000014A (en) 2016-01-07

Similar Documents

Publication Publication Date Title
WO2015190107A1 (en) Method for detecting vibrio parahaemolyticus, and carrier for detecting vibrio parahaemolyticus
JP5830461B2 (en) PCR primer set, PCR reaction solution, detection method for food poisoning bacteria
JP5851395B2 (en) Carrier for detecting food poisoning bacteria, and method for detecting food poisoning bacteria
WO2017212904A1 (en) Method for rapid detection of african swine fever virus using lamp method in which multiple primer sets are combined
US20100075305A1 (en) Detection of bacterium by utilizing dnaj gene and use thereof
Watterworth et al. Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli
US20170088882A1 (en) Carrier for detecting foodborne-illness-causing bacteria, kit for detecting foodborne-illness-causing bacteria, method for detecting foodborne-illness-causing bacteria, and pcr reaction solution for foodborne-illness-causing bacteria
JP5613971B2 (en) Method for detecting pathogenic Listeria monocytogenes
JP6500773B2 (en) Wide area O serogroup determination method of E. coli using PCR method
JP6446845B2 (en) E. coli detection method and E. coli detection carrier
JP2013188224A (en) Method of detecting pathogenic listeria
JP7479640B2 (en) Method for determining a wide range of Shigella O serogroups using PCR
JP2007124970A (en) Method for detecting bordetella pertussis gene using lamp method and primer set therefor
WO2019221219A1 (en) Method for examining bacterium, microarray for examining bacterium, kit for examining bacterium, probe set for examining bacterium, and primer set for examining bacterium
WO2016129249A1 (en) Method for detecting lactic acid bacteria, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument
WO2008140832A2 (en) Method for rapid detection of clostridium botulinum
JP2016000015A (en) Carriers and kits for detecting food poisoning bacteria
JP5522623B2 (en) Primer set for detection of Listeria monocytogenes
JP6613627B2 (en) Nucleic acid amplification success / failure determination method and nucleic acid amplification test kit
JP2007075017A (en) Method for detecting campylobacter jejuni
JP2013220032A (en) Colon bacillus o-antigenic type inspection method using lamp method and inspection kit
KR101336948B1 (en) Methods for Detecting Shigella sonnei
JP2010115122A (en) Method of detecting aspergillus fumigatus relative
KR101336947B1 (en) Methods for Detecting Shigella flexneri
JP2019201626A (en) Method for examining bacterium, micro array for examining bacterium, kit for examining bacterium, probe set for examining bacterium, and primer set for examining bacterium

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15806183

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15806183

Country of ref document: EP

Kind code of ref document: A1