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WO2023214588A1 - Method for detecting spore-forming bacterium - Google Patents

Method for detecting spore-forming bacterium Download PDF

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WO2023214588A1
WO2023214588A1 PCT/JP2023/017195 JP2023017195W WO2023214588A1 WO 2023214588 A1 WO2023214588 A1 WO 2023214588A1 JP 2023017195 W JP2023017195 W JP 2023017195W WO 2023214588 A1 WO2023214588 A1 WO 2023214588A1
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oligonucleotide
seq
sequence represented
duf1657
duf421
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Japanese (ja)
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遥介 伊藤
惇 佐藤
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Kao Corp
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Kao Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present invention relates to a method for detecting spore-forming bacteria.
  • Spore-forming bacteria form spores that are highly resistant to physical treatments such as heat, desiccation, and ultraviolet light.
  • Many species of spore-forming bacteria are known, including bacteria of the genus Bacillus and bacteria of the genus Clostridium . They form spores when exposed to environments unsuitable for growth.
  • Spore-forming bacteria such as bacteria belonging to the genus Bacillus, widely grow in water and soil, so there is a high risk of them coming into contact with food raw materials or contaminating food and drink products (products) during the food and drink manufacturing process.
  • many bacteria are killed by heat treatment at 75° C. for 30 minutes, for example.
  • spore-forming bacteria form spores that can withstand high-temperature environments around 100°C, and such bacteria may not be sufficiently sterilized by heat treatment at 75°C for 30 minutes. If sterilization of spore-forming bacteria is insufficient, the spore-forming bacteria may proliferate within the product, causing spoilage or deterioration of the product. On the other hand, even spore-forming bacteria that exhibit such high heat resistance can be sterilized by setting the heat treatment temperature higher, but high temperature treatment can cause deterioration of raw materials and products, heat treatment costs, etc. Considering this, it is preferable to judge and take into account the heat resistance of the mixed spore-forming bacteria and appropriately determine the heat treatment conditions.
  • the conventional method for evaluating the spore heat resistance of spore-forming bacteria is to isolate and identify the spore-forming bacteria as described above, and then consider the growth medium, temperature, and time appropriate for the bacterial species.
  • a method is used to evaluate spore heat resistance by forming spores and measuring the survival rate when exposed to various heat treatment conditions.
  • Japanese Patent Application Publication No. 2009-183207 discloses a method for quickly and accurately evaluating and measuring the durability of spores in real time using an atomic force microscope equipped with a cantilever. There is.
  • the present invention provides an oligonucleotide pair consisting of the following oligonucleotides (a) and (c), an oligonucleotide pair consisting of the following oligonucleotides (b) and (c), and an oligonucleotide consisting of the following oligonucleotides (d) and (j).
  • the nucleic acid represented by the partial base sequence of the DUF421-DUF1657 gene is amplified, and depending on the presence or absence of the amplification product, Bacillus genus that can grow under neutral conditions is The present invention relates to a method for detecting spore-forming bacteria belonging to the genus Bacillus.
  • Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus (b) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
  • oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 3 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
  • Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus (e) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 5, or an oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 5, and growing under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
  • oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 6, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
  • Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus (h) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 8 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
  • oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or an oligonucleotide with 80% or more identity with the base sequence represented by SEQ ID NO: 9, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
  • oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 10, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, and which has the DUF421-DUF1657 gene and grows under neutral conditions.
  • Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
  • oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 12 and having the plasmid-specific DUF421-DUF1657 gene.
  • the present invention also relates to a method for evaluating the spore heat resistance of a spore-forming bacterium belonging to the genus Bacillus, which evaluates the spore heat resistance of a spore-forming bacterium belonging to the genus Bacillus that can grow under neutral conditions using the amplified product.
  • the present invention also relates to a method for determining heat treatment conditions for spore-forming bacteria belonging to the genus Bacillus, which determines heat treatment conditions for spore-forming bacteria belonging to the genus Bacillus based on the spore heat resistance evaluated by the method.
  • the present invention relates to a kit and oligonucleotide pair for detecting spore-forming bacteria used in the above method.
  • This is a photograph used as a substitute for a drawing. It is a boxplot showing the D105 °C values of Bacillus coagulans strain groups determined to be negative or positive by Multiplex PCR.
  • the present invention relates to a method for detecting spore-forming bacteria that can detect spore-forming bacteria that form spores with high heat resistance simply, quickly, and at low cost.
  • the present invention also relates to providing a method for evaluating the spore heat resistance of spore-forming bacteria, which evaluates the spore heat resistance of spore-forming bacteria simply, quickly, and at low cost.
  • the present invention also relates to a method for determining heat treatment conditions for spore-forming bacteria, which determines heat treatment conditions for spore-forming bacteria that ensure commercial sterility by evaluating spore heat resistance.
  • the present invention relates to the provision of a kit or oligonucleotide pair for detecting spore-forming bacteria that form spores with high heat resistance that can be suitably used in the above method.
  • the present inventors conducted extensive studies. The present inventors discovered a gene encoding a protein of unknown function that has DUF421 and DUF1657 domains in the transposon-derived spoVA2mob operon, which was found to contribute to improving spore heat resistance through comparative genome analysis of Bacillus subtillus.
  • a partial base sequence of the DUF421-DUF1657 gene is amplified using an oligonucleotide pair that can anneal to the base sequence of a specific region (hereinafter also referred to as "specific region") of the DUF421-DUF1657 gene as a primer pair.
  • specific region a specific region of the DUF421-DUF1657 gene
  • spore-forming bacteria that belong to the genus Bacillus and have a highly heat-resistant spore-forming ability can be detected simply, quickly, and at low cost. Furthermore, according to the method for evaluating spore heat resistance of spore-forming bacteria of the present invention, the heat resistance of spores formed by spore-forming bacteria belonging to the genus Bacillus can be evaluated easily, quickly, and at low cost. Furthermore, according to the method for determining heat treatment conditions for spore-forming bacteria of the present invention, appropriate heat treatment conditions for sterilizing spore-forming bacteria can be determined. Furthermore, the kit and oligonucleotide pair for detecting spore-forming bacteria of the present invention can be suitably used in the above method.
  • the method for detecting spore-forming bacteria of the present invention detects a specific partial base sequence of the DUF421-DUF1657 gene, that is, each species of spore-forming bacteria belonging to the genus Bacillus that has the DUF421-DUF1657 gene.
  • a specific partial base sequence of the DUF421-DUF1657 gene that is, each species of spore-forming bacteria belonging to the genus Bacillus that has the DUF421-DUF1657 gene.
  • variable region is a region in the DUF421-DUF1657 gene where base mutations tend to accumulate, and the base sequence of this region differs greatly between species.
  • stringent conditions include, for example, the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell., Cold Spring Harbor Laboratory Press].
  • x SSC composition of 1 x SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 x Denhardt, and 100 mg/mL herring sperm DNA with probe 65
  • conditions include constant temperature at °C for 8 to 16 hours and hybridization.
  • the method for evaluating the spore heat resistance of spore-forming bacteria of the present invention shows that spore-forming bacteria belonging to the genus Bacillus that have the DUF421-DUF1657 gene have improved spore heat resistance compared to spore-forming bacteria that do not have the DUF421-DUF1657 gene. Therefore, this method evaluates spore heat resistance by confirming the presence or absence of an amplification product of the partial base sequence of the DUF421-DUF1657 gene. Furthermore, the method of determining heat treatment conditions for spore-forming bacteria of the present invention is a method of determining heat treatment conditions suitable for the spore-forming bacteria based on the results of spore heat resistance.
  • the above-mentioned methods of the present invention will also be collectively referred to as the "method of the present invention.”
  • the "DUF421-DUF1657 gene” is a gene encoding a protein having a DUF421 domain and a DUF1657 domain, which is present on the spoVA2mob operon.
  • To determine whether the protein of interest has a DUF421 domain and a DUF1657 domain perform NCBI BLASTp analysis on the protein of interest. .1) and perform analysis using blastp (protein-protein BLAST) as the algorithm, and determine whether the coverage is 90% or more.
  • a gene encoding an amino acid sequence showing 90% or more coverage by the above method is referred to as a "DUF421-DUF1657 gene.”
  • spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions refers to bacteria belonging to the genus Bacillus that forms spores, and under aerobic conditions with a pH of 4.6 or more and less than 8.0. There is no particular restriction as long as it is a spore-forming bacterium that can grow under the following conditions. Examples of such spore-forming bacteria belonging to the genus Bacillus include bacteria belonging to the genus Bacillus that can cause spoilage and spoilage in neutral foods and drinks, such as the Bacillus subtilis group, which includes Bacillus subtilis .
  • Bacillus coagulans Bacillus cereus , Bacillus megaterium , Bacillus pumilus , Bacillus simplex , Bacillus anthracis , Bacillus brevis , Bacillus lentus, Bacillus mycoides , and the like.
  • the spore-forming bacteria are preferably Bacillus subtilis, Bacillus coagulans, and Bacillus cereus.
  • Bacillus subtilis group is a bacterial group defined in the database of the National Center for Biotechnology Information (NCBI) (Taxonomy ID: 653685).
  • the Bacillus subtilis group includes Bacillus subtilis, Bacillus licheniformis , Bacillus amyloliquefaciens , Bacillus siamensis , Bacillus velezensis .
  • Bacillus paralicheniformis Bacillus paralicheniformis
  • Bacillus sonorensis preferably Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus siamensis, and The group consisting of Bacillus berezensis is more preferable, and Bacillus subtilis is even more preferable.
  • the DUF421-DUF1657 genes are There is a correlation between the presence or absence of spores and spore heat resistance. That is, among the three types of bacteria mentioned above, the strain that has the DUF421-DUF1657 gene on its genome forms spores that have higher heat resistance than the strain that does not have the DUF421-DUF1657 gene on its genome.
  • the present inventors performed BLASTp analysis using the amino acid sequence encoded by the DUF421-DUF1657 gene derived from Bacillus subtilis strain B4146, and found that It was also revealed that the DUF421-DUF1657 genes are conserved in the genomes of many of the four strains.
  • nucleotide sequences of two DUF421-DUF1657 genes derived from Bacillus xiamensis strain SCSIO05746 and Bacillus berezensis strain 9912D (the proteins encoded by the genes, GenBank: AUJ76361.1 and GenBank: APA02936.1), -
  • the identity with the base sequences of the DUF421-DUF1657 genes derived from Subtilis B4146 strain, Bacillus licheniformis ATCC9789 strain, and Bacillus amyloliquefaciens SRCM101267 strain is all 99% or more.
  • DUF421- It can be inferred that there is a correlation between the presence or absence of the DUF1657 gene and spore heat resistance. That is, among the four types mentioned above, the strain having the DUF421-DUF1657 gene is thought to form spores with higher heat resistance than the strain not having the DUF421-DUF1657 gene.
  • the DUF421-DUF1657 gene is detected by the method of the present invention using a strain of the Bacillus subtilis group as a test strain (represented by a partial base sequence of the DUF421-DUF1657 gene using the nucleic acid primers described below)
  • a strain of the Bacillus subtilis group represented by a partial base sequence of the DUF421-DUF1657 gene using the nucleic acid primers described below
  • the nucleic acid is amplified, the presence of the amplification product is confirmed and the result is determined to be "positive"
  • the spores formed by the Bacillus subtilis strain having the DUF421-DUF1657 genes have high heat resistance.
  • the test strain does not have the DUF421-DUF1657 gene, and the spores formed
  • the heat resistance is relatively lower than the spore heat resistance of the Bacillus subtilis group strain having the DUF421-DUF1657 genes.
  • the D value (Decimal Reduction Value) of a strain of the Bacillus subtilis group having the DUF421-DUF1657 genes can be appropriately determined with reference to known information.
  • the DUF421-DUF1657 gene is detected by the method of the present invention when a Bacillus coagulans strain is used as the test strain (the nucleic acid expressed by the partial base sequence of the DUF421-DUF1657 gene is detected using the nucleic acid primers described below). (when the presence of the amplification product is confirmed and judged to be "positive"), the spores formed by the Bacillus coagulans strain having the DUF421-DUF1657 genes have high heat resistance.
  • the test strain does not have the DUF421-DUF1657 gene, and the spores formed
  • the heat resistance is relatively lower than the spore heat resistance of the Bacillus coagulans strain having the DUF421-DUF1657 genes.
  • the D value of the Bacillus coagulans strain having the DUF421-DUF1657 genes can also be appropriately determined with reference to the results of the Examples shown below. That is, for example, based on the results of Examples, the D105 °C value of Bacillus coagulans can be set to 10 minutes or more.
  • the D value (D 112.5°C value) of Bacillus subtilis at 112.5°C can be set to 1 minute or more
  • the D 112 of Bacillus coagulans determined to be positive by the method of the present invention .5° C. value can also be similarly evaluated and determined as 1 minute or more.
  • the present inventors performed BLASTp analysis using the amino acid sequence encoded by the DUF421-DUF1657 gene derived from Bacillus subtillus strain B4146, and found that the DUF421-DUF1657 gene is conserved in the genome of almost all Bacillus cereus strains. Furthermore, it was revealed that the DUF421-DUF1657 genes are additionally conserved in emetic toxin-producing strains of Bacillus cereus. This is thought to be because the emetic toxin producing gene and the DUF421-DUF1657 gene are encoded on the same plasmid.
  • the additional DUF421-DUF1657 gene specific to the emetic toxin-producing strain will also be referred to as a "plasmid-specific DUF421-DUF1657 gene.”
  • the identity between the base sequence of the DUF421-DUF1657 gene derived from Bacillus subtillus strain B4146 and the base sequence of the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus strain AH187 is approximately 66.9%.
  • the term "DUF421-DUF1657 gene" for Bacillus cereus means the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus.
  • a plasmid-specific DUF421-DUF1657 gene is detected by the method of the present invention when a Bacillus cereus strain is used as a test strain (a partial base of the plasmid-specific DUF421-DUF1657 gene is detected using a nucleic acid primer described below).
  • a nucleic acid primer described below.
  • the nucleic acid represented by the sequence is amplified, the presence of the amplification product is confirmed and the result is determined as “positive”), the Bacillus cereus strain carrying the plasmid-specific DUF421-DUF1657 genes will cause the spores formed to Has high heat resistance.
  • the test strain does not have the plasmid-specific DUF421-DUF1657 gene and is not formed.
  • the heat resistance of the spores is relatively lower than that of the Bacillus cereus strain having the plasmid-specific DUF421-DUF1657 genes.
  • the D value of the Bacillus cereus strain having the plasmid-specific DUF421-DUF1657 gene can be appropriately determined with reference to known information.
  • the DUF421-DUF1657 genes detected by the method of the present invention are conserved in the genome or plasmid of spore-forming bacteria that form spores with high heat resistance.
  • the nucleotide sequence encoding the DUF421 domain located upstream of the DUF421-DUF1657 gene (5'-end side) is also conserved in the genomes of spore-forming bacteria that do not have high heat resistance. There are cases. Therefore, the oligonucleotide used in the method of the present invention is one that satisfies all of the following conditions.
  • the oligonucleotide used in the method of the present invention has a GC content of 30% to 80%, does not self-anneal, has a Tm value of about 55 to 65°C, and is the origin of the DUF421-DUF1657 gene to be detected.
  • a region specific to the bacterial species in the case of Bacillus cereus, a region specific only to the plasmid-specific DUF421-DUF1657 gene, such that the sequence of the DUF421-DUF1657 gene encoded in the genome is not detected
  • It is an oligonucleotide that anneals to a highly conserved region within a bacterial species, and the length of the DNA fragment amplified by the oligonucleotide pair used in the method of the present invention is 1000 bp or less, and the oligonucleotide pair anneals to the DUF421 region (DUF421 domain
  • the ranges of the DUF421 region and DUF1657 region can be determined, for example, with reference to information
  • Oligonucleotide pair (1) An oligonucleotide pair consisting of the oligonucleotides (a) and (c) (hereinafter also referred to as “oligonucleotide pair (1)"), and an oligonucleotide pair consisting of the oligonucleotides (b) and (c) (hereinafter also referred to as “oligonucleotide pair (1)")
  • Oligonucleotide pair (2)'' can be used to detect the DUF421-DUF1657 genes derived from the Bacillus subtilis group.
  • can be used to detect the DUF421-DUF1657 gene means that it can hybridize to a specific region of the DUF421-DUF1657 gene and when the oligonucleotide pair is used as a primer pair, a portion of the DUF421-DUF1657 gene can be detected.
  • a nucleic acid consisting of a base sequence can be amplified.
  • the identity with the base sequence represented by SEQ ID NO: 1 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be.
  • oligonucleotide (a) one to several (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 1.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • Oligonucleotides that can be used for the detection of subtilis group are also preferred.
  • R in the base sequence represented by SEQ ID NO: 1 means a mixed base of adenine (A) and guanine (G).
  • the identity with the base sequence represented by SEQ ID NO: 2 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be.
  • the oligonucleotide (b) one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 2.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions.
  • Oligonucleotides that can be used for the detection of subtilis group are also preferred.
  • the identity with the base sequence represented by SEQ ID NO: 3 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be.
  • oligonucleotide (c) one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 3.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • Oligonucleotides that can be used for the detection of subtilis group are also preferred.
  • the identity of base sequences uses the Identities value calculated by NCBI Blastn analysis. During analysis, we use Somewhat similar sequences (blastn) for Program selection.
  • Bacillus subtilis B4146 This will be explained with reference to the nucleotide sequence of the DUF421-DUF1657 gene derived from Bacillus subtilis strain ATCC11774.
  • the nucleotide sequence of the DUF421-DUF1657 gene of Bacillus subtilis strain B4146 is shown in SEQ ID NO: 13, and the nucleotide sequence of the DUF421-DUF1657 gene of Bacillus subtilis ATCC 11774 strain is shown in SEQ ID NO: 14.
  • the oligonucleotides (a) and (c) correspond to the regions from positions 364 to 381 and from positions 695 to 712, respectively, of the base sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14.
  • the length of the amplified DNA fragment is approximately 350 bp. becomes.
  • the oligonucleotides (b) and (c) correspond to the regions from positions 658 to 675 and from positions 695 to 712, respectively, of the base sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14. .
  • PCR polymerase chain reaction
  • oligonucleotide pair (3) An oligonucleotide pair consisting of the oligonucleotides (d) and (j) (hereinafter also referred to as “oligonucleotide pair (3)"), an oligonucleotide pair consisting of the oligonucleotides (e) and (j) (hereinafter also referred to as “oligonucleotide pair (3)"), nucleotide pair (4)), an oligonucleotide pair consisting of the oligonucleotides (f) and (j) (hereinafter also referred to as "oligonucleotide pair (5)”), and the oligonucleotides (g) and (j).
  • oligonucleotide pair (6) an oligonucleotide pair consisting of the oligonucleotides (h) and (j) (hereinafter also referred to as “oligonucleotide pair (7)")
  • oligonucleotide pair (8) The oligonucleotide pair consisting of oligonucleotides (i) and (j) (hereinafter also referred to as “oligonucleotide pair (8)" can be used to detect the DUF421-DUF1657 gene derived from Bacillus coagulans.
  • the identity with the base sequence represented by SEQ ID NO: 4 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be.
  • the oligonucleotide (d) one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 4.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • Bacillus spp. that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • oligonucleotides that can be used to detect coagulance.
  • the identity with the base sequence represented by SEQ ID NO: 5 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be.
  • oligonucleotide (e) one to several (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 5.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • DUF421-DUF1657 genes is capable of growing under neutral conditions.
  • oligonucleotides that can be used to detect coagulance.
  • the identity with the base sequence represented by SEQ ID NO: 6 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Furthermore, as the oligonucleotide (f), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 6.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • Bacillus spp. that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • oligonucleotides that can be used to detect coagulance.
  • the identity with the base sequence represented by SEQ ID NO: 7 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be.
  • oligonucleotide (g) one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 7.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • DUF421-DUF1657 genes capable of growing under neutral conditions.
  • oligonucleotides that can be used to detect coagulance.
  • the identity with the base sequence represented by SEQ ID NO: 8 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be.
  • the oligonucleotide (h) one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 8.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • Bacillus spp. that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • oligonucleotides that can be used to detect coagulance.
  • the identity with the base sequence represented by SEQ ID NO: 9 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be.
  • oligonucleotide (i) one to several (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 9.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • DUF421-DUF1657 genes are capable of growing under neutral conditions.
  • oligonucleotides that can be used to detect coagulance.
  • the identity with the base sequence represented by SEQ ID NO: 10 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Further, as the oligonucleotide (j), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 10.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • Bacillus spp. that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • DUF421-DUF1657 genes capable of growing under neutral conditions.
  • oligonucleotides that can be used to detect coagulance.
  • the oligonucleotides (d) and (j) correspond to the regions from positions 223 to 240 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (3) and a DNA containing the Bacillus coagulans-derived DUF421-DUF1657 gene as a template, the length of the amplified DNA fragment is approximately 490 bp.
  • the oligonucleotides (e) and (j) correspond to the regions from positions 246 to 263 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (4) and a DNA having the Bacillus coagulans-derived DUF421-DUF1657 gene as a template, the length of the amplified DNA fragment is approximately 465 bp. Furthermore, the oligonucleotides (f) and (j) correspond to the regions from positions 558 to 575 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15.
  • the length of the amplified DNA fragment will be about 155 bp.
  • the oligonucleotides (g) and (j) correspond to the regions from positions 577 to 594 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15.
  • the length of the amplified DNA fragment will be about 135 bp.
  • the oligonucleotides (h) and (j) correspond to the regions from positions 592 to 609 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15.
  • the length of the amplified DNA fragment will be about 120 bp.
  • the oligonucleotides (i) and (j) correspond to the regions from positions 634 to 651 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (8) and DNA having the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be about 80 bp.
  • oligonucleotide pair consisting of oligonucleotides (k) and (l) (hereinafter also referred to as “oligonucleotide pair (9)") can be used to detect the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus. can.
  • the identity with the base sequence represented by SEQ ID NO: 11 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be.
  • oligonucleotide (k) one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 11.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • DUF421-DUF1657 genes capable of growing under neutral conditions.
  • oligonucleotides that can be used for the detection of C. cereus).
  • the identity with the base sequence represented by SEQ ID NO: 12 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Furthermore, as the oligonucleotide (l), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 12.
  • a spore-forming bacterium belonging to the genus Bacillus preferably Bacillus spp.
  • Bacillus spp. that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions.
  • nucleotides more preferably 1 nucleotide, more preferably 1 nucleotide
  • DUF421-DUF1657 genes capable of growing under neutral conditions.
  • oligonucleotides that can be used for the detection of C. cereus).
  • the oligonucleotides (k) and (l) correspond to the regions from positions 457 to 474 and from positions 699 to 721, respectively, of the base sequence set forth in SEQ ID NO: 16.
  • the length of the amplified DNA fragment is approximately 265 bp.
  • PCR Ribonucleic Acid Sequence-based Amplification
  • LCR Low Density C
  • SDA Strand Displacement Amplification
  • NASBA Nucleic Acid Sequence-based Amplification
  • RCA Rolling-circle
  • Conventional nucleic acid amplification methods can be used, such as the LAMP (Loop mediated isothermal amplification) method and the LAMP (Loop mediated isothermal amplification) method.
  • LAMP Loop mediated isothermal amplification
  • LAMP Loop mediated isothermal amplification
  • PCR conditions are not particularly limited as long as the target nucleic acid (amplified DNA fragment) can be amplified to a detectable extent.
  • Preferred examples of PCR reaction conditions include, for example, the oligonucleotide pair (1), the oligonucleotide pair (2), the oligonucleotide pair (3), the oligonucleotide pair (4), and the oligonucleotide pair (5). ), the oligonucleotide pair (6), the oligonucleotide pair (7), the oligonucleotide pair (8), and the oligonucleotide pair (9) as a nucleic acid primer.
  • a heat denaturation reaction to convert double-stranded DNA into single-stranded DNA is performed at 94 to 98°C, preferably 94°C for 10 to 60 seconds, and the primer pair is hybridized to the single-stranded DNA.
  • the annealing reaction is performed at 50 to 62°C, preferably 58 to 60°C for 30 to 60 seconds, and the elongation reaction with DNA polymerase is performed at approximately 72°C for 30 to 60 seconds. One cycle of these is approximately 30 to 60 seconds. Do 35 cycles.
  • PCR is performed using the oligonucleotide pair of the present invention as a primer pair, and the obtained PCR product By performing electrophoresis on the DNA fragments, amplification of DNA fragments having a specific size is observed. By performing such operations, it is possible to confirm whether the sample contains spore-forming bacteria belonging to the genus Bacillus that have the DUF421-DUF1657 genes and can grow under neutral conditions, and also to determine the length of the amplified product.
  • the bacterial species can be identified from
  • PCR may be performed using the above oligonucleotide pair alone as a primer pair, or PCR (Multiplex PCR) may be performed using a mixture of multiple types of oligonucleotide pairs as a primer pair. It's okay.
  • Each oligonucleotide pair used in the method of the present invention is designed to have a different amplification product length for each bacterial species so that it can also be used as an oligonucleotide pair for multiplex PCR.
  • the oligonucleotide pair (1) and the oligonucleotide pair (2) are oligonucleotide pairs that can specifically amplify the partial base sequence of the DUF421-DUF1657 gene derived from the Bacillus subtilis group;
  • the partial base sequence of the DUF421-DUF1657 gene derived from Bacillus cereus (for Bacillus cereus, the DUF421-DUF1657 gene encoded on the genome derived from Bacillus cereus and the plasmid-specific DUF421-DUF1657 gene) is not amplified.
  • the oligonucleotide pair (3), the oligonucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair (7), and the oligonucleotide pair (8) is an oligonucleotide pair that can specifically amplify the partial base sequence of the DUF421-DUF1657 gene derived from Bacillus coagulans.
  • the partial base sequences of the DUF421-DUF1657 gene encoded on the genome derived from B. cereus and the plasmid-specific DUF421-DUF1657 gene are not amplified.
  • the oligonucleotide pair (9) is an oligonucleotide pair that can specifically amplify the partial base sequence of the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus, and is encoded on the genome derived from Bacillus cereus.
  • DUF421-DUF1657 genes and partial base sequences of DUF421-DUF1657 genes derived from Bacillus subtilis group and Bacillus coagulans are not amplified.
  • the preferred combination of each oligonucleotide pair is: a pair of oligonucleotides (preferably the oligonucleotide pair (1)) selected from the group consisting of the oligonucleotide pair (1) and the oligonucleotide pair (2); At least one pair of oligonucleotides selected from the group consisting of the nucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair (7), and the oligonucleotide pair (8). and the oligonucleotide pair (9).
  • confirmation of amplification of DNA fragments can be performed by conventional methods.
  • examples include a method of performing electrophoresis on the amplified product to confirm the presence or absence of a band corresponding to the size of the amplified gene, a method of measuring the amount of the amplified product over time, and a method of decoding the base sequence of the amplified product.
  • the present invention is not limited to these methods.
  • a preferred method is to perform electrophoresis after amplification of a DNA fragment and confirm the presence or absence of a band corresponding to the size of the amplified DNA fragment.
  • detection of amplification products can be performed by conventional methods.
  • methods that incorporate nucleotides labeled with radioactive substances during amplification reactions methods that use primers labeled with fluorescent substances, etc., and methods that increase fluorescence intensity by binding DNA such as ethidium bromide between the amplified DNA double strands.
  • methods that incorporate nucleotides labeled with radioactive substances during amplification reactions methods that use primers labeled with fluorescent substances, etc., and methods that increase fluorescence intensity by binding DNA such as ethidium bromide between the amplified DNA double strands.
  • Examples include a method of introducing a fluorescent substance that increases the intensity, but the present invention is not limited to these methods.
  • a method is preferred in which a fluorescent substance that increases fluorescence intensity by binding to DNA is inserted between the amplified DNA double strands.
  • the method for preparing the above oligonucleotides (a) to (l) used in the method of the present invention is not particularly limited.
  • it can be chemically synthesized based on a designed sequence or purchased from a reagent manufacturer.
  • chemical synthesis based on a designed sequence it can be synthesized using an oligonucleotide synthesizer or the like.
  • oligonucleotides having base sequences in which one or several bases are substituted, deleted, inserted, or added can also be synthesized using conventional methods.
  • you can use, for example, FASMAC's DNA/RNA contract synthesis service or Thermo Fisher Scientific's GeneArt artificial gene synthesis service.
  • test substance used in the present invention there are no particular restrictions on the test substance used in the present invention, and food and drink products themselves, raw materials for food and drink products, isolated bacterial cells, cultured bacterial cells, and the like can be used.
  • neutral food and drink products and raw materials used therein can be used. It is preferable to use it as an analyte.
  • neutral foods and drinks include neutral tea drinks such as green tea drinks, black tea drinks, barley tea drinks, coffee drinks, milk drinks, retort foods, and raw materials used therein.
  • the method for preparing DNA from the specimen is not particularly limited as long as DNA can be obtained with sufficient purity and amount to detect spore-forming bacteria, and it can be used in an unpurified state, but It can also be used after pretreatment such as separation, extraction, concentration, and purification.
  • the nucleic acid can be purified using phenol and chloroform extraction or purified using a commercially available extraction kit to increase the purity of the nucleic acid before use.
  • DNA obtained by reverse transcription of RNA in a subject can also be used.
  • the method for isolating bacteria from foods or raw materials is not particularly limited, and for example, bacteria can be isolated by the method described in Examples below.
  • the spore-forming bacteria When spore-forming bacteria having DUF421-DUF1657 genes are detected in raw materials or products by the method of the present invention, the spore-forming bacteria form highly heat-resistant spores. Therefore, for raw materials or products in which such spore-forming bacteria have been detected, the heat treatment temperature must be set to conditions that can sufficiently kill the spore-forming bacteria (conditions that ensure commercial sterility of heat-treated foods). ).
  • the term "commercial sterility" of heat-treated food refers to the application of heat to eliminate microorganisms that can grow in the food under non-refrigerated conditions during normal storage, distribution, etc., and that are harmful to public health.
  • heat treatment conditions can be appropriately set according to the evaluation results of spore heat resistance (presence or absence of DUF421-DUF1657 genes) and the bacterial species and strain to be identified. For example, if it is confirmed that all spore-forming bacteria belonging to the genus Bacillus detected from various raw materials of neutral food and drink products do not have the DUF421-DUF1657 gene, or when spore-forming bacteria belonging to the genus Bacillus extracted from raw materials If the PCR results for detecting the DUF421-DUF1657 genes on bacterial-derived DNA are negative, this indicates that there is an extremely low possibility that the raw material contains highly heat-resistant spores.
  • "spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions” was detected in products or intermediate products that underwent a specific sterilization process, and furthermore, the method of the present invention detected the spore-forming bacteria with the DUF421-DUF1657 gene. If it is found that the sterilization conditions are insufficient, it can be determined that the sterilization conditions were insufficient.
  • the heat treatment conditions temperature, time, pressure, etc.
  • sufficient to sterilize each bacterial species and strain can be determined, for example, based on the usual methods in this technical field, or by the methods described in the Examples. can also be determined.
  • the steps from the preparation of the specimen to the step of detecting spore-forming bacteria that form highly heat-resistant spores can be carried out in a short time.
  • the specimen is a suspension inoculated with colonies of each strain, it will take about half a day; as in Example 7, the raw material will be the specimen contained in the raw material.
  • the raw material will be the specimen contained in the raw material.
  • it can be carried out in a short time of about 2 days.
  • the kit for detecting spore-forming bacteria having the DUF421-DUF1657 genes of the present invention contains the detection oligonucleotide pair of the present invention as a primer pair.
  • This kit can be used in the method of the invention.
  • the kit of the present invention may contain, depending on the purpose, labeled detection substances, buffers, nucleic acid synthases (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), enzyme substrates (dNTPs, rNTPs, etc.), etc. Contains substances commonly used to detect fungi.
  • the kit of the present invention may contain a positive control for confirming that a detection reaction is possible using the detection oligonucleotide of the present invention. Examples of positive controls include DNA containing the region amplified by the method of the present invention.
  • the present invention further discloses the following detection method, method for evaluating spore heat resistance, method for determining heat treatment conditions, kit for detecting spore-forming bacteria, and oligonucleotide pair.
  • An oligonucleotide pair (1) consisting of the following oligonucleotides (a) and (c), An oligonucleotide pair (2) consisting of the following oligonucleotides (b) and (c), An oligonucleotide pair (3) consisting of the following oligonucleotides (d) and (j), An oligonucleotide pair (4) consisting of the following oligonucleotides (e) and (j), An oligonucleotide pair (5) consisting of the following oligonucleotides (f) and (j), An oligonucleotide pair (6) consisting of the following oligonucleotides (g) and (j), An oligonucleotide pair (7) consisting of the following oligonucleotides (h) and (j), An oligonucleotide pair (8) consisting of the following oligonucleotides (i) and (
  • a method for detecting spore-forming bacteria belonging to the genus Bacillus (a) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 1, or an identity with the base sequence represented by SEQ ID NO: 1 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus subtilis group, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
  • An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an identity with the base sequence represented by SEQ ID NO: 3 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus subtilis group, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
  • An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or an identity with the base sequence represented by SEQ ID NO: 4 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
  • An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5, or an identity with the base sequence represented by SEQ ID NO: 5 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
  • An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6, or an identity with the base sequence represented by SEQ ID NO: 6 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
  • An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or an identity with the base sequence represented by SEQ ID NO: 9 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
  • An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 11, or an identity with the base sequence represented by SEQ ID NO: 11 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus cereus, which is preferably 95% or more and has a plasmid-specific DUF421-DUF1657 gene and can grow under neutral conditions.
  • An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12, or an identity with the base sequence represented by SEQ ID NO: 12 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus cereus, which is preferably 95% or more and has a plasmid-specific DUF421-DUF1657 gene and can grow under neutral conditions.
  • ⁇ 2> The method according to ⁇ 1> above, wherein the spore-forming bacterium is identified by the amplification product.
  • ⁇ 3> The method according to ⁇ 1> or ⁇ 2>, wherein the spore heat resistance of the spore-forming bacteria is evaluated based on the presence or absence of the amplification product.
  • a partial base sequence of the DUF421-DUF1657 gene is prepared using at least one oligonucleotide pair selected from the group consisting of the nucleotide pair (7), the oligonucleotide pair (8), and the oligonucleotide pair (9) as a nucleic acid primer.
  • spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions are spore-forming bacteria that can grow under aerobic conditions with a pH of 4.6 or more and less than 8.0.
  • the spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions include Bacillus subtilis group, Bacillus coagulans, Bacillus cereus, Bacillus megaterium, Bacillus pumilus, Bacillus simplex, Bacillus anthracis, and Bacillus.
  • Bacillus subtilis group is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus siamensis, Bacillus belezensis, Bacillus paralicheniformis, and Bacillus sonorensis.
  • At least one species preferably at least one species selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus siamensis, and Bacillus veresensis, and more preferably Bacillus subtilis.
  • Bacillus subtilis Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus siamensis, and Bacillus veresensis, and more preferably Bacillus subtilis.
  • the oligonucleotide (a) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 1, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 1. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (b) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 2. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (c) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 3. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (d) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or one to several, preferably 1 to 4, more preferably 1 to 4 in the base sequence represented by SEQ ID NO: 4. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (e) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 5. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (f) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6, or one to several, preferably 1 to 4, more preferably 1 to 4 in the base sequence represented by SEQ ID NO: 6. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (g) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 7. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (h) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or one to several, preferably one to four, more preferably one to several in the base sequence represented by SEQ ID NO: 8. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (i) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 9. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (j) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 10, or one to several, preferably 1 to 4, more preferably 1 to 4 in the base sequence represented by SEQ ID NO: 10. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions.
  • the oligonucleotide (k) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 11, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 11. is a neutral base in which 1 to 3 bases, more preferably 1 or 2 bases, and still more preferably 1 base is deleted, substituted, inserted or added, and has a plasmid-specific DUF421-DUF1657 gene.
  • the method according to any one of ⁇ 1> to ⁇ 18> above which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions, preferably Bacillus cereus.
  • the oligonucleotide (l) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12, or one to several, preferably 1 to 4, more preferably 1 to 4 in the base sequence represented by SEQ ID NO: 12. is a neutral base in which 1 to 3 bases, more preferably 1 or 2 bases, and still more preferably 1 base is deleted, substituted, inserted or added, and has a plasmid-specific DUF421-DUF1657 gene.
  • ⁇ 1> to ⁇ 19> above which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions, preferably Bacillus cereus.
  • amplify the expressed nucleic acid amplifying the nucleic acid represented by the partial base sequence of the plasmid-specific DUF421-DUF1657 gene by polymerase chain reaction using the oligonucleotide pair (9) as a primer pair; The method according to any one of ⁇ 1> to ⁇ 20> above.
  • oligonucleotide pair (1) consisting of the oligonucleotides (a) and (c)
  • an oligonucleotide pair (2) consisting of the oligonucleotides (b) and (c)
  • an oligonucleotide pair (2) consisting of the oligonucleotides (d) and (j).
  • the oligonucleotide (a) is the oligonucleotide described in ⁇ 9> above
  • the oligonucleotide (b) is the oligonucleotide described in ⁇ 10> above
  • the oligonucleotide (c) is the oligonucleotide described in ⁇ 11> above
  • the oligonucleotide (d) is the oligonucleotide according to ⁇ 12> above
  • the oligonucleotide (e) is the oligonucleotide described in ⁇ 13> above
  • the oligonucleotide (f) is the oligonucleotide described in ⁇ 14> above
  • the oligonucleotide (g) is the oligonucleotide described in ⁇ 15> above
  • the oligonucleotide pair (1) and the oligonucleotide pair (2) are oligonucleotide pairs that can be used for detection of Bacillus subtilis group
  • the oligonucleotide pair (3), the oligonucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair (7), and the oligonucleotide pair (8) are Bacillus ⁇ An oligonucleotide pair that can be used to detect coagulance
  • the oligonucleotide pair (9) is an oligonucleotide pair that can be used for detection of Bacillus cereus;
  • Example 1 Multiplex PCR of 3 bacterial species groups
  • Primer design Based on the base sequence information of the DUF421-DUF1657 gene (SEQ ID NO: 13) derived from Bacillus subtilis strain B4146, a primer (primer 1) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 1, A primer (primer 3) represented by the base sequence of SEQ ID NO: 3 was designed.
  • a primer (primer 5) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 5
  • SEQ ID NO: A primer (primer 10) represented by a 10 base sequence was designed.
  • a primer (primer 11) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 11 and a primer of SEQ ID NO: 12 were prepared.
  • a primer (primer 12) represented by a base sequence was designed. Based on the information on each designed primer, primers for a reversed phase column purified product were obtained from FASMAC's DNA/RNA contract synthesis service. The nucleotide sequence information of the DUF421-DUF1657 genes of each bacterial species was obtained from NCBI (National Center for Biotechnology Information). When each bacterial strain has DUF421-DUF1657 genes, the length of the DNA fragment amplified by the primer pair of primer 1 and primer 3 is about 350 bp, and the length of the DNA fragment amplified by the primer pair of primer 5 and primer 10 is about 350 bp. The length of the DNA fragment amplified by the primer pair of primer 11 and primer 12 is approximately 265 bp.
  • Bacillus subtillus strain JCA1403, Bacillus coagulans strain NBRC12583, and strain JCA1108 each have the DUF421-DUF1657 genes involved in improving spore heat resistance on their genomes, and Bacillus cereus strain JCM17690 on their plasmids.
  • SCD agar medium (trade name: SCD medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and Bacillus subtilis and Bacillus cereus were incubated at 30°C, and Bacillus coagulans was incubated at 30°C. The plate was allowed to stand at a temperature of 45°C and cultured for 1 to 2 days.
  • SCD liquid medium product name: SCD liquid medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • the cells were cultured for 2 to 3 days under temperature conditions.
  • genomic DNA Collect 1.0 mL of the suspension from each SCD liquid medium after culturing, and use a genomic DNA preparation kit (product name: NucleoSpin Tissue, manufactured by Takara Bio Inc.) to lyse the bacterial cells. DNA was extracted from the bacterial cells according to the protocol attached to the kit, except that a lysis method using lysozyme was used. Note that since DNA including plasmids can be extracted with this kit, the extracted DNA will be hereinafter referred to as "extracted DNA”. The concentration of the obtained extracted DNA solution was adjusted to 50 ng/ ⁇ L.
  • a Multiplex PCR solution was prepared using Multiplex PCR Assay Kit Ver. 2 (trade name, manufactured by Takara Bio Inc.). 0.25 ⁇ L of Multiplex PCR Enzyme Mix, 25 ⁇ L of 2x Multiplex PCR Buffer (Mg 2+ , dNTP plus), 1 ⁇ L of extracted DNA solution (50 ng of extracted DNA), and primers 1, 3, 5, 10, 11, and 12, respectively. The concentration was 0.2 ⁇ M, and sterile distilled water was added so that the volume of the Multiplex PCR solution was 50 ⁇ L.
  • Example 2 Multiplex PCR of 3 bacterial species groups (1) Primer design Based on the base sequence information of the DUF421-DUF1657 gene (SEQ ID NO: 13) derived from Bacillus subtilis strain B4146, a primer (primer 2) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 2 was created. Designed. When the Bacillus subtillus B4146 strain has the DUF421-DUF1657 gene, the length of the DNA fragment amplified by the primer pair of primer 2 and primer 3 used in Example 1 is about 55 bp.
  • primers for a reversed phase column purified product were obtained from FASMAC's DNA/RNA contract synthesis service.
  • the length of the DNA fragment amplified by the primer pair of primer 4 and primer 10 is about 490 bp
  • the length of the DNA fragment amplified by the primer pair of primer 6 and primer 10 is about 490 bp.
  • the length of the DNA fragment amplified by the primer pair of primer 7 and primer 10 is about 135 bp
  • the length of the DNA fragment amplified by the primer pair of primer 8 and primer 10 is about 135 bp.
  • the length of the DNA fragment is approximately 120 bp
  • the length of the DNA fragment amplified by the primer pair of primer 9 and primer 10 is approximately 80 bp.
  • Templates used for multiplex PCR include the extracted DNA solution derived from Bacillus subtilis JCA1403 strain obtained in Example 1, the extracted DNA solution derived from Bacillus coagulans NBRC12583 strain, and Bacillus subtilis strain JCA1403 obtained in Example 1. - Extracted DNA solutions derived from the S. cereus JCM17690 strain were used. Multiplex PCR was performed under the conditions described in Example 1 using the combinations of primer pairs shown in Table 2 below, and an agarose gel image was obtained after electrophoresis. Images are shown in FIGS. 2 and 3. Note that lane numbers 11 and 18 are marker 100bp DNA Step Ladder (Promega).
  • the amplification product specific to the DUF421-DUF1657 gene derived from the Bacillus subtilis group is indicated by a left-pointing black triangle
  • the amplification product specific to the DUF421-DUF1657 gene derived from Bacillus coagulans is indicated by an asterisk
  • the plasmid derived from Bacillus cereus Specific DUF421-DUF1657 gene-specific amplification products are indicated by left-pointing arrowheads, respectively.
  • the DUF421-DUF1657 gene could be detected in a bacterial species-specific manner with any combination of primer pairs in the test sample, which is a mixed DNA derived from different bacterial species. Ta.
  • Example 3 Spore heat resistance evaluation test 1 (1) Sample preparation, PCR Bacillus subtilis group strains include JCA strains (Japan Canned and Bottled Retort Food Association) (JCA1402, JCA1403, JCA1404, JCA1405, JCA1407, JCA1408, JCA1409, JCA1410, JCA1411), JCM strains. (National Research and Development Corporation RIKEN Microbial Materials Development Office) (JCM1465 strain), KM strain (environmentally isolated strain) (KM166 strain, KM167 strain), and 168 strain were used. Each of the above strains was cultured in the same manner as in Example 1 to prepare an extracted DNA solution.
  • JCA strains Japan Canned and Bottled Retort Food Association
  • JCM strains National Research and Development Corporation RIKEN Microbial Materials Development Office
  • JCM1465 strain
  • PCR was performed in the same manner as in Example 1, and the presence or absence of DNA bands was confirmed by electrophoresis. The case where a DNA fragment could be detected at the desired position was judged as "positive”, and the case where no DNA fragment could be detected at the desired position was judged as "negative”. The results are shown in Table 3 below, together with the presence or absence of the DUF421-DUF1657 gene in each strain.
  • the collected product was centrifuged at 8,000 ⁇ g, 4° C., and 20 minutes, and the supernatant was removed and washed with ice-cold sterilized water again for a total of 5 times.
  • the collected material (precipitate) after washing was suspended in ice-cold sterilized water to a volume of approximately 3 mL per culture plate, divided into small portions, and stored frozen at -20°C until use. .
  • the number of spores was determined by heat treatment at 80°C for 10 minutes and serially diluted spore solution on SCD agar medium (trade name: SCD agar medium "Daigo", for general bacterial testing, Fuji It was measured by smearing the film on a film (manufactured by Wako Pure Chemical Industries, Ltd.).
  • SCD agar medium trade name: SCD liquid medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • Level 1 Killed by heating at 90°C for 30 minutes
  • Level 2 Not killed by heating at 90°C for 30 minutes, but killed by heating at 95°C for 30 minutes
  • Level 3 Not killed by heating at 95°C for 30 minutes
  • 4 Killed by heating at 100°C for 30 minutes, killed by heating at 105°C for 10 minutes
  • Level 5 Not killed by heating at 105°C for 10 minutes
  • the D value (112.5° C.) of the above-mentioned Bacillus subtilis group strain was entrusted to the Japan Canned and Bottled Retort Foods Association and measured by the following method.
  • the heat resistance of each test bacterial strain spore in M/15 phosphate buffer (PH7.0) was measured.
  • the test method is to serially dilute the spore solution of the test bacterial strain with sterile 0.1% peptone water, suspend it in M/15 phosphate buffer and each test solution to a concentration of approximately 10 5 CFU/mL, and mix. After that, it was dispensed into TDT tubes and sealed. This TDT tube was heat-treated under predetermined conditions. The TDT tube after heating was opened, the number of spores was measured, and the D value was calculated. The results are shown in Table 3 below.
  • strains of the Bacillus subtilis group that do not have the DUF421-DUF1657 genes and were determined to be "negative” by Multiplex PCR had a heat resistance level of level 2 in the simple heat resistance test, and The D112.5°C values of Bacillus subtilis JCA1402 strain and JCM1465 strain, which were determined to be "negative,” were also 0.09 minutes and 0.08 minutes, respectively. From the above, it has become clear that there is a correlation between the results of Multiplex PCR and heat resistance.
  • Example 4 Spore heat resistance evaluation test 2 (1) Sample preparation, PCR Bacillus coagulans strains include NBRC strain (National Institute of Technology and Evaluation) (NBRC12583 strain), JCA strain (Japan Canned and Bottled Retort Food Association) (JCA1108 strain, JCA1109 strain, JCA1116 strain, JCA1117 strain, JCA1120 strain) , JCA1122 strain, JCA1158 strain, JCA1174 strain, JCA1180 strain, JCA1182 strain), DSM strain (Leibniz Institute German Microbial Strain Archive) (DSM2308 strain, DSM2311 strain, DSM2312 strain, DSM2314 strain, DSM2350 strain, DSM2356 strain, DSM2383 strain) , DSM2384 strain, DSM2385) were used.
  • NBRC12583 strain National Institute of Technology and Evaluation
  • JCA strain Japan Canned and Bottled Retort Food Association
  • JCA1108 strain JCA1109 strain, JCA1116 strain
  • Example 1 Each of the above strains was cultured in the same manner as in Example 1 to prepare an extracted DNA solution. Furthermore, PCR was performed in the same manner as in Example 1, and the presence or absence of DNA bands was confirmed by electrophoresis. The case where a DNA fragment could be detected at the desired position was judged as "positive”, and the case where no DNA fragment could be detected at the desired position was judged as "negative”. The results are shown in Table 4 below, together with the presence or absence of the DUF421-DUF1657 gene in each strain.
  • Example 5 Spore heat resistance evaluation test 3
  • JCM2152 strain, JCM17690 strain was used as the Bacillus cereus strain.
  • JCM2152 strain, JCM17690 strain was used as the Bacillus cereus strain.
  • Each of the above strains was cultured in the same manner as in Example 1 to prepare an extracted DNA solution.
  • PCR was performed in the same manner as in Example 1, and the presence or absence of DNA bands was confirmed by electrophoresis. The case where a DNA fragment could be detected at the desired position was judged as "positive", and the case where no DNA fragment could be detected at the desired position was judged as "negative”.
  • Table 5 The results are shown in Table 5 below, together with the presence or absence of the plasmid-specific DUF421-DUF1657 gene in each strain.
  • the Bacillus cereus strain determined to be positive by Multiplex PCR had a D 90°C value of 82.9 minutes. Furthermore, for the Bacillus cereus strain that was determined to be negative by Multiplex PCR, the D90 °C value was 24.3 minutes. Thus, a correlation was observed between the results of Multiplex PCR and heat resistance (D value).
  • Example 6 Preparation of specimens for high-throughput analysis of raw material isolates
  • MicroSEQ 500 16S rDNA Sequencing Kit manufactured by Thermo Fisher Scientific
  • Bacterial strains identified as Bacillus subtilis group, Bacillus coagulans, and Bacillus cereus were targeted for analysis.
  • Each of the identified bacterial species was cultured in the same manner as in Example 1 to prepare an extracted DNA solution.
  • PCR was performed in the same manner as in Example 1, and the presence or absence of DNA bands was confirmed by electrophoresis. The case where a DNA fragment could be detected at the desired position was judged as "positive", and the case where no DNA fragment could be detected at the desired position was judged as "negative”.
  • the results of Multiplex PCR are shown in Table 6 below.
  • Example 7 Proposal of sensitivity and heat treatment conditions for comprehensive inspection of heat-resistant spore bacteria on raw materials (1) Preparation of highly heat-resistant spore-attached raw material 10 g of raw material with raw material ID: 28 used in Example 6 was heated at 80°C for 10 minutes.
  • the spores of Bacillus subtilis strain JCA1403 (a strain that forms highly heat-resistant spores) whose vegetative cells have been killed by heat treatment are 5.0 ⁇ 10 7 , 5.0 ⁇ 10 5 , 5.0 ⁇ 10 3 , 5.0 ⁇ 10 per gram of raw material. 1 , 5.0 ⁇ 10 -1 spores.
  • As a control test a raw material to which no spores were attached was used. Thereafter, the raw material was allowed to stand until it was sufficiently dry, to prepare a raw material to which highly heat-resistant spores were attached in a pseudo manner.
  • Bacterial body recovery step 90 g of physiological saline was added to the above raw materials, and pulverized and homogenized using a stomacher to collect 30 mL of suspension. Thereafter, centrifugation was performed at 100 ⁇ g for 5 minutes to discard the raw material residue, and further centrifugation was performed at 8000 ⁇ g for 20 minutes to collect precipitated bacterial cells. The collected bacterial cells were resuspended in 3 mL of physiological saline.
  • Results The results of Multiplex PCR and the results of beverage spoilage after heat treatment are shown in Table 7 below.
  • the "estimated number of spores (spores/ml)" in the table indicates the estimated number of spores in the neutral tea beverage and SCD liquid medium in the heating step and the Multiplex PCR test.
  • the method of the present invention can remove the contamination. This shows that it can be detected with high sensitivity.
  • the method of the present invention it is possible to confirm the presence or absence of the DUF421-DUF1657 gene in spore-forming bacteria that can grow under neutral conditions, and it is also possible to detect spore-forming bacteria based on the amplification product. Furthermore, by confirming the presence or absence of the DUF421-DUF1657 gene, the spore heat resistance of spore-forming bacteria can be evaluated, and appropriate heat treatment conditions can be determined for the raw material in which spore-forming bacteria are detected.

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Abstract

A method for detecting a spore-forming bacterium belonging to the genus Bacillus, said method comprising amplifying a nucleic acid represented by a partial base sequence of DUF421-DUF1657 gene using a pair of oligonucleotides having specific sequences as nucleic acid primers, and detecting a spore-forming bacterium that can grow under neutral conditions on the basis of the presence or absence of an amplification product.

Description

芽胞形成細菌の検出方法Method for detecting spore-forming bacteria

　本発明は、芽胞形成細菌の検出方法に関する。 The present invention relates to a method for detecting spore-forming bacteria.

　芽胞形成細菌（spore-forming bacteria）は、熱、乾燥、紫外線等の物理的処理に対し高い耐久性を示す芽胞を形成する。芽胞形成細菌としては、例えばバチルス（Bacillus）属細菌やクロストリジウム（Clostridium）属細菌を代表とした多くの菌種が知られており、このような菌種は特に栄養飢餓状態や乾燥、高温条件下などの生育に適さない環境に晒されることで芽胞を形成する。
　バチルス属に属する細菌等の芽胞形成細菌は水中や土壌等に広く生育しているため、食品原料との接触や飲食品の製造工程において飲食品（製品）内に混入する危険性が高い。通常、多くの菌は例えば７５℃で３０分間の加熱処理を行うことで死滅する。しかし、芽胞形成細菌の中には１００℃付近の高温環境にも耐えうる芽胞を形成するものがあり、このような菌は７５℃で３０分間の加熱処理条件では十分に殺菌できない場合がある。芽胞形成細菌の殺菌が不十分である場合、芽胞形成細菌が製品内で増殖し、製品の腐敗や変敗を引き起こす原因となり得る。他方、このような高い耐熱性を示す芽胞形成細菌であっても、加熱処理温度をより高く設定することで殺菌することは可能であるが、高温処理による原料や製品の劣化、加熱処理コスト等を考慮すると、混入した芽胞形成細菌の耐熱性を判断、考慮して加熱処理条件を適宜決定することが好ましい。 Spore-forming bacteria form spores that are highly resistant to physical treatments such as heat, desiccation, and ultraviolet light. Many species of spore-forming bacteria are known, including bacteria of the genus Bacillus and bacteria of the genus Clostridium . They form spores when exposed to environments unsuitable for growth.
Spore-forming bacteria, such as bacteria belonging to the genus Bacillus, widely grow in water and soil, so there is a high risk of them coming into contact with food raw materials or contaminating food and drink products (products) during the food and drink manufacturing process. Usually, many bacteria are killed by heat treatment at 75° C. for 30 minutes, for example. However, some spore-forming bacteria form spores that can withstand high-temperature environments around 100°C, and such bacteria may not be sufficiently sterilized by heat treatment at 75°C for 30 minutes. If sterilization of spore-forming bacteria is insufficient, the spore-forming bacteria may proliferate within the product, causing spoilage or deterioration of the product. On the other hand, even spore-forming bacteria that exhibit such high heat resistance can be sterilized by setting the heat treatment temperature higher, but high temperature treatment can cause deterioration of raw materials and products, heat treatment costs, etc. Considering this, it is preferable to judge and take into account the heat resistance of the mixed spore-forming bacteria and appropriately determine the heat treatment conditions.

　これまで芽胞形成細菌の同定方法は、芽胞形成細菌を単離して培地で培養した後に、顕微鏡でその形態を観察し同定することにより行われてきた。しかし、この方法は、判定の指標となる各菌種の特徴的な形態が形成されるまでに２週間程度の期間が必要であり、また、正確な判定には熟練を要する等の問題があり、迅速かつ正確な検出を行うことが難しかった。
　また、例えば同一菌種の芽胞形成細菌であっても、その芽胞耐熱性は株ごとに異なることが明らかとなっている。例えば、Berendsen et al., Food Microbiol., 2015, Vol. 45, 18-25には、芽胞形成細菌の１種である枯草菌（バチルス・サブチルス（Bacillus subtilis））の芽胞が、多様な耐熱性を示すことが開示されている。また、Berendsen et al., ISME J., 2016, Vol. 10, 2633-2642には、バチルス・サブチルスの比較ゲノム解析により見出されたトランスポゾン由来spoVA2mobオペロンの中の、DUF421ドメイン及びDUF1657ドメインを有する機能未知のタンパク質をコードする遺伝子が、芽胞耐熱性の向上に寄与することが報告されている。 Up until now, spore-forming bacteria have been identified by isolating the spore-forming bacteria, culturing them in a medium, and then observing and identifying their morphology under a microscope. However, this method requires approximately two weeks for the characteristic morphology of each bacterial species to form, which can be used as an indicator for determination, and there are other problems such as the need for skill to make accurate determinations. , it was difficult to perform quick and accurate detection.
Furthermore, it has been revealed that, even among spore-forming bacteria of the same species, the spore heat resistance varies depending on the strain. For example, in Berendsen et al., Food Microbiol., 2015, Vol. 45, 18-25, spores of Bacillus subtilis , a type of spore-forming bacterium, have a variety of heat-resistant It is disclosed that it shows. In addition, Berendsen et al., ISME J., 2016, Vol. 10, 2633-2642 describes a transposon-derived spoVA2mob operon that contains the DUF421 domain and DUF1657 domain, which was discovered through comparative genome analysis of Bacillus subtilis. It has been reported that a gene encoding a protein of unknown function contributes to improving spore heat resistance.

　芽胞形成細菌の芽胞耐熱性の評価方法としては、例えば従来より、上記のように芽胞形成細菌の単離・同定を行い、さらにその菌種に適した生育培地、温度、時間を考慮した上で芽胞を形成させ、種々の加熱処理条件に晒した際の生存率を測定することにより芽胞耐熱性を評価する方法が用いられている。
　また、例えば、特開２００９－１８３２０７号公報には、芽胞の硬度をカンチレバーが装備された原子間力顕微鏡等により、リアルタイムで迅速かつ正確な芽胞の耐久性評価・測定を行う方法が開示されている。 For example, the conventional method for evaluating the spore heat resistance of spore-forming bacteria is to isolate and identify the spore-forming bacteria as described above, and then consider the growth medium, temperature, and time appropriate for the bacterial species. A method is used to evaluate spore heat resistance by forming spores and measuring the survival rate when exposed to various heat treatment conditions.
Furthermore, for example, Japanese Patent Application Publication No. 2009-183207 discloses a method for quickly and accurately evaluating and measuring the durability of spores in real time using an atomic force microscope equipped with a cantilever. There is.

　本発明は、下記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、下記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対、下記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、下記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、下記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、下記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、下記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、下記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対、並びに下記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対、からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を核酸プライマーとして用いて、DUF421-DUF1657遺伝子の部分塩基配列で表される核酸を増幅し、増幅産物の有無により中性条件で増殖可能なバチルス属に属する芽胞形成細菌を検出する、バチルス属に属する芽胞形成細菌の検出方法に関する。
 
（ａ）配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｂ）配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｃ）配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｄ）配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｅ）配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｆ）配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｇ）配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｈ）配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｉ）配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｊ）配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｋ）配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｌ）配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。 The present invention provides an oligonucleotide pair consisting of the following oligonucleotides (a) and (c), an oligonucleotide pair consisting of the following oligonucleotides (b) and (c), and an oligonucleotide consisting of the following oligonucleotides (d) and (j). an oligonucleotide pair consisting of the following oligonucleotides (e) and (j), an oligonucleotide pair consisting of the following oligonucleotides (f) and (j), an oligonucleotide pair consisting of the following oligonucleotides (g) and (j), From an oligonucleotide pair consisting of the following oligonucleotides (h) and (j), an oligonucleotide pair consisting of the following oligonucleotides (i) and (j), and an oligonucleotide pair consisting of the following oligonucleotides (k) and (l). Using at least one oligonucleotide pair selected from the group as a nucleic acid primer, the nucleic acid represented by the partial base sequence of the DUF421-DUF1657 gene is amplified, and depending on the presence or absence of the amplification product, Bacillus genus that can grow under neutral conditions is The present invention relates to a method for detecting spore-forming bacteria belonging to the genus Bacillus.

(a) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(b) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(c) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 3 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(d) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 4 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(e) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 5, or an oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 5, and growing under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(f) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 6, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(g) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 7 and grows under neutral conditions and has the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(h) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 8 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or an oligonucleotide with 80% or more identity with the base sequence represented by SEQ ID NO: 9, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(j) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 10, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(k) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 11, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 11 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions.
(l) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 12 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions.

　また本発明は、前記増幅産物により中性条件で増殖可能なバチルス属に属する芽胞形成細菌の芽胞耐熱性を評価する、バチルス属に属する芽胞形成細菌の芽胞耐熱性の評価方法に関する。
　また本発明は、前記方法により評価した芽胞耐熱性に基づいて、バチルス属に属する芽胞形成細菌の加熱処理条件を決定する、バチルス属に属する芽胞形成細菌の加熱処理条件の決定方法に関する。
　さらに本発明は、上記方法に用いる芽胞形成細菌検出用のキット及びオリゴヌクレオチド対に関する。 The present invention also relates to a method for evaluating the spore heat resistance of a spore-forming bacterium belonging to the genus Bacillus, which evaluates the spore heat resistance of a spore-forming bacterium belonging to the genus Bacillus that can grow under neutral conditions using the amplified product.
The present invention also relates to a method for determining heat treatment conditions for spore-forming bacteria belonging to the genus Bacillus, which determines heat treatment conditions for spore-forming bacteria belonging to the genus Bacillus based on the spore heat resistance evaluated by the method.
Furthermore, the present invention relates to a kit and oligonucleotide pair for detecting spore-forming bacteria used in the above method.

オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、並びにオリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対を用いて増幅したＰＣＲ産物の電気泳動の結果を示した図面代用写真である。Amplified using an oligonucleotide pair consisting of oligonucleotides (a) and (c), an oligonucleotide pair consisting of oligonucleotides (e) and (j), and an oligonucleotide pair consisting of oligonucleotides (k) and (l). This is a photograph substituted for a drawing showing the results of electrophoresis of PCR products. オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対と、オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、並びにオリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対からなる群より選ばれる１対のヌクレオチド対と、オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対とを、それぞれ組み合わせて用いて増幅したＰＣＲ産物の電気泳動の結果を示した図面代用写真である。An oligonucleotide pair consisting of oligonucleotides (a) and (c), an oligonucleotide pair consisting of oligonucleotides (d) and (j), an oligonucleotide pair consisting of oligonucleotides (e) and (j), an oligonucleotide pair consisting of oligonucleotides (f) ) and (j), an oligonucleotide pair consisting of oligonucleotides (g) and (j), an oligonucleotide pair consisting of oligonucleotides (h) and (j), and an oligonucleotide pair consisting of oligonucleotides (i) and (j). Electrophoresis results of PCR products amplified using a combination of a pair of nucleotides selected from the group consisting of oligonucleotide pairs consisting of ) and an oligonucleotide pair consisting of oligonucleotides (k) and (l), respectively. This is a photograph used as a substitute for a drawing. オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対と、オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、並びにオリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対からなる群より選ばれる１対のヌクレオチド対と、オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対とを、それぞれ組み合わせて用いて増幅したＰＣＲ産物の電気泳動の結果を示した図面代用写真である。An oligonucleotide pair consisting of oligonucleotides (b) and (c), an oligonucleotide pair consisting of oligonucleotides (d) and (j), an oligonucleotide pair consisting of oligonucleotides (e) and (j), an oligonucleotide pair consisting of oligonucleotides (f) ) and (j), an oligonucleotide pair consisting of oligonucleotides (g) and (j), an oligonucleotide pair consisting of oligonucleotides (h) and (j), and an oligonucleotide pair consisting of oligonucleotides (i) and (j). Electrophoresis results of PCR products amplified using a combination of a pair of nucleotides selected from the group consisting of oligonucleotide pairs consisting of ) and an oligonucleotide pair consisting of oligonucleotides (k) and (l), respectively. This is a photograph used as a substitute for a drawing. Multiplex PCRによって陰性又は陽性と判定されたバチルス・コアグランス（Bacillus coagulans）の菌株群におけるＤ１０５℃値を示した箱ひげ図である。It is a boxplot showing the D105 °C values of Bacillus coagulans strain groups determined to be negative or positive by Multiplex PCR.

発明の詳細な説明Detailed description of the invention

　上記のような従来法により芽胞形成細菌の芽胞耐熱性を評価する場合、芽胞形成細菌を培養して芽胞を形成させた後に芽胞耐熱性を測定する必要があるため、芽胞耐熱性の評価には長時間を要する。さらに、原料中に存在する芽胞の芽胞耐熱性を直接評価することができないため、別途原料中から芽胞形成細菌を単離する必要があり、手間とコストがかかるといった問題もある。
　また特開２００９－１８３２０７号公報に記載の方法で芽胞耐熱性を測定する場合には、測定用の大がかりな設備が必要となり、設備コストが高くなるというデメリットがある。 When evaluating the spore heat resistance of spore-forming bacteria using the conventional method described above, it is necessary to measure the spore heat resistance after culturing the spore-forming bacteria and forming spores. It takes a long time. Furthermore, since the spore heat resistance of the spores present in the raw material cannot be directly evaluated, it is necessary to separately isolate the spore-forming bacteria from the raw material, which is a problem in that it is time-consuming and costly.
Furthermore, when measuring spore heat resistance by the method described in JP-A-2009-183207, large-scale measurement equipment is required, which has the disadvantage of increasing equipment cost.

　本発明は、簡便かつ迅速に、また低コストに、高い耐熱性を有する芽胞を形成する芽胞形成細菌を検出できる、芽胞形成細菌の検出方法の提供に関する。
　また本発明は、簡便かつ迅速に、また低コストに、芽胞形成細菌の芽胞耐熱性を評価する、芽胞形成細菌の芽胞耐熱性の評価方法の提供に関する。
　また本発明は、芽胞耐熱性を評価することにより商業的無菌性が確保される芽胞形成細菌の加熱処理条件を決定する、芽胞形成細菌の加熱処理条件の決定方法の提供に関する。
　さらに本発明は、上記方法に好適に用いることができる高い耐熱性を有する芽胞を形成する芽胞形成細菌検出用のキットないしオリゴヌクレオチド対の提供に関する。 The present invention relates to a method for detecting spore-forming bacteria that can detect spore-forming bacteria that form spores with high heat resistance simply, quickly, and at low cost.
The present invention also relates to providing a method for evaluating the spore heat resistance of spore-forming bacteria, which evaluates the spore heat resistance of spore-forming bacteria simply, quickly, and at low cost.
The present invention also relates to a method for determining heat treatment conditions for spore-forming bacteria, which determines heat treatment conditions for spore-forming bacteria that ensure commercial sterility by evaluating spore heat resistance.
Furthermore, the present invention relates to the provision of a kit or oligonucleotide pair for detecting spore-forming bacteria that form spores with high heat resistance that can be suitably used in the above method.

　本発明者らは上記に鑑み、鋭意検討を行った。本発明者らは、バチルス・サブチルスの比較ゲノム解析により芽胞耐熱性の向上に寄与するとして見出されたトランスポゾン由来spoVA2mobオペロンの中のDUF421ドメイン及びDUF1657ドメインを有する機能未知のタンパク質をコードする遺伝子（以下、「DUF421-DUF1657遺伝子」ともいう。）に着目し、当該遺伝子をＤＮＡマーカーとして用いる芽胞耐熱性の評価方法の開発を試みた。その結果、DUF421-DUF1657遺伝子の特定の領域（以下、「特異的な領域」ともいう）の塩基配列とアニールできるオリゴヌクレオチド対をプライマー対として用いて、DUF421-DUF1657遺伝子の部分塩基配列を増幅することで、芽胞耐熱性に関与するDUF421-DUF1657遺伝子の有無を判別できるだけでなく、増幅した核酸のサイズ情報から菌種を同定できることを明らかにした。また、DUF421-DUF1657遺伝子の増幅産物の有無と芽胞耐熱性に相関が見られ、芽胞形成細菌について上記のプライマーを用いてDUF421-DUF1657遺伝子の増幅産物を得ることができる場合、当該芽胞形成細菌が形成する芽胞は高い耐熱性を有することを見出した。また、当該遺伝子を検出することにより、原料中に存在する芽胞形成細菌の芽胞耐熱性を評価・判断することができ、これによって原料の適切な加熱処理条件を決定できることを見出した。
　本発明はこれらの知見に基づき完成されるに至ったものである。 In view of the above, the present inventors conducted extensive studies. The present inventors discovered a gene encoding a protein of unknown function that has DUF421 and DUF1657 domains in the transposon-derived spoVA2mob operon, which was found to contribute to improving spore heat resistance through comparative genome analysis of Bacillus subtillus. We focused on the ``DUF421-DUF1657 gene'' (hereinafter also referred to as the "DUF421-DUF1657 gene") and attempted to develop a method for evaluating spore heat resistance using this gene as a DNA marker. As a result, a partial base sequence of the DUF421-DUF1657 gene is amplified using an oligonucleotide pair that can anneal to the base sequence of a specific region (hereinafter also referred to as "specific region") of the DUF421-DUF1657 gene as a primer pair. By doing this, they were able to not only determine the presence or absence of the DUF421-DUF1657 genes involved in spore heat resistance, but also identify the bacterial species based on the size information of the amplified nucleic acid. In addition, there is a correlation between the presence or absence of the amplification product of the DUF421-DUF1657 gene and spore heat resistance, and if the amplification product of the DUF421-DUF1657 gene can be obtained using the above primers for spore-forming bacteria, the spore-forming bacterium is It was found that the spores formed have high heat resistance. We also discovered that by detecting this gene, it is possible to evaluate and determine the spore heat resistance of the spore-forming bacteria present in the raw material, and thereby determine the appropriate heat treatment conditions for the raw material.
The present invention has been completed based on these findings.

　本発明の芽胞形成細菌の検出方法によれば、高耐熱性の芽胞形成能を有するバチルス属に属する芽胞形成細菌を、簡便かつ迅速に、また低コストに検出することができる。また、本発明の芽胞形成細菌の芽胞耐熱性の評価方法によれば、簡便かつ迅速に、また低コストにバチルス属に属する芽胞形成細菌の形成する芽胞の耐熱性を評価することができる。さらに本発明の芽胞形成細菌の加熱処理条件の決定方法によれば、芽胞形成細菌を殺菌するための適切な加熱処理条件を決定することができる。さらに、本発明の芽胞形成細菌検出用のキット及びオリゴヌクレオチド対は、上記方法に好適に用いることができる。 According to the method for detecting spore-forming bacteria of the present invention, spore-forming bacteria that belong to the genus Bacillus and have a highly heat-resistant spore-forming ability can be detected simply, quickly, and at low cost. Furthermore, according to the method for evaluating spore heat resistance of spore-forming bacteria of the present invention, the heat resistance of spores formed by spore-forming bacteria belonging to the genus Bacillus can be evaluated easily, quickly, and at low cost. Furthermore, according to the method for determining heat treatment conditions for spore-forming bacteria of the present invention, appropriate heat treatment conditions for sterilizing spore-forming bacteria can be determined. Furthermore, the kit and oligonucleotide pair for detecting spore-forming bacteria of the present invention can be suitably used in the above method.

　本発明の芽胞形成細菌の検出方法は、DUF421-DUF1657遺伝子の特定の部分塩基配列、すなわちバチルス属に属する芽胞形成細菌のうちDUF421-DUF1657遺伝子を有するバチルス属に属する芽胞形成細菌の各菌種にそれぞれ特異的な領域（可変領域）にストリンジェントな条件でハイブリダイズ可能なオリゴヌクレオチド対を用いてDUF421-DUF1657遺伝子の部分塩基配列を増幅させて増幅産物の有無を確認することにより、DUF421-DUF1657遺伝子を有する芽胞形成細菌を種レベルで特異的に識別・検出する方法である。ここで、「可変領域」とは、DUF421-DUF1657遺伝子中で塩基変異が蓄積しやすい領域であり、この領域の塩基配列は種間で大きく異なる。なお、本明細書において「ストリンジェントな条件」としては、例えばMolecular　Cloning－A　LABORATORY　MANUAL　THIRD　EDITION［Joseph　Sambrook,　David　W.　Russell.,　Cold　Spring　Harbor　Laboratory　Press］記載の方法が挙げられ、例えば、６×ＳＳＣ（１×ＳＳＣの組成：０.１５M塩化ナトリウム、０．０１５Mクエン酸ナトリウム、pH７．０）、０．５％ＳＤＳ、５×デンハート及び１００mg／mLニシン精子ＤＮＡを含む溶液にプローブとともに６５℃で８～１６時間恒温し、ハイブリダイズさせる条件が挙げられる。
　また本発明の芽胞形成細菌の芽胞耐熱性の評価方法は、DUF421-DUF1657遺伝子を有するバチルス属に属する芽胞形成細菌がDUF421-DUF1657遺伝子を有しない芽胞形成細菌と比較して芽胞耐熱性が向上していることから、DUF421-DUF1657遺伝子の部分塩基配列の増幅産物の有無を確認することで、芽胞耐熱性を評価する方法である。また、本発明の芽胞形成細菌の加熱処理条件の決定方法は、芽胞耐熱性の結果に基づき、その芽胞形成細菌に適した加熱処理条件を決定する方法である。
　以下、上記本発明の各方法をまとめて、「本発明の方法」ともいう。 The method for detecting spore-forming bacteria of the present invention detects a specific partial base sequence of the DUF421-DUF1657 gene, that is, each species of spore-forming bacteria belonging to the genus Bacillus that has the DUF421-DUF1657 gene. By amplifying the partial base sequence of the DUF421-DUF1657 gene using a pair of oligonucleotides that can hybridize to each specific region (variable region) under stringent conditions and confirming the presence or absence of the amplified product, DUF421-DUF1657 This is a method for specifically identifying and detecting spore-forming bacteria containing genes at the species level. Here, the "variable region" is a region in the DUF421-DUF1657 gene where base mutations tend to accumulate, and the base sequence of this region differs greatly between species. In this specification, "stringent conditions" include, for example, the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell., Cold Spring Harbor Laboratory Press]. x SSC (composition of 1 x SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 x Denhardt, and 100 mg/mL herring sperm DNA with probe 65 Examples of conditions include constant temperature at ℃ for 8 to 16 hours and hybridization.
Furthermore, the method for evaluating the spore heat resistance of spore-forming bacteria of the present invention shows that spore-forming bacteria belonging to the genus Bacillus that have the DUF421-DUF1657 gene have improved spore heat resistance compared to spore-forming bacteria that do not have the DUF421-DUF1657 gene. Therefore, this method evaluates spore heat resistance by confirming the presence or absence of an amplification product of the partial base sequence of the DUF421-DUF1657 gene. Furthermore, the method of determining heat treatment conditions for spore-forming bacteria of the present invention is a method of determining heat treatment conditions suitable for the spore-forming bacteria based on the results of spore heat resistance.
Hereinafter, the above-mentioned methods of the present invention will also be collectively referred to as the "method of the present invention."

　本発明ないし本明細書において、「DUF421-DUF1657遺伝子」とは、spoVA2mobオペロン上に存在する、DUF421ドメイン及びDUF1657ドメインを有するタンパク質をコードする遺伝子である。目的のタンパク質がDUF421ドメイン及びDUF1657ドメインを有しているかは、目的のタンパク質についてNCBI BLASTp解析を行い、query配列にはバチルス・サブチルス　B4146株由来のDUF421-DUF1657遺伝子がコードするアミノ酸配列（GenBank: KIN50158.1）を入力し、Algorithmにはblastp（protein-protein BLAST）を用いて解析を実行し、90%以上のcoverageを有するか否かにより決定することができる。本発明ないし本明細書では、上記の方法により９０％以上のcoverageを示すアミノ酸配列をコードする遺伝子を、「DUF421-DUF1657遺伝子」とする。 In the present invention and the present specification, the "DUF421-DUF1657 gene" is a gene encoding a protein having a DUF421 domain and a DUF1657 domain, which is present on the spoVA2mob operon. To determine whether the protein of interest has a DUF421 domain and a DUF1657 domain, perform NCBI BLASTp analysis on the protein of interest. .1) and perform analysis using blastp (protein-protein BLAST) as the algorithm, and determine whether the coverage is 90% or more. In the present invention and this specification, a gene encoding an amino acid sequence showing 90% or more coverage by the above method is referred to as a "DUF421-DUF1657 gene."

　本発明において、「中性条件で増殖可能なバチルス属に属する芽胞形成細菌」とは、芽胞を形成するバチルス属に属する細菌であって、ｐＨ４．６以上、８．０未満の好気的条件下で増殖可能な芽胞形成細菌であれば特に制限されない。このようなバチルス属に属する芽胞形成細菌としては、中性飲食品において腐敗、変敗の原因となり得るバチルス属に属する菌が挙げられ、例えばバチルス・サブチルス等が含まれるバチルス・サブチルス群（Bacillus subtilis group）、バチルス・コアグランス（Bacillus coagulans）、バチルス・セレウス（Bacillus cereus）、バチルス・メガテリウム（Bacillus megaterium）、バチルス・ピュミルス（Bacillus pumilus）、バチルス・シンプレックス（Bacillus simplex）、バチルス・アンスラシス（Bacillus anthracis）、バチルス・ブレヴィス（Bacillus brevis）、バチルス・レンタス（Bacillus lentus）、バチルス・ミコイデス（Bacillus mycoides）、等が挙げられる。中でも、前記芽胞形成細菌はバチルス・サブチルス群、バチルス・コアグランス及びバチルス・セレウスであることが好ましい。 In the present invention, "spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions" refers to bacteria belonging to the genus Bacillus that forms spores, and under aerobic conditions with a pH of 4.6 or more and less than 8.0. There is no particular restriction as long as it is a spore-forming bacterium that can grow under the following conditions. Examples of such spore-forming bacteria belonging to the genus Bacillus include bacteria belonging to the genus Bacillus that can cause spoilage and spoilage in neutral foods and drinks, such as the Bacillus subtilis group, which includes Bacillus subtilis . group), Bacillus coagulans , Bacillus cereus , Bacillus megaterium , Bacillus pumilus , Bacillus simplex , Bacillus anthracis , Bacillus brevis , Bacillus lentus, Bacillus mycoides , and the like. Among these, the spore-forming bacteria are preferably Bacillus subtilis, Bacillus coagulans, and Bacillus cereus.

＜バチルス・サブチルス群＞
　本発明ないし本明細書において、前記「バチルス・サブチルス群」とは、National Center for Biotechnology Information（NCBI）のデータベースにおいて規定（Taxonomy ID：653685）される細菌群である。本発明の方法において、前記バチルス・サブチルス群は、バチルス・サブチルス、バチルス・リケニフォルミス（Bacillus licheniformis）、バチルス・アミロリケファシエンス（Bacillus amyloliquefaciens）、バチルス・シアメンシス（Bacillus siamensis）、バチルス・ベレゼンシス（Bacillus velezensis）、バチルス・パラリケニフォルミス（Bacillus paralicheniformis）、及びバチルス・ソノレンシス（Bacillus sonorensis）からなる群であることが好ましく、バチルス・サブチルス、バチルス・リケニフォルミス、バチルス・アミロリケファシエンス、バチルス・シアメンシス、及びバチルス・ベレゼンシスからなる群であることがより好ましく、バチルス・サブチルスであることがさらに好ましい。 <Bacillus subtilis group>
In the present invention and the present specification, the "Bacillus subtilis group" is a bacterial group defined in the database of the National Center for Biotechnology Information (NCBI) (Taxonomy ID: 653685). In the method of the present invention, the Bacillus subtilis group includes Bacillus subtilis, Bacillus licheniformis , Bacillus amyloliquefaciens , Bacillus siamensis , Bacillus velezensis . ), Bacillus paralicheniformis , and Bacillus sonorensis , preferably Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus siamensis, and The group consisting of Bacillus berezensis is more preferable, and Bacillus subtilis is even more preferable.

　バチルス・サブチルス、バチルス・リケニフォルミス、バチルス・アミロリケファシエンスの３種については、Berendsen et al., ISME J., 2016, Vol. 10, 2633-2642に開示されているように、DUF421-DUF1657遺伝子の有無と芽胞耐熱性は相関関係にある。すなわち上記３種の菌において、ゲノム上にDUF421-DUF1657遺伝子を有する菌株は、ゲノム上にDUF421-DUF1657遺伝子を有しない菌株と比較して、高い耐熱性を有する芽胞を形成する。
　また、本発明者らはバチルス・サブチルスB4146株由来のDUF421-DUF1657遺伝子がコードするアミノ酸配列を用いてBLASTp解析を行い、バチルス・シアメンシス、バチルス・ベレゼンシス、バチルス・パラリケニフォルミス、及びバチルス・ソノレンシスの４種についても、多くの菌株でゲノム上にDUF421-DUF1657遺伝子が保存されていることを明らかにした。なお、バチルス・シアメンシスSCSIO05746株、及びバチルス・ベレゼンシス9912D株由来の２種のDUF421-DUF1657遺伝子の塩基配列（当該遺伝子がコードするタンパク質、GenBank: AUJ76361.1、及びGenBank: APA02936.1）と、バチルス・サブチルスB4146株、バチルス・リケニフォルミスATCC9789株、及びバチルス・アミロリケファシエンスSRCM101267株由来のDUF421-DUF1657遺伝子の塩基配列との同一性は、いずれも９９％以上である。よって、バチルス・シアメンシス、バチルス・ベレゼンシス、バチルス・パラリケニフォルミス、及びバチルス・ソノレンシスの４種においても、バチルス・サブチルス、バチルス・リケニフォルミス、バチルス・アミロリケファシエンスの３種と同様に、DUF421-DUF1657遺伝子の有無と芽胞耐熱性は相関関係にあると推測することができる。すなわち、上記の４種においても、DUF421-DUF1657遺伝子を有する菌株は、DUF421-DUF1657遺伝子を有しない菌株と比較して、高い耐熱性を有する芽胞を形成すると考えられる。 For the three species Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, the DUF421-DUF1657 genes are There is a correlation between the presence or absence of spores and spore heat resistance. That is, among the three types of bacteria mentioned above, the strain that has the DUF421-DUF1657 gene on its genome forms spores that have higher heat resistance than the strain that does not have the DUF421-DUF1657 gene on its genome.
In addition, the present inventors performed BLASTp analysis using the amino acid sequence encoded by the DUF421-DUF1657 gene derived from Bacillus subtilis strain B4146, and found that It was also revealed that the DUF421-DUF1657 genes are conserved in the genomes of many of the four strains. In addition, the nucleotide sequences of two DUF421-DUF1657 genes derived from Bacillus xiamensis strain SCSIO05746 and Bacillus berezensis strain 9912D (the proteins encoded by the genes, GenBank: AUJ76361.1 and GenBank: APA02936.1), - The identity with the base sequences of the DUF421-DUF1657 genes derived from Subtilis B4146 strain, Bacillus licheniformis ATCC9789 strain, and Bacillus amyloliquefaciens SRCM101267 strain is all 99% or more. Therefore, DUF421- It can be inferred that there is a correlation between the presence or absence of the DUF1657 gene and spore heat resistance. That is, among the four types mentioned above, the strain having the DUF421-DUF1657 gene is thought to form spores with higher heat resistance than the strain not having the DUF421-DUF1657 gene.

　本発明の方法により、供試菌株としてバチルス・サブチルス群の菌株を用いた際にDUF421-DUF1657遺伝子が検出される場合（後述の核酸プライマーを用いてDUF421-DUF1657遺伝子の部分塩基配列で表される核酸の増幅したとき、増幅産物の存在が確認され、「陽性」と判断される場合）、当該DUF421-DUF1657遺伝子を有するバチルス・サブチルス群の菌株は、形成される芽胞が高い耐熱性を有する。一方、後述の核酸プライマーを用いて増幅反応を行っても増幅産物が確認できず「陰性」と判断される場合、供試菌株はDUF421-DUF1657遺伝子を有しておらず、形成される芽胞の耐熱性は、当該DUF421-DUF1657遺伝子を有するバチルス・サブチルス群の菌株の芽胞耐熱性よりも相対的に低い。
　前記DUF421-DUF1657遺伝子を有するバチルス・サブチルス群の菌株のＤ値（Decimal Reduction Value）は、公知の情報を参照して適宜決定することができる。例えば、Berendsen et al., The ISME Journal (2016) 10, 2633-2642には、トランスポゾン由来spoVA2mobオペロンを少なくとも１コピー以上有するバチルス・サブチルスの１１２．５℃におけるＤ値（Ｄ１１２．５℃値）が１分間以上であることが示されている。当該トランスポゾン由来spoVA2mobオペロンにはDUF421-DUF1657遺伝子が含まれていることから、例えば本発明の方法により陽性と判断されたバチルス・サブチルス群のＤ１１２．５℃値を１分間以上と評価・決定することができる。
　なお、本明細書において、例えば「Ｄ１００℃値」とあるのは、１００℃の加熱処理により、対象菌数を加熱処理前から１／１０にまで減少させるのに要する時間を意味する。 When the DUF421-DUF1657 gene is detected by the method of the present invention using a strain of the Bacillus subtilis group as a test strain (represented by a partial base sequence of the DUF421-DUF1657 gene using the nucleic acid primers described below) When the nucleic acid is amplified, the presence of the amplification product is confirmed and the result is determined to be "positive"), the spores formed by the Bacillus subtilis strain having the DUF421-DUF1657 genes have high heat resistance. On the other hand, if the amplification reaction is performed using the nucleic acid primers described below and the amplification product is not confirmed and the test result is negative, the test strain does not have the DUF421-DUF1657 gene, and the spores formed The heat resistance is relatively lower than the spore heat resistance of the Bacillus subtilis group strain having the DUF421-DUF1657 genes.
The D value (Decimal Reduction Value) of a strain of the Bacillus subtilis group having the DUF421-DUF1657 genes can be appropriately determined with reference to known information. For example, Berendsen et al., The ISME Journal (2016) 10, 2633-2642 describes the D value at 112.5°C of Bacillus subtilis, which has at least one copy of the transposon-derived spoVA2mob operon (D 112.5°C value). It has been shown that the time is 1 minute or more. Since the transposon-derived spoVA2mob operon contains the DUF421-DUF1657 gene, for example, the D112.5°C value of the Bacillus subtilis group determined to be positive by the method of the present invention is evaluated and determined to be 1 minute or more. be able to.
In addition, in this specification, for example, "D 100°C value" means the time required to reduce the number of target bacteria to 1/10 from before the heat treatment by heat treatment at 100°C.

＜バチルス・コアグランス＞
　また本発明者らはバチルス・サブチルスB4146株由来のDUF421-DUF1657遺伝子がコードするアミノ酸配列を用いてBLASTp解析を行い、バチルス・コアグランスについても、多くの菌株でゲノム上にDUF421-DUF1657遺伝子を保存していることを明らかにした。なお、バチルス・サブチルスB4146株由来のDUF421-DUF1657遺伝子の塩基配列と、バチルス・コアグランスDSM1=ATCC7050株（NBRC12583株と同一株）由来のDUF421-DUF1657遺伝子の塩基配列との同一性は、約74.0％である。 <Bacillus coagulans>
The present inventors also performed BLASTp analysis using the amino acid sequence encoded by the DUF421-DUF1657 gene derived from Bacillus subtillus strain B4146, and found that the DUF421-DUF1657 gene is conserved in the genome of many strains of Bacillus coagulans. It was revealed that The identity between the base sequence of the DUF421-DUF1657 gene derived from Bacillus subtillus B4146 strain and the base sequence of the DUF421-DUF1657 gene derived from Bacillus coagulans DSM1=ATCC7050 strain (same strain as NBRC12583 strain) is approximately 74.0%. It is.

　本発明の方法により、供試菌株としてバチルス・コアグランスの菌株を用いた際にDUF421-DUF1657遺伝子が検出される場合（後述の核酸プライマーを用いてDUF421-DUF1657遺伝子の部分塩基配列で表される核酸の増幅したとき、増幅産物の存在が確認され、「陽性」と判断される場合）、当該DUF421-DUF1657遺伝子を有するバチルス・コアグランスの菌株は、形成される芽胞が高い耐熱性を有する。一方、後述の核酸プライマーを用いて増幅反応を行っても増幅産物が確認できず「陰性」と判断される場合、供試菌株はDUF421-DUF1657遺伝子を有しておらず、形成される芽胞の耐熱性は、当該DUF421-DUF1657遺伝子を有するバチルス・コアグランスの菌株の芽胞耐熱性よりも相対的に低い。
　前記DUF421-DUF1657遺伝子を有するバチルス・コアグランスの菌株のＤ値は、下記に示す実施例の結果を参照して適宜決定することもできる。すなわち、例えば実施例の結果より、バチルス・コアグランスのＤ１０５℃値を１０分以上とすることができる。また、例えば上記のようにバチルス・サブチルスの１１２．５℃におけるＤ値（Ｄ１１２．５℃値）を１分間以上とできることから、本発明の方法により陽性と判断されたバチルス・コアグランスのＤ１１２．５℃値も、同様に１分間以上と評価・決定することもできる。 When the DUF421-DUF1657 gene is detected by the method of the present invention when a Bacillus coagulans strain is used as the test strain (the nucleic acid expressed by the partial base sequence of the DUF421-DUF1657 gene is detected using the nucleic acid primers described below). (when the presence of the amplification product is confirmed and judged to be "positive"), the spores formed by the Bacillus coagulans strain having the DUF421-DUF1657 genes have high heat resistance. On the other hand, if the amplification reaction is performed using the nucleic acid primers described below and the result is "negative" because no amplification product is confirmed, the test strain does not have the DUF421-DUF1657 gene, and the spores formed The heat resistance is relatively lower than the spore heat resistance of the Bacillus coagulans strain having the DUF421-DUF1657 genes.
The D value of the Bacillus coagulans strain having the DUF421-DUF1657 genes can also be appropriately determined with reference to the results of the Examples shown below. That is, for example, based on the results of Examples, the D105 °C value of Bacillus coagulans can be set to 10 minutes or more. In addition, for example, as mentioned above, since the D value (D 112.5°C value) of Bacillus subtilis at 112.5°C can be set to 1 minute or more, the D 112 of Bacillus coagulans determined to be positive by the method of the present invention .5° C. value can also be similarly evaluated and determined as 1 minute or more.

＜バチルス・セレウス＞
　また本発明者らはバチルス・サブチルスB4146株由来のDUF421-DUF1657遺伝子がコードするアミノ酸配列を用いてBLASTp解析を行い、バチルス・セレウスについては、ほぼすべての菌株のゲノムにDUF421-DUF1657遺伝子が保存されており、さらにバチルス・セレウスの菌株のうち嘔吐毒産生株特異的に付加的にDUF421-DUF1657遺伝子が保存されていることを明らかにした。これは嘔吐毒産生遺伝子とDUF421-DUF1657遺伝子が同一プラスミド上にコードされていることが理由であると考えられる。以下、当該嘔吐毒産生株特異的に付加的なDUF421-DUF1657遺伝子を「プラスミド特異的DUF421-DUF1657遺伝子」とも称す。なお、バチルス・サブチルスB4146株由来のDUF421-DUF1657遺伝子の塩基配列と、バチルス・セレウスAH187株由来のプラスミド特異的DUF421-DUF1657遺伝子の塩基配列との同一性は、約66.9％である。
　なお、本発明ないし本明細書において、特段の断りがない場合、「DUF421-DUF1657遺伝子」とは、バチルス・セレウスについては、バチルス・セレウス由来のプラスミド特異的DUF421-DUF1657遺伝子のことを意味する。 <Bacillus cereus>
In addition, the present inventors performed BLASTp analysis using the amino acid sequence encoded by the DUF421-DUF1657 gene derived from Bacillus subtillus strain B4146, and found that the DUF421-DUF1657 gene is conserved in the genome of almost all Bacillus cereus strains. Furthermore, it was revealed that the DUF421-DUF1657 genes are additionally conserved in emetic toxin-producing strains of Bacillus cereus. This is thought to be because the emetic toxin producing gene and the DUF421-DUF1657 gene are encoded on the same plasmid. Hereinafter, the additional DUF421-DUF1657 gene specific to the emetic toxin-producing strain will also be referred to as a "plasmid-specific DUF421-DUF1657 gene." The identity between the base sequence of the DUF421-DUF1657 gene derived from Bacillus subtillus strain B4146 and the base sequence of the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus strain AH187 is approximately 66.9%.
In the present invention and this specification, unless otherwise specified, the term "DUF421-DUF1657 gene" for Bacillus cereus means the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus.

　本発明の方法により、供試菌株としてバチルス・セレウスの菌株を用いた際にプラスミド特異的DUF421-DUF1657遺伝子が検出される場合（後述の核酸プライマーを用いてプラスミド特異的DUF421-DUF1657遺伝子の部分塩基配列で表される核酸の増幅したとき、増幅産物の存在が確認され、「陽性」と判断される場合）、プラスミド特異的DUF421-DUF1657遺伝子を有するバチルス・セレウスの菌株は、形成される芽胞が高い耐熱性を有する。一方、後述の核酸プライマーを用いて増幅反応を行っても増幅産物が確認できず「陰性」と判断される場合、供試菌株はプラスミド特異的DUF421-DUF1657遺伝子を有しておらず、形成される芽胞の耐熱性は、当該プラスミド特異的DUF421-DUF1657遺伝子を有するバチルス・セレウスの菌株の芽胞耐熱性よりも相対的に低い。
　前記プラスミド特異的DUF421-DUF1657遺伝子を有するバチルス・セレウスの菌株のＤ値は、公知の情報を参照して適宜決定することができる。例えば、Carlin et al., Food Microbiology (2006) 109, 132-138、には、非嘔吐毒産生株は全てＤ９０℃値が３００分以下であることが示されている。上記の通り、本発明者らの解析により、非嘔吐毒産生株はプラスミド特異的DUF421-DUF1657遺伝子を持たないと考えられるため、本発明の方法により陰性と判断されたバチルス・セレウスのＤ９０℃値を３００分間以下と評価・決定することができる。また、事前にバチルス・セレウスのＤ値を測定し（例えば下記実施例に記載の方法等によりＤ値を測定し）、測定結果に従って本発明の方法により陰性と判断されたバチルス・セレウスのＤ値を特定の値以下として評価・決定することもできる。 When a plasmid-specific DUF421-DUF1657 gene is detected by the method of the present invention when a Bacillus cereus strain is used as a test strain (a partial base of the plasmid-specific DUF421-DUF1657 gene is detected using a nucleic acid primer described below). When the nucleic acid represented by the sequence is amplified, the presence of the amplification product is confirmed and the result is determined as “positive”), the Bacillus cereus strain carrying the plasmid-specific DUF421-DUF1657 genes will cause the spores formed to Has high heat resistance. On the other hand, if an amplification product cannot be confirmed even if the amplification reaction is performed using the nucleic acid primers described below and the test result is negative, the test strain does not have the plasmid-specific DUF421-DUF1657 gene and is not formed. The heat resistance of the spores is relatively lower than that of the Bacillus cereus strain having the plasmid-specific DUF421-DUF1657 genes.
The D value of the Bacillus cereus strain having the plasmid-specific DUF421-DUF1657 gene can be appropriately determined with reference to known information. For example, Carlin et al., Food Microbiology (2006) 109, 132-138, shows that all non-emetic toxin producing strains have a D 90°C value of 300 minutes or less. As mentioned above, our analysis shows that non-emetic toxin - producing strains do not have the plasmid-specific DUF421-DUF1657 genes. The value can be evaluated and determined to be 300 minutes or less. In addition, the D value of Bacillus cereus is measured in advance (for example, the D value is measured by the method described in the example below), and the D value of Bacillus cereus that is determined to be negative by the method of the present invention according to the measurement results. It is also possible to evaluate and determine that the value is less than or equal to a specific value.

　本発明の方法によって検出するDUF421-DUF1657遺伝子は、高い耐熱性を有する芽胞を形成する芽胞形成細菌のゲノム又はプラスミドに保存されている。一方で、DUF421-DUF1657遺伝子の上流部分（５’－末端側）に存在するDUF421ドメインをコードする塩基配列は、高耐熱性を有しない芽胞を形成する芽胞形成細菌のゲノムにも保存されている場合がある。そのため、本発明の方法に用いるオリゴヌクレオチドは、下記の条件の全てを満たすオリゴヌクレオチドである。
　すなわち、本発明の方法に用いるオリゴヌクレオチドは、ＧＣ含有率が３０％～８０％であり、自己アニールせず、Ｔｍ値が約５５～６５℃であり、検出するDUF421-DUF1657遺伝子の由来となる菌種に特異的な領域（バチルス・セレウスの場合は、さらにゲノムにコードされたDUF421-DUF1657遺伝子の配列を検出しないような、プラスミド特異的DUF421-DUF1657遺伝子のみに特異的な領域）で、かつ菌種内では保存性の高い領域にアニールするオリゴヌクレオチドであり、本発明の方法に用いるオリゴヌクレオチド対によって増幅されるＤＮＡ断片の長さが１０００ｂｐ以下であり、オリゴヌクレオチド対がDUF421領域（DUF421ドメインをコードする塩基配列）とDUF1657領域（DUF1657ドメインをコードする塩基配列）を跨ぐような位置に設計されているオリゴヌクレオチドである。
　なお、前記DUF421領域とDUF1657領域の範囲は、例えば各タンパク質について、NCBIのデータベースに記載の情報を参照して決定することができる。 The DUF421-DUF1657 genes detected by the method of the present invention are conserved in the genome or plasmid of spore-forming bacteria that form spores with high heat resistance. On the other hand, the nucleotide sequence encoding the DUF421 domain located upstream of the DUF421-DUF1657 gene (5'-end side) is also conserved in the genomes of spore-forming bacteria that do not have high heat resistance. There are cases. Therefore, the oligonucleotide used in the method of the present invention is one that satisfies all of the following conditions.
That is, the oligonucleotide used in the method of the present invention has a GC content of 30% to 80%, does not self-anneal, has a Tm value of about 55 to 65°C, and is the origin of the DUF421-DUF1657 gene to be detected. A region specific to the bacterial species (in the case of Bacillus cereus, a region specific only to the plasmid-specific DUF421-DUF1657 gene, such that the sequence of the DUF421-DUF1657 gene encoded in the genome is not detected), and It is an oligonucleotide that anneals to a highly conserved region within a bacterial species, and the length of the DNA fragment amplified by the oligonucleotide pair used in the method of the present invention is 1000 bp or less, and the oligonucleotide pair anneals to the DUF421 region (DUF421 domain This is an oligonucleotide designed to span the DUF1657 region (nucleotide sequence encoding the DUF1657 domain) and the DUF1657 region (nucleotide sequence encoding the DUF1657 domain).
The ranges of the DUF421 region and DUF1657 region can be determined, for example, with reference to information listed in the NCBI database for each protein.

　前記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対（以下、「オリゴヌクレオチド対（１）」ともいう）、並びに前記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対（以下、「オリゴヌクレオチド対（２）」ともいう）は、バチルス・サブチルス群由来のDUF421-DUF1657遺伝子の検出に使用できる。なお、本明細書において、「DUF421-DUF1657遺伝子の検出に使用できる」とは、DUF421-DUF1657遺伝子の特定領域にハイブリダイズでき、オリゴヌクレオチド対をプライマー対として使用した際にDUF421-DUF1657遺伝子の部分塩基配列からなる核酸を増幅できることを意味する。
　前記オリゴヌクレオチド（ａ）において、アニーリングの観点から、配列番号１で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ａ）として、配列番号１で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・サブチルス群）の検出に使用できるオリゴヌクレオチドも好ましい。なお、前記オリゴヌクレオチド（ａ）において、配列番号１で表される塩基配列中の「Ｒ」とは、アデニン（Ａ）とグアニン（Ｇ）の混合塩基を意味する。
　前記オリゴヌクレオチド（ｂ）において、アニーリングの観点から、配列番号２で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｂ）として、配列番号２で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・サブチルス群）の検出に使用できるオリゴヌクレオチドも好ましい。
　前記オリゴヌクレオチド（ｃ）において、アニーリングの観点から、配列番号３で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｃ）として、配列番号３で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・サブチルス群）の検出に使用できるオリゴヌクレオチドも好ましい。
　なお本明細書において、塩基配列の同一性は、NCBI Blastn解析によって計算されたIdentitiesの値を用いている。解析の際、Program selectionにはSomewhat similar sequences(blastn)を用いている。 An oligonucleotide pair consisting of the oligonucleotides (a) and (c) (hereinafter also referred to as "oligonucleotide pair (1)"), and an oligonucleotide pair consisting of the oligonucleotides (b) and (c) (hereinafter also referred to as "oligonucleotide pair (1)") Oligonucleotide pair (2)'') can be used to detect the DUF421-DUF1657 genes derived from the Bacillus subtilis group. In this specification, "can be used to detect the DUF421-DUF1657 gene" means that it can hybridize to a specific region of the DUF421-DUF1657 gene and when the oligonucleotide pair is used as a primer pair, a portion of the DUF421-DUF1657 gene can be detected. This means that a nucleic acid consisting of a base sequence can be amplified.
In the oligonucleotide (a), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 1 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. In addition, as the oligonucleotide (a), one to several (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 1. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Oligonucleotides that can be used for the detection of subtilis group) are also preferred. In addition, in the oligonucleotide (a), "R" in the base sequence represented by SEQ ID NO: 1 means a mixed base of adenine (A) and guanine (G).
In the oligonucleotide (b), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 2 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Further, as the oligonucleotide (b), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 2. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Oligonucleotides that can be used for the detection of subtilis group) are also preferred.
In the oligonucleotide (c), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 3 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Furthermore, as the oligonucleotide (c), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 3. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Oligonucleotides that can be used for the detection of subtilis group) are also preferred.
Note that in this specification, the identity of base sequences uses the Identities value calculated by NCBI Blastn analysis. During analysis, we use Somewhat similar sequences (blastn) for Program selection.

　前記オリゴヌクレオチド対（１）及び前記オリゴヌクレオチド対（２）をそれぞれ用いてバチルス・サブチルス群由来のゲノム上のDUF421-DUF1657遺伝子の部分塩基配列を増幅して得られる増幅産物について、バチルス・サブチルスB4146株、及びバチルス・サブチルスATCC11774株由来のDUF421-DUF1657遺伝子の塩基配列を参照して説明する。
　バチルス・サブチルスB4146株のDUF421-DUF1657遺伝子の塩基配列を配列番号１３に、バチルス・サブチルスATCC11774株のDUF421-DUF1657遺伝子の塩基配列を配列番号１４にそれぞれ示す。前記オリゴヌクレオチド（ａ）及び（ｃ）は、配列番号１３及び配列番号１４に記載の塩基配列のうち、いずれもそれぞれ364位～381位まで、及び695位～712位までの領域に対応する。よって、例えば前記オリゴヌクレオチド対（１）、及びテンプレートとしてバチルス・サブチルス群由来のDUF421-DUF1657遺伝子を有するＤＮＡを用いてポリメラーゼ連鎖反応（ＰＣＲ）を行った場合、増幅ＤＮＡ断片の長さは約３５０ｂｐとなる。
　また前記オリゴヌクレオチド（ｂ）及び（ｃ）は、配列番号１３及び配列番号１４に記載の塩基配列のうち、いずれもそれぞれ658位～675位まで、及び695位～712位までの領域に対応する。よって、例えば前記オリゴヌクレオチド対（２）、及びテンプレートとしてバチルス・サブチルス群由来のDUF421-DUF1657遺伝子を有するＤＮＡを用いてポリメラーゼ連鎖反応（ＰＣＲ）を行った場合、増幅ＤＮＡ断片の長さは約５５ｂｐとなる。 Regarding the amplification product obtained by amplifying the partial base sequence of the DUF421-DUF1657 gene on the genome derived from the Bacillus subtilis group using the oligonucleotide pair (1) and the oligonucleotide pair (2), respectively, Bacillus subtilis B4146 This will be explained with reference to the nucleotide sequence of the DUF421-DUF1657 gene derived from Bacillus subtilis strain ATCC11774.
The nucleotide sequence of the DUF421-DUF1657 gene of Bacillus subtilis strain B4146 is shown in SEQ ID NO: 13, and the nucleotide sequence of the DUF421-DUF1657 gene of Bacillus subtilis ATCC 11774 strain is shown in SEQ ID NO: 14. The oligonucleotides (a) and (c) correspond to the regions from positions 364 to 381 and from positions 695 to 712, respectively, of the base sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14. Therefore, for example, when polymerase chain reaction (PCR) is performed using the oligonucleotide pair (1) and DNA having the DUF421-DUF1657 gene derived from Bacillus subtilis group as a template, the length of the amplified DNA fragment is approximately 350 bp. becomes.
Furthermore, the oligonucleotides (b) and (c) correspond to the regions from positions 658 to 675 and from positions 695 to 712, respectively, of the base sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14. . Therefore, for example, when polymerase chain reaction (PCR) is performed using the oligonucleotide pair (2) and DNA having the DUF421-DUF1657 gene derived from Bacillus subtilis group as a template, the length of the amplified DNA fragment is approximately 55 bp. becomes.

　前記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対（以下、「オリゴヌクレオチド対（３）」ともいう）、前記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対（以下、「オリゴヌクレオチド対（４）」ともいう）、前記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対（以下、「オリゴヌクレオチド対（５）」ともいう）、前記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対（以下、「オリゴヌクレオチド対（６）」ともいう）、前記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対（以下、「オリゴヌクレオチド対（７）」ともいう）、並びに前記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対（以下、「オリゴヌクレオチド対（８）」ともいう）は、バチルス・コアグランス由来のDUF421-DUF1657遺伝子の検出に使用することができる。
　前記オリゴヌクレオチド（ｄ）において、アニーリングの観点から、配列番号４で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｄ）として、配列番号４で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・コアグランス）の検出に使用できるオリゴヌクレオチドも好ましい。
　前記オリゴヌクレオチド（ｅ）において、アニーリングの観点から、配列番号５で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｅ）として、配列番号５で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・コアグランス）の検出に使用できるオリゴヌクレオチドも好ましい。
　前記オリゴヌクレオチド（ｆ）において、アニーリングの観点から、配列番号６で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｆ）として、配列番号６で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・コアグランス）の検出に使用できるオリゴヌクレオチドも好ましい。
　前記オリゴヌクレオチド（ｇ）において、アニーリングの観点から、配列番号７で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｇ）として、配列番号７で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・コアグランス）の検出に使用できるオリゴヌクレオチドも好ましい。
　前記オリゴヌクレオチド（ｈ）において、アニーリングの観点から、配列番号８で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｈ）として、配列番号８で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・コアグランス）の検出に使用できるオリゴヌクレオチドも好ましい。
　前記オリゴヌクレオチド（ｉ）において、アニーリングの観点から、配列番号９で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｉ）として、配列番号９で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・コアグランス）の検出に使用できるオリゴヌクレオチドも好ましい。
　前記オリゴヌクレオチド（ｊ）において、アニーリングの観点から、配列番号１０で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｊ）として、配列番号１０で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・コアグランス）の検出に使用できるオリゴヌクレオチドも好ましい。 An oligonucleotide pair consisting of the oligonucleotides (d) and (j) (hereinafter also referred to as "oligonucleotide pair (3)"), an oligonucleotide pair consisting of the oligonucleotides (e) and (j) (hereinafter also referred to as "oligonucleotide pair (3)"), nucleotide pair (4)), an oligonucleotide pair consisting of the oligonucleotides (f) and (j) (hereinafter also referred to as "oligonucleotide pair (5)"), and the oligonucleotides (g) and (j). (hereinafter also referred to as "oligonucleotide pair (6)"), an oligonucleotide pair consisting of the oligonucleotides (h) and (j) (hereinafter also referred to as "oligonucleotide pair (7)"), The oligonucleotide pair consisting of oligonucleotides (i) and (j) (hereinafter also referred to as "oligonucleotide pair (8)") can be used to detect the DUF421-DUF1657 gene derived from Bacillus coagulans.
In the oligonucleotide (d), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 4 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. In addition, as the oligonucleotide (d), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 4. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Also preferred are oligonucleotides that can be used to detect coagulance.
In the oligonucleotide (e), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 5 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. In addition, as the oligonucleotide (e), one to several (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 5. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Also preferred are oligonucleotides that can be used to detect coagulance.
In the oligonucleotide (f), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 6 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Furthermore, as the oligonucleotide (f), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 6. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Also preferred are oligonucleotides that can be used to detect coagulance.
In the oligonucleotide (g), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 7 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Furthermore, as the oligonucleotide (g), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 7. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Also preferred are oligonucleotides that can be used to detect coagulance.
In the oligonucleotide (h), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 8 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. In addition, as the oligonucleotide (h), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 8. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Also preferred are oligonucleotides that can be used to detect coagulance.
In the oligonucleotide (i), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 9 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Furthermore, as the oligonucleotide (i), one to several (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 9. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Also preferred are oligonucleotides that can be used to detect coagulance.
In the oligonucleotide (j), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 10 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Further, as the oligonucleotide (j), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 10. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Also preferred are oligonucleotides that can be used to detect coagulance.

　前記オリゴヌクレオチド対（３）、前記オリゴヌクレオチド対（４）、前記オリゴヌクレオチド対（５）、前記オリゴヌクレオチド対（６）、前記オリゴヌクレオチド対（７）、及び前記オリゴヌクレオチド対（８）をそれぞれ用いてバチルス・コアグランス由来のゲノム上のDUF421-DUF1657遺伝子の部分塩基配列を増幅して得られる増幅産物について、バチルス・コアグランスDSM1=ATCC7050株由来のDUF421-DUF1657遺伝子の塩基配列を参照して説明する。
　バチルス・コアグランスDSM1=ATCC7050株由来のDUF421-DUF1657遺伝子の塩基配列を配列番号１５に示す。前記オリゴヌクレオチド（ｄ）及び（ｊ）は、配列番号１５に記載の塩基配列のうち、それぞれ223位～240位まで、及び695位～712位までの領域に対応する。よって、例えば前記オリゴヌクレオチド対（３）、及びテンプレートとしてバチルス・コアグランス由来のDUF421-DUF1657遺伝子を有するＤＮＡを用いてＰＣＲを行った場合、増幅ＤＮＡ断片の長さは約４９０ｂｐとなる。
　また前記オリゴヌクレオチド（ｅ）及び（ｊ）は、配列番号１５に記載の塩基配列のうち、それぞれ246位～263位まで、及び695位～712位までの領域に対応する。よって、例えば前記オリゴヌクレオチド対（４）、及びテンプレートとしてバチルス・コアグランス由来のDUF421-DUF1657遺伝子を有するＤＮＡを用いてＰＣＲを行った場合、増幅ＤＮＡ断片の長さは約４６５ｂｐとなる。
　また前記オリゴヌクレオチド（ｆ）及び（ｊ）は、配列番号１５に記載の塩基配列のうち、それぞれ558位～575位まで、及び695位～712位までの領域に対応する。よって、例えば前記オリゴヌクレオチド対（５）、及びテンプレートとしてバチルス・コアグランス由来のDUF421-DUF1657遺伝子を有するＤＮＡを用いてＰＣＲを行った場合、増幅ＤＮＡ断片の長さは約１５５ｂｐとなる。
　また前記オリゴヌクレオチド（ｇ）及び（ｊ）は、配列番号１５に記載の塩基配列のうち、それぞれ577位～594位まで、及び695位～712位までの領域に対応する。よって、例えば前記オリゴヌクレオチド対（６）、及びテンプレートとしてバチルス・コアグランス由来のDUF421-DUF1657遺伝子を有するＤＮＡを用いてＰＣＲを行った場合、増幅ＤＮＡ断片の長さは約１３５ｂｐとなる。
　また前記オリゴヌクレオチド（ｈ）及び（ｊ）は、配列番号１５に記載の塩基配列のうち、それぞれ592位～609位まで、及び695位～712位までの領域に対応する。よって、例えば前記オリゴヌクレオチド対（７）、及びテンプレートとしてバチルス・コアグランス由来のDUF421-DUF1657遺伝子を有するＤＮＡを用いてＰＣＲを行った場合、増幅ＤＮＡ断片の長さは約１２０ｂｐとなる。
　また前記オリゴヌクレオチド（ｉ）及び（ｊ）は、配列番号１５に記載の塩基配列のうち、それぞれ634位～651位まで、及び695位～712位までの領域に対応する。よって、例えば前記オリゴヌクレオチド対（８）、及びテンプレートとしてバチルス・コアグランス由来のDUF421-DUF1657遺伝子を有するＤＮＡを用いてＰＣＲを行った場合、増幅ＤＮＡ断片の長さは約８０ｂｐとなる。 The oligonucleotide pair (3), the oligonucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair (7), and the oligonucleotide pair (8), respectively. The amplification product obtained by amplifying the partial base sequence of the DUF421-DUF1657 gene on the genome derived from Bacillus coagulans using the following method will be explained with reference to the base sequence of the DUF421-DUF1657 gene derived from Bacillus coagulans strain DSM1=ATCC7050. .
The base sequence of the DUF421-DUF1657 gene derived from Bacillus coagulans DSM1=ATCC7050 strain is shown in SEQ ID NO: 15. The oligonucleotides (d) and (j) correspond to the regions from positions 223 to 240 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (3) and a DNA containing the Bacillus coagulans-derived DUF421-DUF1657 gene as a template, the length of the amplified DNA fragment is approximately 490 bp.
Furthermore, the oligonucleotides (e) and (j) correspond to the regions from positions 246 to 263 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (4) and a DNA having the Bacillus coagulans-derived DUF421-DUF1657 gene as a template, the length of the amplified DNA fragment is approximately 465 bp.
Furthermore, the oligonucleotides (f) and (j) correspond to the regions from positions 558 to 575 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (5) and DNA having the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be about 155 bp.
Furthermore, the oligonucleotides (g) and (j) correspond to the regions from positions 577 to 594 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (6) and a DNA having the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be about 135 bp.
Furthermore, the oligonucleotides (h) and (j) correspond to the regions from positions 592 to 609 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (7) and a DNA having the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be about 120 bp.
Furthermore, the oligonucleotides (i) and (j) correspond to the regions from positions 634 to 651 and from positions 695 to 712, respectively, of the base sequence set forth in SEQ ID NO: 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (8) and DNA having the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be about 80 bp.

　前記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対（以下、「オリゴヌクレオチド対（９）」ともいう）は、バチルス・セレウス由来のプラスミド特異的DUF421-DUF1657遺伝子の検出に使用することができる。
　前記オリゴヌクレオチド（ｋ）において、アニーリングの観点から、配列番号１１で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｋ）として、配列番号１１で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・セレウス）の検出に使用できるオリゴヌクレオチドも好ましい。
　前記オリゴヌクレオチド（ｌ）において、アニーリングの観点から、配列番号１２で表される塩基配列との同一性は８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。また前記オリゴヌクレオチド（ｌ）として、配列番号１２で表される塩基配列において１個ないし数個（例えば、１個以上４個以下、好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個）、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌（好ましくはバチルス・セレウス）の検出に使用できるオリゴヌクレオチドも好ましい。 The oligonucleotide pair consisting of oligonucleotides (k) and (l) (hereinafter also referred to as "oligonucleotide pair (9)") can be used to detect the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus. can.
In the oligonucleotide (k), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 11 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Furthermore, as the oligonucleotide (k), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 11. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Also preferred are oligonucleotides that can be used for the detection of C. cereus).
In the oligonucleotide (l), from the viewpoint of annealing, the identity with the base sequence represented by SEQ ID NO: 12 is preferably 85% or more, more preferably 90% or more, and 95% or more. It is even more preferable that there be. Furthermore, as the oligonucleotide (l), one to several oligonucleotides (for example, 1 to 4, preferably 1 to 3, more preferably 1 or 2) in the base sequence represented by SEQ ID NO: 12. a spore-forming bacterium belonging to the genus Bacillus (preferably Bacillus spp.) that has a deletion, substitution, insertion, or addition of nucleotides (more preferably 1 nucleotide, more preferably 1 nucleotide) and has DUF421-DUF1657 genes and is capable of growing under neutral conditions. Also preferred are oligonucleotides that can be used for the detection of C. cereus).

　前記オリゴヌクレオチド対（９）を用いてバチルス・セレウスのプラスミド特異的DUF421-DUF1657遺伝子の部分塩基配列を増幅して得られる増幅産物について、バチルス・セレウスAH187株由来のプラスミド特異的DUF421-DUF1657遺伝子の塩基配列を参照して説明する。
　バチルス・セレウスAH187株由来のプラスミド特異的DUF421-DUF1657遺伝子の塩基配列を配列番号１６に示す。前記オリゴヌクレオチド（ｋ）及び（ｌ）は、配列番号１６に記載の塩基配列のうち、それぞれ457位～474位まで、及び699位～721位までの領域に対応する。よって、例えばオリゴヌクレオチド対（９）、及びテンプレートとしてバチルス・セレウス由来のプラスミド特異的DUF421-DUF1657遺伝子を有するＤＮＡを用いてＰＣＲを行った場合、増幅ＤＮＡ断片の長さは約２６５ｂｐとなる。 Regarding the amplification product obtained by amplifying the partial base sequence of the plasmid-specific DUF421-DUF1657 gene of Bacillus cereus using the oligonucleotide pair (9), This will be explained with reference to the base sequence.
The nucleotide sequence of the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus strain AH187 is shown in SEQ ID NO: 16. The oligonucleotides (k) and (l) correspond to the regions from positions 457 to 474 and from positions 699 to 721, respectively, of the base sequence set forth in SEQ ID NO: 16. Therefore, for example, when PCR is performed using the oligonucleotide pair (9) and a DNA containing the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus as a template, the length of the amplified DNA fragment is approximately 265 bp.

　核酸を増幅する方法として特に制限はなく、ＰＣＲ法、リアルタイムＰＣＲ法、ＬＣＲ（Ligase　Chain　Reaction）法、ＳＤＡ（Strand　Displacement　Amplification）法、ＮＡＳＢＡ（Nucleic　Acid　Sequence-based　Amplification）法、ＲＣＡ（Rolling-circle　amplification）法、ＬＡＭＰ（Loop　mediated　isothermal　amplification）法など、通常の核酸増幅法を用いることができる。本発明においては、迅速性の点からＰＣＲ法を用いるのが好ましい。
　ＰＣＲの条件は、目的の核酸（増幅ＤＮＡ断片）を検出可能な程度に増幅することができれば特に制限されない。ＰＣＲの反応条件の好ましい一例としては、例えば、前記オリゴヌクレオチド対（１）、前記オリゴヌクレオチド対（２）、前記オリゴヌクレオチド対（３）、前記オリゴヌクレオチド対（４）、前記オリゴヌクレオチド対（５）、前記オリゴヌクレオチド対（６）、前記オリゴヌクレオチド対（７）、前記オリゴヌクレオチド対（８）、及び前記オリゴヌクレオチド対（９）からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を核酸プライマーとして用いてＰＣＲ法を行う場合、２本鎖ＤＮＡを１本鎖にする熱変性反応を９４～９８℃、好ましくは９４℃で１０～６０秒間行い、プライマー対を１本鎖ＤＮＡにハイブリダイズさせるアニーリング反応を５０～６２℃、好ましくは５８～６０℃で３０～６０秒間行い、ＤＮＡポリメラーゼを作用させる伸長反応を約７２℃で３０～６０秒間行い、これらを１サイクルとしたものを約３０～３５サイクル行う。
　被検体にDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌が含まれる場合、本発明のオリゴヌクレオチド対をプライマー対として使用してＰＣＲを行い、得られたＰＣＲ産物について電気泳動を行うことで、特定のサイズを有するＤＮＡ断片の増幅が認められる。このような操作を行うことにより、被検体にDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌が含まれているかを確認することができ、さらに増幅産物の長さから菌種を同定することができる。 There are no particular restrictions on the method for amplifying nucleic acids, and examples include PCR, real-time PCR, LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification), NASBA (Nucleic Acid Sequence-based Amplification), and RCA (Rolling-circle). Conventional nucleic acid amplification methods can be used, such as the LAMP (Loop mediated isothermal amplification) method and the LAMP (Loop mediated isothermal amplification) method. In the present invention, it is preferable to use the PCR method from the viewpoint of rapidity.
PCR conditions are not particularly limited as long as the target nucleic acid (amplified DNA fragment) can be amplified to a detectable extent. Preferred examples of PCR reaction conditions include, for example, the oligonucleotide pair (1), the oligonucleotide pair (2), the oligonucleotide pair (3), the oligonucleotide pair (4), and the oligonucleotide pair (5). ), the oligonucleotide pair (6), the oligonucleotide pair (7), the oligonucleotide pair (8), and the oligonucleotide pair (9) as a nucleic acid primer. When performing the PCR method using as a primer, a heat denaturation reaction to convert double-stranded DNA into single-stranded DNA is performed at 94 to 98°C, preferably 94°C for 10 to 60 seconds, and the primer pair is hybridized to the single-stranded DNA. The annealing reaction is performed at 50 to 62°C, preferably 58 to 60°C for 30 to 60 seconds, and the elongation reaction with DNA polymerase is performed at approximately 72°C for 30 to 60 seconds. One cycle of these is approximately 30 to 60 seconds. Do 35 cycles.
When the subject contains a spore-forming bacterium belonging to the genus Bacillus that has the DUF421-DUF1657 gene and can grow under neutral conditions, PCR is performed using the oligonucleotide pair of the present invention as a primer pair, and the obtained PCR product By performing electrophoresis on the DNA fragments, amplification of DNA fragments having a specific size is observed. By performing such operations, it is possible to confirm whether the sample contains spore-forming bacteria belonging to the genus Bacillus that have the DUF421-DUF1657 genes and can grow under neutral conditions, and also to determine the length of the amplified product. The bacterial species can be identified from

　本発明の方法では、上記のオリゴヌクレオチド対を単独でプライマー対として用いてPCRを行っても良いし、複数種のオリゴヌクレオチド対を混合したものをプライマー対として用いてPCR（Multiplex PCR）を行っても良い。Multiplex PCR用のオリゴヌクレオチド対としても使用できるよう、本発明の方法に用いる各オリゴヌクレオチド対では、各菌種における増幅産物長が異なるように設計されている。
　なお、前記オリゴヌクレオチド対（１）、及び前記オリゴヌクレオチド対（２）は、バチルス・サブチルス群由来のDUF421-DUF1657遺伝子の部分塩基配列を特異的に増幅できるオリゴヌクレオチド対であり、バチルス・コアグランス及びバチルス・セレウス由来のDUF421-DUF1657遺伝子（バチルス・セレウスについては、バチルス・セレウス由来のゲノム上にコードされるDUF421-DUF1657遺伝子、及びプラスミド特異的DUF421-DUF1657遺伝子）の部分塩基配列は増幅しない。また、前記オリゴヌクレオチド対（３）、前記オリゴヌクレオチド対（４）、前記オリゴヌクレオチド対（５）、前記オリゴヌクレオチド対（６）、前記オリゴヌクレオチド対（７）、及び前記オリゴヌクレオチド対（８）は、バチルス・コアグランス由来のDUF421-DUF1657遺伝子の部分塩基配列を特異的に増幅できるオリゴヌクレオチド対であり、バチルス・サブチルス群及びバチルス・セレウス由来のDUF421-DUF1657遺伝子（バチルス・セレウスについては、バチルス・セレウス由来のゲノム上にコードされるDUF421-DUF1657遺伝子、及びプラスミド特異的DUF421-DUF1657遺伝子）の部分塩基配列は増幅しない。さらに、前記オリゴヌクレオチド対（９）は、バチルス・セレウス由来のプラスミド特異的DUF421-DUF1657遺伝子の部分塩基配列を特異的に増幅できるオリゴヌクレオチド対であり、バチルス・セレウス由来のゲノム上にコードされるDUF421-DUF1657遺伝子、並びにバチルス・サブチルス群及びバチルス・コアグランス由来のDUF421-DUF1657遺伝子の部分塩基配列は増幅しない。 In the method of the present invention, PCR may be performed using the above oligonucleotide pair alone as a primer pair, or PCR (Multiplex PCR) may be performed using a mixture of multiple types of oligonucleotide pairs as a primer pair. It's okay. Each oligonucleotide pair used in the method of the present invention is designed to have a different amplification product length for each bacterial species so that it can also be used as an oligonucleotide pair for multiplex PCR.
The oligonucleotide pair (1) and the oligonucleotide pair (2) are oligonucleotide pairs that can specifically amplify the partial base sequence of the DUF421-DUF1657 gene derived from the Bacillus subtilis group; The partial base sequence of the DUF421-DUF1657 gene derived from Bacillus cereus (for Bacillus cereus, the DUF421-DUF1657 gene encoded on the genome derived from Bacillus cereus and the plasmid-specific DUF421-DUF1657 gene) is not amplified. Further, the oligonucleotide pair (3), the oligonucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair (7), and the oligonucleotide pair (8) is an oligonucleotide pair that can specifically amplify the partial base sequence of the DUF421-DUF1657 gene derived from Bacillus coagulans. The partial base sequences of the DUF421-DUF1657 gene encoded on the genome derived from B. cereus and the plasmid-specific DUF421-DUF1657 gene are not amplified. Furthermore, the oligonucleotide pair (9) is an oligonucleotide pair that can specifically amplify the partial base sequence of the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus, and is encoded on the genome derived from Bacillus cereus. DUF421-DUF1657 genes and partial base sequences of DUF421-DUF1657 genes derived from Bacillus subtilis group and Bacillus coagulans are not amplified.

　前記各オリゴヌクレオチド対を複数対組み合わせて用いてMultiplex PCRを行う場合（前記各オリゴヌクレオチド対を複数対組み合わせて混合プライマー対として用いてMultiplex PCRを行う場合）、好ましい各オリゴヌクレオチド対の組み合わせは、前記オリゴヌクレオチド対（１）及び前記オリゴヌクレオチド対（２）からなる群より選ばれる１対のオリゴヌクレオチド対（好ましくは前記オリゴヌクレオチド対（１））と、前記オリゴヌクレオチド対（３）、前記オリゴヌクレオチド対（４）、前記オリゴヌクレオチド対（５）、前記オリゴヌクレオチド対（６）、前記オリゴヌクレオチド対（７）、及び前記オリゴヌクレオチド対（８）からなる群より選ばれる少なくとも１対のオリゴヌクレオチド対と、前記オリゴヌクレオチド対（９）との組み合わせである。 When performing Multiplex PCR using a combination of multiple pairs of each of the aforementioned oligonucleotide pairs (when performing Multiplex PCR using a combination of multiple pairs of each of the aforementioned oligonucleotides as a mixed primer pair), the preferred combination of each oligonucleotide pair is: a pair of oligonucleotides (preferably the oligonucleotide pair (1)) selected from the group consisting of the oligonucleotide pair (1) and the oligonucleotide pair (2); At least one pair of oligonucleotides selected from the group consisting of the nucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair (7), and the oligonucleotide pair (8). and the oligonucleotide pair (9).

　本発明において、ＤＮＡ断片の増幅の確認は通常の方法で行うことができる。例えば増幅産物について電気泳動を行い増幅した遺伝子の大きさに対応するバンドの有無を確認する方法、増幅産物量を経時的に計測する方法、増幅産物の塩基配列を解読する方法等が挙げられるが、本発明はこれらの方法に限定されるものではない。本発明においては、ＤＮＡ断片の増幅処理後に電気泳動を行い、増幅したＤＮＡ断片の大きさに対応するバンドの有無を確認する方法が好ましい。また、本発明において、増幅産物の検出は通常の方法で行うことができる。例えば増幅反応時に放射性物質などで標識されたヌクレオチドを取り込ませる方法、蛍光物質などで標識されたプライマーを用いる方法、増幅したＤＮＡ２本鎖の間にエチジウムブロマイドなどのＤＮＡと結合することにより蛍光強度が強くなる蛍光物質を入り込ませる方法等が挙げられるが、本発明はこれらの方法に限定されるものではない。本発明においては、増幅したＤＮＡ２本鎖の間にＤＮＡと結合することにより蛍光強度が強くなる蛍光物質を入り込ませる方法が好ましい。 In the present invention, confirmation of amplification of DNA fragments can be performed by conventional methods. Examples include a method of performing electrophoresis on the amplified product to confirm the presence or absence of a band corresponding to the size of the amplified gene, a method of measuring the amount of the amplified product over time, and a method of decoding the base sequence of the amplified product. However, the present invention is not limited to these methods. In the present invention, a preferred method is to perform electrophoresis after amplification of a DNA fragment and confirm the presence or absence of a band corresponding to the size of the amplified DNA fragment. Furthermore, in the present invention, detection of amplification products can be performed by conventional methods. For example, methods that incorporate nucleotides labeled with radioactive substances during amplification reactions, methods that use primers labeled with fluorescent substances, etc., and methods that increase fluorescence intensity by binding DNA such as ethidium bromide between the amplified DNA double strands. Examples include a method of introducing a fluorescent substance that increases the intensity, but the present invention is not limited to these methods. In the present invention, a method is preferred in which a fluorescent substance that increases fluorescence intensity by binding to DNA is inserted between the amplified DNA double strands.

　本発明の方法に用いる上記オリゴヌクレオチド（ａ）～（ｌ）の調製方法は特に限定されない。例えば、設計した配列を基にして化学合成したり、試薬メーカーから購入することができる。設計した配列を基にして化学合成する場合には、オリゴヌクレオチド合成装置等を用いて合成することができる。また、合成後、吸着カラム、高速液体クロマトグラフィーや電気泳動法を用いて精製したものを用いることもできる。また、１個ないし数個の塩基が置換、欠失、挿入若しくは付加された塩基配列を有するオリゴヌクレオチドについても、通常の方法を使用して合成できる。
　また、試薬メーカーから購入する場合は、例えばFASMAC社のDNA/RNA受託合成サービスや、Thermo Fisher Scientific社のGeneArt人工遺伝子合成サービスを利用することができる。 The method for preparing the above oligonucleotides (a) to (l) used in the method of the present invention is not particularly limited. For example, it can be chemically synthesized based on a designed sequence or purchased from a reagent manufacturer. In the case of chemical synthesis based on a designed sequence, it can be synthesized using an oligonucleotide synthesizer or the like. Further, it is also possible to use a product purified using an adsorption column, high performance liquid chromatography, or electrophoresis after synthesis. Furthermore, oligonucleotides having base sequences in which one or several bases are substituted, deleted, inserted, or added can also be synthesized using conventional methods.
In addition, when purchasing from a reagent manufacturer, you can use, for example, FASMAC's DNA/RNA contract synthesis service or Thermo Fisher Scientific's GeneArt artificial gene synthesis service.

　本発明において使用される被検体としては特に制限はなく、飲食品自体、飲食品の原材料、単離菌体、培養菌体等を用いることができ、中でも中性飲食品及びそれに用いられる原料を被検体として用いることが好ましい。このような中性飲食品の具体例としては、例えば緑茶飲料・紅茶飲料・麦茶飲料等の中性茶系飲料、コーヒー飲料、乳飲料、レトルト食品、及びそれらに用いられる原料等が挙げられる。
　前記被検体からＤＮＡを調製する方法としては、芽胞形成細菌の検出を行うのに十分な精製度及び量のＤＮＡが得られるのであれば特に制限されず、未精製の状態でも使用できるが、さらに分離、抽出、濃縮、精製等の前処理をして使用することもできる。例えば、フェノール及びクロロホルム抽出を行って精製したり、市販の抽出キットを用いて精製して、核酸の純度を高めて使用することができる。また、被検体中のＲＮＡを逆転写して得られるＤＮＡを用いることもできる。また食品や原料中から菌を分離する方法は特に限定されず、例えば後述の実施例に記載の方法によって菌を分離することもできる。 There are no particular restrictions on the test substance used in the present invention, and food and drink products themselves, raw materials for food and drink products, isolated bacterial cells, cultured bacterial cells, and the like can be used. Among them, neutral food and drink products and raw materials used therein can be used. It is preferable to use it as an analyte. Specific examples of such neutral foods and drinks include neutral tea drinks such as green tea drinks, black tea drinks, barley tea drinks, coffee drinks, milk drinks, retort foods, and raw materials used therein.
The method for preparing DNA from the specimen is not particularly limited as long as DNA can be obtained with sufficient purity and amount to detect spore-forming bacteria, and it can be used in an unpurified state, but It can also be used after pretreatment such as separation, extraction, concentration, and purification. For example, the nucleic acid can be purified using phenol and chloroform extraction or purified using a commercially available extraction kit to increase the purity of the nucleic acid before use. Furthermore, DNA obtained by reverse transcription of RNA in a subject can also be used. Furthermore, the method for isolating bacteria from foods or raw materials is not particularly limited, and for example, bacteria can be isolated by the method described in Examples below.

　本発明の方法により原料又は製品中にDUF421-DUF1657遺伝子を有する芽胞形成細菌が検出される場合、当該芽胞形成細菌は高耐熱性の芽胞を形成する。そのため、このような芽胞形成細菌が検出された原料又は製品に対しては、加熱処理温度を当該芽胞形成細菌が十分に殺菌できるような条件（加熱処理食品の商業的無菌性が確保される条件）に設定することができる。なお、本明細書において加熱処理食品の「商業的無菌性」とは、熱を加えることで、通常の貯蔵、流通等の際の非冷蔵状態において食品中で繁殖しうる微生物や公衆衛生上有害な生存微生物（胞子を含む）を除去すること、又は水分活性管理と熱を加えて、貯蔵、流通等の際の通常の非冷蔵状態において食品中で繁殖しうる微生物を食品から除去すること、により達成される状態を意味する。 When spore-forming bacteria having DUF421-DUF1657 genes are detected in raw materials or products by the method of the present invention, the spore-forming bacteria form highly heat-resistant spores. Therefore, for raw materials or products in which such spore-forming bacteria have been detected, the heat treatment temperature must be set to conditions that can sufficiently kill the spore-forming bacteria (conditions that ensure commercial sterility of heat-treated foods). ). In this specification, the term "commercial sterility" of heat-treated food refers to the application of heat to eliminate microorganisms that can grow in the food under non-refrigerated conditions during normal storage, distribution, etc., and that are harmful to public health. the removal of viable microorganisms (including spores) from food, or the removal of microorganisms that can grow in food under normal non-refrigerated conditions during storage, distribution, etc., by water activity management and the application of heat; means the state achieved by

　本発明ないし本明細書において、加熱処理条件は、芽胞耐熱性（DUF421-DUF1657遺伝子の有無）の評価結果や、同定される菌種及び菌株に応じて適宜設定することができる。例えば、中性飲食品の各種原料から検出されるバチルス属に属する芽胞形成細菌の全てでDUF421-DUF1657遺伝子を持っていないことが確認された場合、あるいは原料から抽出されたバチルス属に属する芽胞形成細菌由来のＤＮＡに対してDUF421-DUF1657遺伝子を検出するためのPCRの結果が陰性である場合は、当該原料中に高耐熱性の芽胞が含まれている可能性が極めて低いことを示しており、殺菌工程においてＤ１１２．５℃値＝１分以下のように菌を十分に殺滅できる加熱条件を設定可能である。また、特定の殺菌工程を経た製品または製造中間品において、「中性条件で増殖可能なバチルス属に属する芽胞形成細菌」が検出され、さらに本発明の方法により当該芽胞形成細菌がDUF421-DUF1657遺伝子を有することが認められた場合、当該殺菌条件が不十分であったと判断することができる。
　また、予め各菌種、菌株の芽胞耐熱性を評価し、加熱処理条件を決定しておくことで、同一の菌種、菌株が検出された場合に当該条件をそのまま、或いは適宜変更して適用することもできる。各菌種、菌株を殺菌するのに十分な加熱処理条件（温度、時間、圧力等）は、例えば本技術分野における通常の方法に基づいて決定することもでき、また実施例に記載の方法によっても決定することができる。 In the present invention and the present specification, heat treatment conditions can be appropriately set according to the evaluation results of spore heat resistance (presence or absence of DUF421-DUF1657 genes) and the bacterial species and strain to be identified. For example, if it is confirmed that all spore-forming bacteria belonging to the genus Bacillus detected from various raw materials of neutral food and drink products do not have the DUF421-DUF1657 gene, or when spore-forming bacteria belonging to the genus Bacillus extracted from raw materials If the PCR results for detecting the DUF421-DUF1657 genes on bacterial-derived DNA are negative, this indicates that there is an extremely low possibility that the raw material contains highly heat-resistant spores. In the sterilization process, it is possible to set heating conditions that can sufficiently kill bacteria, such as D 112.5°C value = 1 minute or less. In addition, "spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions" was detected in products or intermediate products that underwent a specific sterilization process, and furthermore, the method of the present invention detected the spore-forming bacteria with the DUF421-DUF1657 gene. If it is found that the sterilization conditions are insufficient, it can be determined that the sterilization conditions were insufficient.
In addition, by evaluating the spore heat resistance of each bacterial species and strain in advance and determining the heat treatment conditions, if the same bacterial species or strain is detected, the conditions can be applied as is or with appropriate changes. You can also. The heat treatment conditions (temperature, time, pressure, etc.) sufficient to sterilize each bacterial species and strain can be determined, for example, based on the usual methods in this technical field, or by the methods described in the Examples. can also be determined.

　本発明の方法によれば、被検体の調製工程から高耐熱性の芽胞を形成する芽胞形成細菌の検出工程までを短時間で行うことができる。例えば下記の実施例６のように、各菌株のコロニーを植菌した懸濁液を被検体とする場合であれば半日程度、実施例７のように、原料を被検体として原料中に含まれる前記芽胞形成細菌を検出する場合であれば２日程度という短時間で行うことができる。 According to the method of the present invention, the steps from the preparation of the specimen to the step of detecting spore-forming bacteria that form highly heat-resistant spores can be carried out in a short time. For example, as in Example 6 below, if the specimen is a suspension inoculated with colonies of each strain, it will take about half a day; as in Example 7, the raw material will be the specimen contained in the raw material. In the case of detecting the spore-forming bacteria, it can be carried out in a short time of about 2 days.

　本発明のDUF421-DUF1657遺伝子を有する芽胞形成細菌検出用のキット（以下、「本発明のキット」とも称す。）は、前記本発明の検出用オリゴヌクレオチド対をプライマー対として含有するものである。このキットは、本発明の方法に用いることができる。本発明のキットは、前記プライマー対の他に、目的に応じ、標識検出物質、緩衝液、核酸合成酵素（ＤＮＡポリメラーゼ、ＲＮＡポリメラーゼ、逆転写酵素等）、酵素基質（ｄＮＴＰ，ｒＮＴＰ等）等、菌類の検出に通常用いられる物質を含有する。本発明のキットには、本発明の検出用オリゴヌクレオチドによって検出反応が可能であることを確認するための陽性対照（ポジティブコントロール）を含んでいてもよい。陽性対照としては、例えば、本発明の方法により増幅される領域を含んだＤＮＡが挙げられる。 The kit for detecting spore-forming bacteria having the DUF421-DUF1657 genes of the present invention (hereinafter also referred to as the "kit of the present invention") contains the detection oligonucleotide pair of the present invention as a primer pair. This kit can be used in the method of the invention. In addition to the primer pair, the kit of the present invention may contain, depending on the purpose, labeled detection substances, buffers, nucleic acid synthases (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), enzyme substrates (dNTPs, rNTPs, etc.), etc. Contains substances commonly used to detect fungi. The kit of the present invention may contain a positive control for confirming that a detection reaction is possible using the detection oligonucleotide of the present invention. Examples of positive controls include DNA containing the region amplified by the method of the present invention.

　上述した実施形態に関し、本発明はさらに以下の検出方法、芽胞耐熱性の評価方法、加熱処理条件の決定方法、芽胞形成細菌検出用のキット、及びオリゴヌクレオチド対を開示する。 Regarding the embodiments described above, the present invention further discloses the following detection method, method for evaluating spore heat resistance, method for determining heat treatment conditions, kit for detecting spore-forming bacteria, and oligonucleotide pair.

＜１＞
　下記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対（１）、
　下記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対（２）、
　下記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対（３）、
　下記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対（４）、
　下記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対（５）、
　下記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対（６）、
　下記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対（７）、
　下記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対（８）、並びに
　下記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対（９）
からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を核酸プライマーとして用いて、DUF421-DUF1657遺伝子の部分塩基配列で表される核酸を増幅し、
増幅産物の有無により中性条件で増殖可能なバチルス属に属する芽胞形成細菌を検出する、
バチルス属に属する芽胞形成細菌の検出方法。
 
（ａ）配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・サブチルス群、の検出に使用できるオリゴヌクレオチド。
（ｂ）配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・サブチルス群、の検出に使用できるオリゴヌクレオチド。
（ｃ）配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・サブチルス群、の検出に使用できるオリゴヌクレオチド。
（ｄ）配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチド。
（ｅ）配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチド。
（ｆ）配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチド。
（ｇ）配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチド。
（ｈ）配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチド。
（ｉ）配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチド。
（ｊ）配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチド。
（ｋ）配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・セレウス、の検出に使用できるオリゴヌクレオチド。
（ｌ）配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列との同一性が８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・セレウス、の検出に使用できるオリゴヌクレオチド。 <1>
An oligonucleotide pair (1) consisting of the following oligonucleotides (a) and (c),
An oligonucleotide pair (2) consisting of the following oligonucleotides (b) and (c),
An oligonucleotide pair (3) consisting of the following oligonucleotides (d) and (j),
An oligonucleotide pair (4) consisting of the following oligonucleotides (e) and (j),
An oligonucleotide pair (5) consisting of the following oligonucleotides (f) and (j),
An oligonucleotide pair (6) consisting of the following oligonucleotides (g) and (j),
An oligonucleotide pair (7) consisting of the following oligonucleotides (h) and (j),
An oligonucleotide pair (8) consisting of the following oligonucleotides (i) and (j), and an oligonucleotide pair (9) consisting of the following oligonucleotides (k) and (l)
Amplifying a nucleic acid represented by a partial base sequence of the DUF421-DUF1657 gene using at least one oligonucleotide pair selected from the group consisting of as a nucleic acid primer,
Detects spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions based on the presence or absence of amplification products.
A method for detecting spore-forming bacteria belonging to the genus Bacillus.

(a) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 1, or an identity with the base sequence represented by SEQ ID NO: 1 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus subtilis group, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(b) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or an identity with the base sequence represented by SEQ ID NO: 2 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus subtilis group, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(c) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an identity with the base sequence represented by SEQ ID NO: 3 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus subtilis group, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(d) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or an identity with the base sequence represented by SEQ ID NO: 4 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(e) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5, or an identity with the base sequence represented by SEQ ID NO: 5 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(f) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6, or an identity with the base sequence represented by SEQ ID NO: 6 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(g) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an identity with the base sequence represented by SEQ ID NO: 7 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(h) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or an identity with the base sequence represented by SEQ ID NO: 8 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or an identity with the base sequence represented by SEQ ID NO: 9 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(j) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 10, or an identity with the base sequence represented by SEQ ID NO: 10 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which is preferably 95% or more and has the DUF421-DUF1657 genes and can grow under neutral conditions.
(k) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 11, or an identity with the base sequence represented by SEQ ID NO: 11 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus cereus, which is preferably 95% or more and has a plasmid-specific DUF421-DUF1657 gene and can grow under neutral conditions.
(l) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12, or an identity with the base sequence represented by SEQ ID NO: 12 of 80% or more, preferably 85% or more, more preferably 90% or more, and An oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus cereus, which is preferably 95% or more and has a plasmid-specific DUF421-DUF1657 gene and can grow under neutral conditions.

＜２＞
　前記増幅産物により前記芽胞形成細菌の同定を行う、前記＜１＞に記載の方法。
＜３＞
　前記増幅産物の有無により前記芽胞形成細菌の芽胞耐熱性を評価する、前記＜１＞又は＜２＞に記載の方法。 <2>
The method according to <1> above, wherein the spore-forming bacterium is identified by the amplification product.
<3>
The method according to <1> or <2>, wherein the spore heat resistance of the spore-forming bacteria is evaluated based on the presence or absence of the amplification product.

＜４＞
　前記オリゴヌクレオチド対（１）、前記オリゴヌクレオチド対（２）、前記オリゴヌクレオチド対（３）、前記オリゴヌクレオチド対（４）、前記オリゴヌクレオチド対（５）、前記オリゴヌクレオチド対（６）、前記オリゴヌクレオチド対（７）、前記オリゴヌクレオチド対（８）、及び前記オリゴヌクレオチド対（９）からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を核酸プライマーとして用いて、DUF421-DUF1657遺伝子の部分塩基配列で表される核酸を増幅し、
増幅産物の有無により中性条件で増殖可能なバチルス属に属する芽胞形成細菌の芽胞耐熱性を評価する、
バチルス属に属する芽胞形成細菌の芽胞耐熱性の評価方法。
＜５＞前記＜３＞又は＜４＞の方法によって評価した芽胞耐熱性に基づいて、前記芽胞形成細菌の加熱処理条件を決定することを特徴とする、バチルス属に属する芽胞形成細菌の加熱処理条件の決定方法。 <4>
The oligonucleotide pair (1), the oligonucleotide pair (2), the oligonucleotide pair (3), the oligonucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair A partial base sequence of the DUF421-DUF1657 gene is prepared using at least one oligonucleotide pair selected from the group consisting of the nucleotide pair (7), the oligonucleotide pair (8), and the oligonucleotide pair (9) as a nucleic acid primer. Amplify the nucleic acid represented by
Evaluating the spore heat resistance of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions based on the presence or absence of amplification products.
A method for evaluating spore heat resistance of spore-forming bacteria belonging to the genus Bacillus.
<5> Heat treatment of spore-forming bacteria belonging to the genus Bacillus, characterized in that heat treatment conditions for the spore-forming bacteria are determined based on the spore heat resistance evaluated by the method of <3> or <4> above. How to determine conditions.

＜６＞
　前記中性条件で増殖可能なバチルス属に属する芽胞形成細菌が、ｐＨ４．６以上８．０未満の好気的条件下で増殖可能な芽胞形成細菌である、前記＜１＞～＜５＞のいずれか１項に記載の方法。
＜７＞前記中性条件で増殖可能なバチルス属に属する芽胞形成細菌が、バチルス・サブチルス群、バチルス・コアグランス、バチルス・セレウス、バチルス・メガテリウム、バチルス・ピュミルス、バチルス・シンプレックス、バチルス・アンスラシス、バチルス・ブレヴィス、バチルス・レンタス、及びバチルス・ミコイデスからなる群より選ばれる少なくとも１種、好ましくはバチルス・サブチルス群、バチルス・コアグランス、及びバチルス・セレウスからなる群より選ばれる少なくとも１種、である、前記＜１＞～＜６＞のいずれか１項に記載の方法。
＜８＞前記バチルス・サブチルス群が、バチルス・サブチルス、バチルス・リケニフォルミス、バチルス・アミロリケファシエンス、バチルス・シアメンシス、バチルス・ベレゼンシス、バチルス・パラリケニフォルミス、及びバチルス・ソノレンシスからなる群より選ばれる少なくとも１種、好ましくはバチルス・サブチルス、バチルス・リケニフォルミス、バチルス・アミロリケファシエンス、バチルス・シアメンシス、及びバチルス・ベレゼンシスからなる群より選ばれる少なくとも１種、より好ましくはバチルス・サブチルス、である、前記＜７＞に記載の方法。 <6>
<1> to <5> above, wherein the spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions are spore-forming bacteria that can grow under aerobic conditions with a pH of 4.6 or more and less than 8.0. The method described in any one of the above.
<7> The spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions include Bacillus subtilis group, Bacillus coagulans, Bacillus cereus, Bacillus megaterium, Bacillus pumilus, Bacillus simplex, Bacillus anthracis, and Bacillus. - At least one species selected from the group consisting of Bacillus brevis, Bacillus lentus, and Bacillus mycoides, preferably at least one species selected from the group consisting of Bacillus subtilis, Bacillus coagulans, and Bacillus cereus. The method according to any one of <1> to <6>.
<8> The Bacillus subtilis group is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus siamensis, Bacillus belezensis, Bacillus paralicheniformis, and Bacillus sonorensis. at least one species, preferably at least one species selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus siamensis, and Bacillus veresensis, and more preferably Bacillus subtilis. The method described in <7>.

＜９＞
　前記オリゴヌクレオチド（ａ）が、配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・サブチルス群、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜８＞のいずれか１項に記載の方法。
＜１０＞
　前記オリゴヌクレオチド（ｂ）が、配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・サブチルス群、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜９＞のいずれか１項に記載の方法。
＜１１＞
　前記オリゴヌクレオチド（ｃ）が、配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・サブチルス群、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１０＞のいずれか１項に記載の方法。
＜１２＞
　前記オリゴヌクレオチド（ｄ）が、配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１１＞のいずれか１項に記載の方法。
＜１３＞
　前記オリゴヌクレオチド（ｅ）が、配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１２＞のいずれか１項に記載の方法。
＜１４＞
　前記オリゴヌクレオチド（ｆ）が、配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１３＞のいずれか１項に記載の方法。
＜１５＞
　前記オリゴヌクレオチド（ｇ）が、配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１４＞のいずれか１項に記載の方法。
＜１６＞
　前記オリゴヌクレオチド（ｈ）が、配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１５＞のいずれか１項に記載の方法。
＜１７＞
　前記オリゴヌクレオチド（ｉ）が、配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１６＞のいずれか１項に記載の方法。
＜１８＞
　前記オリゴヌクレオチド（ｊ）が、配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・コアグランス、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１７＞のいずれか１項に記載の方法。
＜１９＞
　前記オリゴヌクレオチド（ｋ）が、配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・セレウス、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１８＞のいずれか１項に記載の方法。
＜２０＞
　前記オリゴヌクレオチド（ｌ）が、配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列において１個ないし数個、好ましくは１個以上４個以下、より好ましくは１個以上３個以下、より好ましくは１個又は２個、さらに好ましくは１個、の塩基が欠失、置換、挿入若しくは付加されており、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌、好ましくはバチルス・セレウス、の検出に使用できるオリゴヌクレオチドである、前記＜１＞～＜１９＞のいずれか１項に記載の方法。 <9>
The oligonucleotide (a) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 1, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 1. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <8>, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably the Bacillus subtilis group.
<10>
The oligonucleotide (b) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 2. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <9> above, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus subtilis group.
<11>
The oligonucleotide (c) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 3. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <10>, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus subtilis group.
<12>
The oligonucleotide (d) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or one to several, preferably 1 to 4, more preferably 1 to 4 in the base sequence represented by SEQ ID NO: 4. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <11>, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans.
<13>
The oligonucleotide (e) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 5. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <12>, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans.
<14>
The oligonucleotide (f) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6, or one to several, preferably 1 to 4, more preferably 1 to 4 in the base sequence represented by SEQ ID NO: 6. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <13>, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans.
<15>
The oligonucleotide (g) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 7. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <14>, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans.
<16>
The oligonucleotide (h) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or one to several, preferably one to four, more preferably one to several in the base sequence represented by SEQ ID NO: 8. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <15>, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans.
<17>
The oligonucleotide (i) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 9. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <16>, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans.
<18>
The oligonucleotide (j) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 10, or one to several, preferably 1 to 4, more preferably 1 to 4 in the base sequence represented by SEQ ID NO: 10. has one or more and three or less, more preferably one or two, and still more preferably one base deleted, substituted, inserted, or added, and has the DUF421-DUF1657 gene and grows under neutral conditions. The method according to any one of <1> to <17>, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans.
<19>
The oligonucleotide (k) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 11, or one to several, preferably one or more and four or less, more preferably one or more in the base sequence represented by SEQ ID NO: 11. is a neutral base in which 1 to 3 bases, more preferably 1 or 2 bases, and still more preferably 1 base is deleted, substituted, inserted or added, and has a plasmid-specific DUF421-DUF1657 gene. The method according to any one of <1> to <18> above, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions, preferably Bacillus cereus.
<20>
The oligonucleotide (l) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12, or one to several, preferably 1 to 4, more preferably 1 to 4 in the base sequence represented by SEQ ID NO: 12. is a neutral base in which 1 to 3 bases, more preferably 1 or 2 bases, and still more preferably 1 base is deleted, substituted, inserted or added, and has a plasmid-specific DUF421-DUF1657 gene. The method according to any one of <1> to <19> above, which is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions, preferably Bacillus cereus.

＜２１＞
　前記オリゴヌクレオチド対（１）、前記オリゴヌクレオチド対（２）、前記オリゴヌクレオチド対（３）、前記オリゴヌクレオチド対（４）、前記オリゴヌクレオチド対（５）、前記オリゴヌクレオチド対（６）、前記オリゴヌクレオチド対（７）、及び前記オリゴヌクレオチド対（８）からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対をプライマー対として用いてポリメラーゼ連鎖反応法によりゲノム上のDUF421-DUF1657遺伝子の部分塩基配列で表される核酸を増幅し、
　前記オリゴヌクレオチド対（９）をプライマー対として用いてポリメラーゼ連鎖反応法によりプラスミド特異的DUF421-DUF1657遺伝子の部分塩基配列で表される核酸を増幅する、
前記＜１＞～＜２０＞のいずれか１項に記載の方法。
＜２２＞
　前記オリゴヌクレオチド対（１）及び前記オリゴヌクレオチド対（２）からなる群より選ばれる１対のオリゴヌクレオチド対、好ましくは前記オリゴヌクレオチド対（１）、
　前記オリゴヌクレオチド対（３）、前記オリゴヌクレオチド対（４）、前記オリゴヌクレオチド対（５）、前記オリゴヌクレオチド対（６）、前記オリゴヌクレオチド対（７）、及び前記オリゴヌクレオチド対（８）からなる群より選ばれる少なくとも１対のオリゴヌクレオチド対、並びに
　前記オリゴヌクレオチド対（９）、
を混合して混合プライマー対として用いる、前記＜２１＞に記載の方法。
＜２３＞
　前記オリゴヌクレオチド対（１）、及び前記オリゴヌクレオチド対（２）からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を用いて増幅産物が確認されたときは、被検体に含まれる芽胞形成細菌をバチルス・サブチルス群と同定し、
　前記オリゴヌクレオチド対（３）対、前記オリゴヌクレオチド対（４）、前記オリゴヌクレオチド対（５）、前記オリゴヌクレオチド対（６）、前記オリゴヌクレオチド対（７）、及び前記オリゴヌクレオチド対（８）からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を用いて増幅産物が確認されたときは、被検体に含まれる芽胞形成細菌をバチルス・コアグランスと同定し、
　前記オリゴヌクレオチド対（９）を用いて増幅産物が確認されたときは、被検体に含まれる芽胞形成細菌をバチルス・セレウスと同定する、
前記＜１＞～＜２２＞のいずれか１項に記載の方法。
＜２４＞
　被検体を中性飲食品及びそれに用いられる原料とする、前記＜１＞～＜２３＞のいずれか１項に記載の方法。 <21>
The oligonucleotide pair (1), the oligonucleotide pair (2), the oligonucleotide pair (3), the oligonucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair Using at least one oligonucleotide pair selected from the group consisting of the nucleotide pair (7) and the oligonucleotide pair (8) as a primer pair, a partial base sequence of the DUF421-DUF1657 gene on the genome is obtained by a polymerase chain reaction method. amplify the expressed nucleic acid;
amplifying the nucleic acid represented by the partial base sequence of the plasmid-specific DUF421-DUF1657 gene by polymerase chain reaction using the oligonucleotide pair (9) as a primer pair;
The method according to any one of <1> to <20> above.
<22>
A pair of oligonucleotides selected from the group consisting of the oligonucleotide pair (1) and the oligonucleotide pair (2), preferably the oligonucleotide pair (1),
Consisting of the oligonucleotide pair (3), the oligonucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair (7), and the oligonucleotide pair (8) at least one oligonucleotide pair selected from the group, and the oligonucleotide pair (9),
The method according to <21> above, wherein the mixture is used as a mixed primer pair.
<23>
When an amplification product is confirmed using at least one oligonucleotide pair selected from the group consisting of the oligonucleotide pair (1) and the oligonucleotide pair (2), the spore-forming bacteria contained in the sample are confirmed. Identified as Bacillus subtilis group,
From the oligonucleotide pair (3), the oligonucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair (7), and the oligonucleotide pair (8) When an amplification product is confirmed using at least one oligonucleotide pair selected from the group consisting of: identifying the spore-forming bacteria contained in the subject as Bacillus coagulans;
When an amplification product is confirmed using the oligonucleotide pair (9), identifying the spore-forming bacteria contained in the subject as Bacillus cereus;
The method according to any one of <1> to <22> above.
<24>
The method according to any one of <1> to <23> above, wherein the subject is a neutral food or drink and a raw material used therein.

＜２５＞
　前記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対（１）、前記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対（２）、前記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対（３）、前記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対（４）、前記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対（５）、前記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対（６）、前記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対（７）、前記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対（８）、並びに前記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対（９）からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を有する、ＤＮＡキット。
＜２６＞
　前記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対（１）、前記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対（２）、前記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対（３）、前記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対（４）、前記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対（５）、前記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対（６）、前記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対（７）、前記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対（８）、並びに前記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対（９）。
＜２７＞
　前記オリゴヌクレオチド（ａ）が、前記＜９＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｂ）が、前記＜１０＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｃ）が、前記＜１１＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｄ）が、前記＜１２＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｅ）が、前記＜１３＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｆ）が、前記＜１４＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｇ）が、前記＜１５＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｈ）が、前記＜１６＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｉ）が、前記＜１７＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｊ）が、前記＜１８＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｋ）が、前記＜１９＞に記載のオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｌ）が、前記＜２０＞に記載のオリゴヌクレオチドである、
前記＜２５＞又は＜２６＞に記載のＤＮＡキット又はオリゴヌクレオチド対。
＜２８＞
　前記オリゴヌクレオチド対（１）、及び前記オリゴヌクレオチド対（２）がバチルス・サブチルス群の検出に使用できるオリゴヌクレオチド対であり、
　前記オリゴヌクレオチド対（３）、前記オリゴヌクレオチド対（４）、前記オリゴヌクレオチド対（５）、前記オリゴヌクレオチド対（６）、前記オリゴヌクレオチド対（７）、及び前記オリゴヌクレオチド対（８）がバチルス・コアグランスの検出に使用できるオリゴヌクレオチド対であり、
　前記オリゴヌクレオチド対（９）がバチルス・セレウスの検出に使用できるオリゴヌクレオチド対である、
前記＜２５＞～＜２７＞のいずれか１項に記載のＤＮＡキット又はオリゴヌクレオチド対。 <25>
An oligonucleotide pair (1) consisting of the oligonucleotides (a) and (c), an oligonucleotide pair (2) consisting of the oligonucleotides (b) and (c), and an oligonucleotide pair (2) consisting of the oligonucleotides (d) and (j). an oligonucleotide pair (3), an oligonucleotide pair (4) consisting of the oligonucleotides (e) and (j), an oligonucleotide pair (5) consisting of the oligonucleotides (f) and (j), and an oligonucleotide pair (5) consisting of the oligonucleotides (f) and (j); ) and (j), an oligonucleotide pair (7) consisting of the oligonucleotides (h) and (j), and an oligonucleotide pair (8) consisting of the oligonucleotides (i) and (j). ), and at least one oligonucleotide pair selected from the group consisting of the oligonucleotide pair (9) consisting of the oligonucleotides (k) and (l).
<26>
An oligonucleotide pair (1) consisting of the oligonucleotides (a) and (c), an oligonucleotide pair (2) consisting of the oligonucleotides (b) and (c), and an oligonucleotide pair (2) consisting of the oligonucleotides (d) and (j). an oligonucleotide pair (3), an oligonucleotide pair (4) consisting of the oligonucleotides (e) and (j), an oligonucleotide pair (5) consisting of the oligonucleotides (f) and (j), and an oligonucleotide pair (5) consisting of the oligonucleotides (f) and (j); ) and (j), an oligonucleotide pair (7) consisting of the oligonucleotides (h) and (j), and an oligonucleotide pair (8) consisting of the oligonucleotides (i) and (j). ), and an oligonucleotide pair (9) consisting of the oligonucleotides (k) and (l).
<27>
The oligonucleotide (a) is the oligonucleotide described in <9> above,
The oligonucleotide (b) is the oligonucleotide described in <10> above,
The oligonucleotide (c) is the oligonucleotide described in <11> above,
The oligonucleotide (d) is the oligonucleotide according to <12> above,
The oligonucleotide (e) is the oligonucleotide described in <13> above,
The oligonucleotide (f) is the oligonucleotide described in <14> above,
The oligonucleotide (g) is the oligonucleotide described in <15> above,
The oligonucleotide (h) is the oligonucleotide described in <16> above,
The oligonucleotide (i) is the oligonucleotide described in <17> above,
The oligonucleotide (j) is the oligonucleotide described in <18> above,
The oligonucleotide (k) is the oligonucleotide described in <19> above,
The oligonucleotide (l) is the oligonucleotide described in <20> above,
The DNA kit or oligonucleotide pair according to <25> or <26> above.
<28>
The oligonucleotide pair (1) and the oligonucleotide pair (2) are oligonucleotide pairs that can be used for detection of Bacillus subtilis group,
The oligonucleotide pair (3), the oligonucleotide pair (4), the oligonucleotide pair (5), the oligonucleotide pair (6), the oligonucleotide pair (7), and the oligonucleotide pair (8) are Bacillus・An oligonucleotide pair that can be used to detect coagulance,
the oligonucleotide pair (9) is an oligonucleotide pair that can be used for detection of Bacillus cereus;
The DNA kit or oligonucleotide pair according to any one of <25> to <27>.

　以下、本発明を実施例に基づきさらに詳細に説明するが、本発明はこれに限定されるものではない。 Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited thereto.

実施例１　３菌種群のMultiplex PCR
（１）プライマーの設計
　バチルス・サブチルスB4146株由来のDUF421-DUF1657遺伝子（配列番号１３）の塩基配列情報を基に、配列番号１の塩基配列で表されるオリゴヌクレオチドからなるプライマー（プライマー１）、及び配列番号３の塩基配列で表されるプライマー（プライマー３）を設計した。また、バチルス・コアグランスDSM1=ATCC7050株由来のDUF421-DUF1657遺伝子（配列番号１５）の塩基配列を基に、配列番号５の塩基配列で表されるオリゴヌクレオチドからなるプライマー（プライマー５）、及び配列番号１０の塩基配列で表されるプライマー（プライマー１０）を設計した。さらに、バチルス・セレウスAH187株由来のDUF421-DUF1657遺伝子（配列番号１６）の塩基配列を基に、配列番号１１の塩基配列で表されるオリゴヌクレオチドからなるプライマー（プライマー１１）、及び配列番号１２の塩基配列で表されるプライマー（プライマー１２）を設計した。設計した各プライマー情報を基に、FASMAC社のDNA/RNA受託合成サービスより、逆相カラム精製品のプライマーを得た。
　なお、各菌種のDUF421-DUF1657遺伝子の塩基配列情報は、NCBI(National Center for Biotechnology Information)より入手した。
　各菌種の菌株がDUF421-DUF1657遺伝子を有している場合、上記プライマー１及びプライマー３のプライマー対によって増幅されるＤＮＡ断片の長さは約３５０ｂｐ、上記プライマー５及びプライマー１０のプライマー対によって増幅されるＤＮＡ断片の長さは約４６５ｂｐ、上記プライマー１１及びプライマー１２のプライマー対によって増幅されるＤＮＡ断片の長さは約２６５ｂｐである。
（２）被検体の調製
　上記で作製した各プライマーの評価には、下記表１に記載のバチルス・サブチルスJCA1402株、JCA1403株及びJCM1465株、バチルス・コアグランスNBRC12583株、JCA1108株及びDSM2314株、並びにバチルス・セレウスJCM2152株及びJCM17690株を用いた。なお、JCA株は日本缶詰びん詰レトルト食品協会、JCM株は国立研究開発法人理化学研究所微生物材料開発室、NBRC株は独立行政法人製品評価技術基盤機構、DSM株はライプニッツ研究所ドイツ微生物株保存機関、よりそれぞれ入手した。なお、バチルス・サブチルスJCA1403株、バチルス・コアグランスNBRC12583株及びJCA1108株はそれぞれゲノム上に、バチルス・セレウスJCM17690株はプラスミド上に、芽胞耐熱性向上にかかわるDUF421-DUF1657遺伝子を有している。
　各菌株をSCD寒天培地（商品名：SCD培地「ダイゴ」、一般細菌検査用、富士フイルム和光純薬株式会社製）に播種し、バチルス・サブチルス、バチルス・セレウスは３０℃で、バチルス・コアグランスは４５℃の温度条件でプレートを静置させて１～２日間培養した。寒天培地で培養後、各コロニーを白金耳でかき取り、SCD液体培地（商品名：SCD液体培地「ダイゴ」、一般細菌検査用、富士フイルム和光純薬株式会社製）に植菌して同様の温度条件下で２～３日間培養した。 Example 1 Multiplex PCR of 3 bacterial species groups
(1) Primer design Based on the base sequence information of the DUF421-DUF1657 gene (SEQ ID NO: 13) derived from Bacillus subtilis strain B4146, a primer (primer 1) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 1, A primer (primer 3) represented by the base sequence of SEQ ID NO: 3 was designed. In addition, based on the base sequence of the DUF421-DUF1657 gene (SEQ ID NO: 15) derived from Bacillus coagulans DSM1=ATCC7050 strain, a primer (primer 5) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 5, and SEQ ID NO: A primer (primer 10) represented by a 10 base sequence was designed. Furthermore, based on the base sequence of the DUF421-DUF1657 gene (SEQ ID NO: 16) derived from Bacillus cereus strain AH187, a primer (primer 11) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 11 and a primer of SEQ ID NO: 12 were prepared. A primer (primer 12) represented by a base sequence was designed. Based on the information on each designed primer, primers for a reversed phase column purified product were obtained from FASMAC's DNA/RNA contract synthesis service.
The nucleotide sequence information of the DUF421-DUF1657 genes of each bacterial species was obtained from NCBI (National Center for Biotechnology Information).
When each bacterial strain has DUF421-DUF1657 genes, the length of the DNA fragment amplified by the primer pair of primer 1 and primer 3 is about 350 bp, and the length of the DNA fragment amplified by the primer pair of primer 5 and primer 10 is about 350 bp. The length of the DNA fragment amplified by the primer pair of primer 11 and primer 12 is approximately 265 bp.
(2) Preparation of test samples For evaluation of each primer prepared above, Bacillus subtillus strains JCA1402, JCA1403, and JCM1465, Bacillus coagulans NBRC12583, JCA1108, and DSM2314, and Bacillus coagulans listed in Table 1 below were used. - B. cereus JCM2152 strain and JCM17690 strain were used. The JCA strain is from the Japan Canned and Bottled Retort Foods Association, the JCM strain is from the RIKEN Microbial Materials Development Office, the NBRC strain is from the National Institute of Technology and Evaluation, and the DSM strain is from the Leibniz Institute German Microbial Strain Storage. They were obtained from each institution. In addition, Bacillus subtillus strain JCA1403, Bacillus coagulans strain NBRC12583, and strain JCA1108 each have the DUF421-DUF1657 genes involved in improving spore heat resistance on their genomes, and Bacillus cereus strain JCM17690 on their plasmids.
Each strain was seeded on an SCD agar medium (trade name: SCD medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and Bacillus subtilis and Bacillus cereus were incubated at 30°C, and Bacillus coagulans was incubated at 30°C. The plate was allowed to stand at a temperature of 45°C and cultured for 1 to 2 days. After culturing on an agar medium, each colony was scraped off with a platinum loop and inoculated into an SCD liquid medium (product name: SCD liquid medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in the same way. The cells were cultured for 2 to 3 days under temperature conditions.

（３）ゲノムＤＮＡの調製
　培養後の各SCD液体培地から懸濁液を１．０ｍＬ回収し、ゲノムＤＮＡ調製用キット（商品名：NucleoSpin Tissue、タカラバイオ株式会社製）を用い、菌体溶解時にリゾチームによる溶解法を採用した以外は当該キットに付属のプロトコルに準拠して菌体からＤＮＡを抽出した。なお、当該キットではプラスミドを含めたＤＮＡが抽出可能であるため、以下抽出したＤＮＡを「抽出ＤＮＡ」と記載する。得られた抽出ＤＮＡ溶液の濃度は５０ｎｇ/μＬに調整した。 (3) Preparation of genomic DNA Collect 1.0 mL of the suspension from each SCD liquid medium after culturing, and use a genomic DNA preparation kit (product name: NucleoSpin Tissue, manufactured by Takara Bio Inc.) to lyse the bacterial cells. DNA was extracted from the bacterial cells according to the protocol attached to the kit, except that a lysis method using lysozyme was used. Note that since DNA including plasmids can be extracted with this kit, the extracted DNA will be hereinafter referred to as "extracted DNA". The concentration of the obtained extracted DNA solution was adjusted to 50 ng/μL.

（４）Multiplex PCR反応
　上記で得られた抽出ＤＮＡ溶液をＤＮＡテンプレートとして、Multiplex PCR Assay Kit Ver.2 （商品名、タカラバイオ株式会社社製）を用いてMultiplex PCR溶液を調製した。Multiplex PCR Enzyme Mixを０．２５μＬ、2x Multiplex PCR Buffer (Mg2+、dNTP plus)を２５μＬ、抽出ＤＮＡ溶液を１μＬ（抽出ＤＮＡ５０ｎｇ分）、プライマー１、３、５、１０、１１及び１２をそれぞれ終濃度が０．２μＭとなるように加え、無菌蒸留水をMultiplex PCR溶液の容量が５０μＬとなるように加えた。なお、抽出ＤＮＡ溶液としてバシルス・サブチリスJCA1402株、バチルス・コアグランスNBRC12583株、及びバシルス・セレウスJCM17690株の抽出ＤＮＡ溶液の混合液も使用した。
　サーマルサイクラーとしてProflex PCR system（Thermo Fisher Scientific社製）を用いた。反応サイクルは、(i)９４℃、６０秒間の熱変性反応、(ii)９４℃、３０秒間の熱変性反応、（iii）６０℃、３０秒間のアニーリング反応、（iv）７２℃、３０秒間の伸長反応、(v)７２℃、３００秒間の伸長反応、とし、（ii）～(iv)を１サイクルとして３０サイクル繰り返した。 (4) Multiplex PCR reaction Using the extracted DNA solution obtained above as a DNA template, a Multiplex PCR solution was prepared using Multiplex PCR Assay Kit Ver. 2 (trade name, manufactured by Takara Bio Inc.). 0.25 μL of Multiplex PCR Enzyme Mix, 25 μL of 2x Multiplex PCR Buffer (Mg 2+ , dNTP plus), 1 μL of extracted DNA solution (50 ng of extracted DNA), and primers 1, 3, 5, 10, 11, and 12, respectively. The concentration was 0.2 μM, and sterile distilled water was added so that the volume of the Multiplex PCR solution was 50 μL. Additionally, a mixture of extracted DNA solutions of Bacillus subtilis JCA1402 strain, Bacillus coagulans NBRC12583 strain, and Bacillus cereus JCM17690 strain was also used as the extracted DNA solution.
A Proflex PCR system (manufactured by Thermo Fisher Scientific) was used as a thermal cycler. The reaction cycle was (i) heat denaturation reaction at 94°C for 60 seconds, (ii) heat denaturation reaction at 94°C for 30 seconds, (iii) annealing reaction at 60°C for 30 seconds, (iv) 72°C for 30 seconds. (v) an elongation reaction at 72° C. for 300 seconds, and 30 cycles were repeated with (ii) to (iv) as one cycle.

（５）電気泳動及び撮影
　ＰＣＲ後、ＰＣＲ溶液から２μＬを分取してローディングバッファーと十分に混和し、２％アガロースゲルを用いてMupid-exU (タカラバイオ株式会社製)により電気泳動（１００Ｖ、３０分間）を行った。DNAマーカーには100bp DNA Step Ladder（Promega社製）を使用した。電気泳動終了後、アガロースゲルをSYBR Safe (Thermo Fisher Scientific社製)で３０分間染色し、BioDoc-It Imaging Systems（UVP社製）を使用して254 nmの紫外線を照射し、３～５秒の露光時間で撮影し、増幅されたＤＮＡ断片の有無を確認した。アガロースゲルの撮影画像を図１に示す。
　なお、図１中の番号１～１０は、下記表１記載の対応する試料番号の試料から抽出したＤＮＡを用いて反応を行ったサンプルであることを示している。図１の各レーン番号と、アプライしたサンプルとの対応は下記表１の通りである。 (5) Electrophoresis and photography After PCR, aliquot 2 μL from the PCR solution, mix well with loading buffer, and perform electrophoresis (100 V, 30 minutes). 100bp DNA Step Ladder (manufactured by Promega) was used as a DNA marker. After electrophoresis, the agarose gel was stained with SYBR Safe (Thermo Fisher Scientific) for 30 minutes, irradiated with 254 nm ultraviolet light using BioDoc-It Imaging Systems (UVP), and stained for 3 to 5 seconds. Photographs were taken at different exposure times to confirm the presence or absence of amplified DNA fragments. A captured image of the agarose gel is shown in Figure 1.
Note that numbers 1 to 10 in FIG. 1 indicate samples in which reactions were performed using DNA extracted from samples with corresponding sample numbers listed in Table 1 below. The correspondence between each lane number in FIG. 1 and the applied sample is shown in Table 1 below.

Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001

　図１より、DUF421-DUF1657遺伝子を有するバチルス・サブチルスJCA1403株、バチルス・コアグランスNBRC12583株及びJCA1108株、並びにバチルス・セレウスJCM17690株を被検体としてＰＣＲを行った場合は、それぞれ各菌種に特有のサイズのＤＮＡ断片が検出された。また、バチルス・サブチルスJCA1403株、バチルス・コアグランスNBRC12583株及びバチルス・セレウスJCM17690株の抽出ＤＮＡ溶液の混合物をテンプレートとして用いた場合であっても、それぞれ各菌種に特有のサイズのＤＮＡ断片が検出された。
　これに対し、バチルス・サブチルスJCA1402株、JCM1465株、バチルス・コアグランスDSM2314株、バチルス・セレウスJCM2152株を被検体としてＰＣＲを行った場合は、これらの菌株はDUF421-DUF1657遺伝子を有していないため、ＤＮＡ断片が検出されなかった。
　以上より、本発明の方法により、被検体が異なる菌種の混合である場合であっても、DUF421-DUF1657遺伝子を菌種特異的に検出できることが示された。 From Figure 1, when PCR was performed using Bacillus subtilis strain JCA1403, Bacillus coagulans strain NBRC12583 and JCA1108, and Bacillus cereus strain JCM17690, which have the DUF421-DUF1657 genes, the sizes specific to each bacterial species were determined. DNA fragments were detected. Furthermore, even when a mixture of extracted DNA solutions of Bacillus subtillus strain JCA1403, Bacillus coagulans strain NBRC12583, and Bacillus cereus strain JCM17690 was used as a template, DNA fragments with sizes specific to each bacterial species were detected. Ta.
On the other hand, when PCR was performed using Bacillus subtillus strain JCA1402, JCM1465 strain, Bacillus coagulans strain DSM2314, and Bacillus cereus strain JCM2152 as test samples, these strains did not have the DUF421-DUF1657 gene, so No DNA fragments were detected.
From the above, it was shown that the method of the present invention allows the DUF421-DUF1657 gene to be detected in a bacterial species-specific manner even when the subject is a mixture of different bacterial species.

実施例２　３菌種群のMultiplex PCR
（１）プライマーの設計
　バチルス・サブチルスB4146株由来のDUF421-DUF1657遺伝子（配列番号１３）の塩基配列情報を基に、配列番号２の塩基配列で表されるオリゴヌクレオチドからなるプライマー（プライマー２）を設計した。
　バチルス・サブチルスB4146株がDUF421-DUF1657遺伝子を有している場合、上記プライマー２及び実施例１で用いたプライマー３のプライマー対によって増幅されるＤＮＡ断片の長さは約５５ｂｐである。 Example 2 Multiplex PCR of 3 bacterial species groups
(1) Primer design Based on the base sequence information of the DUF421-DUF1657 gene (SEQ ID NO: 13) derived from Bacillus subtilis strain B4146, a primer (primer 2) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 2 was created. Designed.
When the Bacillus subtillus B4146 strain has the DUF421-DUF1657 gene, the length of the DNA fragment amplified by the primer pair of primer 2 and primer 3 used in Example 1 is about 55 bp.

　さらに、バチルス・コアグランスDSM1=ATCC7050株由来のDUF421-DUF1657遺伝子（配列番号１５）の塩基配列を基に、配列番号１０の塩基配列で表されるプライマー（プライマー１０）と対をなすプライマーとして、配列番号４の塩基配列で表されるオリゴヌクレオチドからなるプライマー（プライマー４）、配列番号６の塩基配列で表されるオリゴヌクレオチドからなるプライマー（プライマー６）、配列番号７の塩基配列で表されるオリゴヌクレオチドからなるプライマー（プライマー７）、配列番号８の塩基配列で表されるオリゴヌクレオチドからなるプライマー（プライマー８）、及び配列番号９の塩基配列で表されるオリゴヌクレオチドからなるプライマー（プライマー９）をそれぞれ設計した。設計した各プライマー情報を基に、FASMAC社のDNA/RNA受託合成サービスより、逆相カラム精製品のプライマーを得た。
　バチルス・コアグランスDSM1=ATCC7050株がDUF421-DUF1657遺伝子を有している場合、上記プライマー４及びプライマー１０のプライマー対によって増幅されるＤＮＡ断片の長さは約４９０ｂｐ、上記プライマー６及びプライマー１０のプライマー対によって増幅されるＤＮＡ断片の長さは約１５５ｂｐ、上記プライマー７及びプライマー１０のプライマー対によって増幅されるＤＮＡ断片の長さは約１３５ｂｐ、上記プライマー８及びプライマー１０のプライマー対によって増幅されるＤＮＡ断片の長さは約１２０ｂｐ、上記プライマー９及びプライマー１０のプライマー対によって増幅されるＤＮＡ断片の長さは約８０ｂｐである。 Furthermore, based on the base sequence of the DUF421-DUF1657 gene (SEQ ID NO: 15) derived from Bacillus coagulans DSM1=ATCC7050 strain, the sequence A primer consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 4 (primer 4), a primer consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 6 (primer 6), an oligo represented by the base sequence of SEQ ID NO: 7 A primer (primer 7) consisting of a nucleotide, a primer (primer 8) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 8, and a primer (primer 9) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 9. Each was designed. Based on the information on each designed primer, primers for a reversed phase column purified product were obtained from FASMAC's DNA/RNA contract synthesis service.
When the Bacillus coagulans DSM1=ATCC7050 strain has the DUF421-DUF1657 genes, the length of the DNA fragment amplified by the primer pair of primer 4 and primer 10 is about 490 bp, and the length of the DNA fragment amplified by the primer pair of primer 6 and primer 10 is about 490 bp. The length of the DNA fragment amplified by the primer pair of primer 7 and primer 10 is about 135 bp, and the length of the DNA fragment amplified by the primer pair of primer 8 and primer 10 is about 135 bp. The length of the DNA fragment is approximately 120 bp, and the length of the DNA fragment amplified by the primer pair of primer 9 and primer 10 is approximately 80 bp.

（２）Multiplex PCR反応、電気泳動及び撮影
　Multiplex PCRに用いるテンプレートとしては、実施例１で得られたバチルス・サブチルスJCA1403株由来の抽出ＤＮＡ溶液、バチルス・コアグランスNBRC12583株由来の抽出ＤＮＡ溶液、及びバチルス・セレウスJCM17690株由来の抽出ＤＮＡ溶液をそれぞれ用いた。
　用いるプライマー対の組合せを下記表２の組合せとして、実施例１に記載の条件でMultiplex PCRを行い、電気泳動後にアガロースゲルの撮影画像を得た。画像を図２及び図３に示す。
　なお、レーン番号１１及び１８は、マーカー 100bp DNA Step Ladder (Promega)である。 (2) Multiplex PCR reaction, electrophoresis and photography Templates used for multiplex PCR include the extracted DNA solution derived from Bacillus subtilis JCA1403 strain obtained in Example 1, the extracted DNA solution derived from Bacillus coagulans NBRC12583 strain, and Bacillus subtilis strain JCA1403 obtained in Example 1. - Extracted DNA solutions derived from the S. cereus JCM17690 strain were used.
Multiplex PCR was performed under the conditions described in Example 1 using the combinations of primer pairs shown in Table 2 below, and an agarose gel image was obtained after electrophoresis. Images are shown in FIGS. 2 and 3.
Note that lane numbers 11 and 18 are marker 100bp DNA Step Ladder (Promega).

Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002

　図２及び図３中、バチルス・サブチルス群由来のDUF421-DUF1657遺伝子特異的な増幅産物を左向き黒三角、バチルス・コアグランス由来のDUF421-DUF1657遺伝子特異的な増幅産物をアスタリスク、バチルス・セレウス由来のプラスミド特異的DUF421-DUF1657遺伝子特異的な増幅産物を左向き矢頭でそれぞれ示す。
　図２及び図３より明らかなように、いずれのプライマー対の組合せであっても、異なる菌種由来の混合ＤＮＡである被検体について、DUF421-DUF1657遺伝子を菌種特異的に検出できることが示された。 In Figures 2 and 3, the amplification product specific to the DUF421-DUF1657 gene derived from the Bacillus subtilis group is indicated by a left-pointing black triangle, the amplification product specific to the DUF421-DUF1657 gene derived from Bacillus coagulans is indicated by an asterisk, and the plasmid derived from Bacillus cereus Specific DUF421-DUF1657 gene-specific amplification products are indicated by left-pointing arrowheads, respectively.
As is clear from Figures 2 and 3, it was shown that the DUF421-DUF1657 gene could be detected in a bacterial species-specific manner with any combination of primer pairs in the test sample, which is a mixed DNA derived from different bacterial species. Ta.

実施例３　芽胞耐熱性評価試験１
（１）検体の調製、ＰＣＲ
　バチルス・サブチルス群の菌株として、JCA株(日本缶詰びん詰レトルト食品協会)（JCA1402株、JCA1403株、JCA1404株、JCA1405株、JCA1407株、JCA1408株、JCA1409株、JCA1410株、JCA1411株）、JCM株(国立研究開発法人理化学研究所微生物材料開発室)（JCM1465株）、KM株(環境分離株)（KM166株、KM167株）、及び１６８株を用いた。
　上記各菌株を、実施例１と同様の方法で培養して抽出ＤＮＡ溶液を調製した。さらに、実施例１と同様の方法でＰＣＲを行い、電気泳動によりＤＮＡのバンドの有無を確認した。目的の位置にＤＮＡ断片が検出できた場合を「陽性」、目的の位置にＤＮＡ断片が検出できなかった場合を「陰性」とした。結果を、各菌株におけるDUF421-DUF1657遺伝子の有無と併せて下記表３に示す。 Example 3 Spore heat resistance evaluation test 1
(1) Sample preparation, PCR
Bacillus subtilis group strains include JCA strains (Japan Canned and Bottled Retort Food Association) (JCA1402, JCA1403, JCA1404, JCA1405, JCA1407, JCA1408, JCA1409, JCA1410, JCA1411), JCM strains. (National Research and Development Corporation RIKEN Microbial Materials Development Office) (JCM1465 strain), KM strain (environmentally isolated strain) (KM166 strain, KM167 strain), and 168 strain were used.
Each of the above strains was cultured in the same manner as in Example 1 to prepare an extracted DNA solution. Furthermore, PCR was performed in the same manner as in Example 1, and the presence or absence of DNA bands was confirmed by electrophoresis. The case where a DNA fragment could be detected at the desired position was judged as "positive", and the case where no DNA fragment could be detected at the desired position was judged as "negative". The results are shown in Table 3 below, together with the presence or absence of the DUF421-DUF1657 gene in each strain.

（２）簡易耐熱性試験
　上記の菌株を、DSM寒天培地１Ｌ（０．８％Ｎｕｔｒｉｅｎｔ　Ｂｒｏｔｈ　（Ｄｉｆｃｏ）、０．１％ＫＣｌ、０．０１２％ＭｇＳＯ４・７Ｈ２Ｏ及び５％Ａｇａｒを４００ｍＬで作製し、１Ｎ　ＮａＯＨを２００μＬ加え、ｐＨ７．０に調整をした。オートクレーブ後に１Ｍ　Ｃａ（ＮＯ３）２を０．４ｍＬ、０．０１Ｍ　ＭｎＣｌ２を０．４ｍＬ、及び１ｍＭ　ＦｅＳＯ４を０．４ｍＬ加えた）にて適温で１週間培養し、位相差顕微鏡にて芽胞形成を確認した後、氷冷滅菌水にて芽胞を回収した。回収物を８０００×ｇ、４℃、２０分の条件で遠心分離を行い、上澄みを除去して再び氷冷滅菌水を加える洗浄を計５回行った。洗浄後の回収物（沈殿物）に対し、培養プレート１枚あたり約３ｍＬ分となるように氷冷滅菌水を加えて懸濁し、小分けに分注して使用まで－２０℃で冷凍保存させた。芽胞数（懸濁液１ｍＬあたりの芽胞数）は８０℃で１０分間の加熱処理を行い、段階希釈した芽胞液をSCD寒天培地（商品名：SCD寒天培地「ダイゴ」、一般細菌検査用、富士フイルム和光純薬株式会社製）に塗抹することにより測定した。
　得られた芽胞をSCD液体培地（商品名：SCD液体培地「ダイゴ」、一般細菌検査用、富士フイルム和光純薬株式会社製）１０ｍＬ中に１０６～１０７　ｓｐｏｒｅｓ／ｍＬとなるように添加し、９０℃、９５℃、１００℃で３０分間、又は１０５℃で１０分間加熱後、３０℃で７日間静置培養を行った。静置培養後の菌株の生育を確認することで、耐熱性を下記評価基準を基に判定した。本試験は菌株ごとに３回の反復試験を行い、１回の試験でも死滅が認められた場合に「加熱で死滅した」と判定した。なお、菌株が死滅したか否かは、静置培養後に目視による濁りが観察できなかった場合を「加熱で死滅した」と判断し、目視による濁りが観察できた場合を「加熱では死滅しなかった」と判断した。結果を下記表３に示す。
 
－評価基準－
レベル１：９０℃３０分の加熱で死滅した
レベル２：９０℃３０分の加熱では死滅せず、９５℃３０分の加熱で死滅した
レベル３：９５℃３０分の加熱では死滅せず、１００℃３０分の加熱で死滅した
レベル４：１００℃３０分の加熱では死滅せず、１０５℃１０分の加熱で死滅した
レベル５：１０５℃１０分の加熱でも死滅しなかった (2) Simple heat resistance test The above strain was grown in 1 L of DSM agar medium (400 mL of 0.8% Nutrient Broth (Difco), 0.1% KCl, 0.012% MgSO 4.7H 2 O and 5% Agar). After autoclaving, 0.4 mL of 1M Ca(NO 3 ) 2 , 0.4 mL of 0.01M MnCl 2 , and 0.4 mL of 1 mM FeSO 4 were added. The cells were cultured for one week at an appropriate temperature for 1 week, and spore formation was confirmed using a phase contrast microscope. The spores were collected using ice-cold sterilized water. The collected product was centrifuged at 8,000×g, 4° C., and 20 minutes, and the supernatant was removed and washed with ice-cold sterilized water again for a total of 5 times. The collected material (precipitate) after washing was suspended in ice-cold sterilized water to a volume of approximately 3 mL per culture plate, divided into small portions, and stored frozen at -20°C until use. . The number of spores (number of spores per mL of suspension) was determined by heat treatment at 80°C for 10 minutes and serially diluted spore solution on SCD agar medium (trade name: SCD agar medium "Daigo", for general bacterial testing, Fuji It was measured by smearing the film on a film (manufactured by Wako Pure Chemical Industries, Ltd.).
The obtained spores were added to 10 mL of SCD liquid medium (trade name: SCD liquid medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 10 6 to 10 7 spores/mL. After heating at 90°C, 95°C, 100°C for 30 minutes, or 105°C for 10 minutes, static culture was performed at 30°C for 7 days. By confirming the growth of the bacterial strain after static culture, heat resistance was determined based on the following evaluation criteria. This test was repeated three times for each strain, and if killing was observed even in one test, it was determined that the strain had been killed by heating. As for whether the strain was killed or not, if no turbidity was visually observed after static culture, it was judged as "killed by heating", and if turbidity was visually observed, it was judged as "not killed by heating". The decision was made. The results are shown in Table 3 below.

-Evaluation criteria-
Level 1: Killed by heating at 90°C for 30 minutes Level 2: Not killed by heating at 90°C for 30 minutes, but killed by heating at 95°C for 30 minutes Level 3: Not killed by heating at 95°C for 30 minutes, 100 Level 4: Killed by heating at 100°C for 30 minutes, killed by heating at 105°C for 10 minutes Level 5: Not killed by heating at 105°C for 10 minutes

（３）耐熱性試験（Ｄ値）
　上記バチルス・サブチルス群の菌株について、日本缶詰びん詰レトルト食品協会に委託し、以下の方法にてＤ値（１１２．５℃）を測定した。
　各供試菌株芽胞のＭ／１５リン酸緩衝液（ＰＨ７．０）中の耐熱性を測定した。試験方法は、供試菌株の芽胞液を滅菌０．１％ペプトン水で段階希釈し、Ｍ／１５リン酸緩衝液および各試験液中に約１０５　ＣＦＵ／ｍＬになるように懸濁し、混和後、ＴＤＴチューブに分注および溶封した。このＴＤＴチューブを所定の条件で加熱処理した。加熱後のＴＤＴチューブを開管し、芽胞数を測定してＤ値を算出した。結果を下記表３に示す。 (3) Heat resistance test (D value)
The D value (112.5° C.) of the above-mentioned Bacillus subtilis group strain was entrusted to the Japan Canned and Bottled Retort Foods Association and measured by the following method.
The heat resistance of each test bacterial strain spore in M/15 phosphate buffer (PH7.0) was measured. The test method is to serially dilute the spore solution of the test bacterial strain with sterile 0.1% peptone water, suspend it in M/15 phosphate buffer and each test solution to a concentration of approximately 10 5 CFU/mL, and mix. After that, it was dispensed into TDT tubes and sealed. This TDT tube was heat-treated under predetermined conditions. The TDT tube after heating was opened, the number of spores was measured, and the D value was calculated. The results are shown in Table 3 below.

Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003

　表３より明らかなように、DUF421-DUF1657遺伝子を有しており、Multiplex PCRによって「陽性」と判定されたバチルス・サブチルス群の菌株では、簡易耐熱性試験における耐熱性レベルがいずれもレベル５であり、また「陽性」と判定されたバチルス・サブチルスJCA1403株のＤ１１２．５℃値は１．１８分であった。これに対し、DUF421-DUF1657遺伝子を有しておらず、Multiplex PCRによって「陰性」と判定されたバチルス・サブチルス群の菌株では、簡易耐熱性試験における耐熱性レベルがいずれもレベル２であり、また「陰性」と判定されたバチルス・サブチルスJCA1402株及びJCM1465株のＤ１１２．５℃値も、それぞれ０．０９分及び０．０８分であった。以上より、Multiplex PCRの結果と耐熱性に相関が見られることが明らかになった。
　また上記結果より、Multiplex PCRの結果が陽性であれば、１０５℃で１０分間の加熱であっても十分量（１０６～１０７　ｓｐｏｒｅｓ／ｍＬ）の芽胞は死滅せず、Multiplex PCRの結果が陰性であれば、９５℃で３０分間の加熱で十分量（１０６～１０７　ｓｐｏｒｅｓ／ｍＬ）の芽胞を死滅できることが示された。 As is clear from Table 3, all strains of the Bacillus subtilis group that have the DUF421-DUF1657 genes and were determined to be "positive" by Multiplex PCR had a heat resistance level of level 5 in the simple heat resistance test. The D112.5°C value of Bacillus subtillus JCA1403 strain, which was determined to be "positive", was 1.18 minutes. On the other hand, strains of the Bacillus subtilis group that do not have the DUF421-DUF1657 genes and were determined to be "negative" by Multiplex PCR had a heat resistance level of level 2 in the simple heat resistance test, and The D112.5°C values of Bacillus subtilis JCA1402 strain and JCM1465 strain, which were determined to be "negative," were also 0.09 minutes and 0.08 minutes, respectively. From the above, it has become clear that there is a correlation between the results of Multiplex PCR and heat resistance.
In addition, from the above results, if the Multiplex PCR result is positive, even heating at 105°C for 10 minutes will not kill a sufficient amount of spores (10 6 to 10 7 spores/mL), and the Multiplex PCR result will be negative. If negative, it was shown that a sufficient amount (10 6 to 10 7 spores/mL) of spores could be killed by heating at 95° C. for 30 minutes.

実施例４　芽胞耐熱性評価試験２
（１）検体の調製、ＰＣＲ
　バチルス・コアグランスの菌株として、NBRC株（独立行政法人製品評価技術基盤機構）（NBRC12583株）、JCA株(日本缶詰びん詰レトルト食品協会)（JCA1108株、JCA1109株、JCA1116株、JCA1117株、JCA1120株、JCA1122株、JCA1158株、JCA1174株、JCA1180株、JCA1182株）、DSM株（ライプニッツ研究所ドイツ微生物株保存機関)（DSM2308株、DSM2311株、DSM2312株、DSM2314株、DSM2350株、DSM2356株、DSM2383株、DSM2384株、DSM2385）を用いた。
　上記各菌株を、実施例１と同様の方法で培養して抽出ＤＮＡ溶液を調製した。さらに、実施例１と同様の方法でＰＣＲを行い、電気泳動によりＤＮＡのバンドの有無を確認した。目的の位置にＤＮＡ断片が検出できた場合を「陽性」、目的の位置にＤＮＡ断片が検出できなかった場合を「陰性」とした。結果を、各菌株におけるDUF421-DUF1657遺伝子の有無と併せて下記表４に示す。 Example 4 Spore heat resistance evaluation test 2
(1) Sample preparation, PCR
Bacillus coagulans strains include NBRC strain (National Institute of Technology and Evaluation) (NBRC12583 strain), JCA strain (Japan Canned and Bottled Retort Food Association) (JCA1108 strain, JCA1109 strain, JCA1116 strain, JCA1117 strain, JCA1120 strain) , JCA1122 strain, JCA1158 strain, JCA1174 strain, JCA1180 strain, JCA1182 strain), DSM strain (Leibniz Institute German Microbial Strain Archive) (DSM2308 strain, DSM2311 strain, DSM2312 strain, DSM2314 strain, DSM2350 strain, DSM2356 strain, DSM2383 strain) , DSM2384 strain, DSM2385) were used.
Each of the above strains was cultured in the same manner as in Example 1 to prepare an extracted DNA solution. Furthermore, PCR was performed in the same manner as in Example 1, and the presence or absence of DNA bands was confirmed by electrophoresis. The case where a DNA fragment could be detected at the desired position was judged as "positive", and the case where no DNA fragment could be detected at the desired position was judged as "negative". The results are shown in Table 4 below, together with the presence or absence of the DUF421-DUF1657 gene in each strain.

（２）耐熱性試験（Ｄ値）
　上記バチルス・コアグランスの菌株について、日本缶詰びん詰レトルト食品協会に委託し、上位実施例３と同様の方法にてＤ値を測定した。結果を下記表４に示す。さらに、下記表４の結果を箱ひげ図で示した図を、図４に示す。 (2) Heat resistance test (D value)
Regarding the above Bacillus coagulans strain, the D value was measured in the same manner as in Upper Example 3, outsourced to the Japan Canned and Bottled Retort Foods Association. The results are shown in Table 4 below. Further, FIG. 4 shows a box plot of the results of Table 4 below.

Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004

　上記表４及び図４より、DUF421-DUF1657遺伝子を有しており、Multiplex PCRによって「陽性」と判定されたバチルス・コアグランスの菌株では、いずれもＤ１０５℃値が１０分以上であった。また、DUF421-DUF1657遺伝子を有しておらず、Multiplex PCRによって「陰性」と判定されたバチルス・コアグランスの菌株では、いずれもＤ１０５℃値が１０分未満であった。
　このように、Multiplex PCRの結果と耐熱性（Ｄ値）に相関が見られ、Multiplex PCRの結果が陽性であればＤ１０５℃値が１０分以上、陰性であればＤ１０５℃値が１０分未満と評価できることが示された。 From Table 4 and FIG. 4 above, all of the Bacillus coagulans strains that had the DUF421-DUF1657 genes and were determined to be "positive" by Multiplex PCR had a D 105°C value of 10 minutes or more. Furthermore, all of the Bacillus coagulans strains that did not have the DUF421-DUF1657 gene and were determined to be "negative" by Multiplex PCR had a D 105°C value of less than 10 minutes.
In this way, there is a correlation between the Multiplex PCR results and heat resistance (D value); if the Multiplex PCR result is positive, the D105℃ value is 10 minutes or more, and if the Multiplex PCR result is negative, the D105℃ value is 10 minutes. It was shown that it can be evaluated as below.

実施例５　芽胞耐熱性評価試験３
（１）検体の調製、ＰＣＲ
　バチルス・セレウスの菌株として、JCM株（国立研究開発法人理化学研究所微生物材料開発室）（JCM2152株、JCM17690株）を用いた。
　上記各菌株を、実施例１と同様の方法で培養して抽出ＤＮＡ溶液を調製した。さらに、実施例１と同様の方法でＰＣＲを行い、電気泳動によりＤＮＡのバンドの有無を確認した。目的の位置にＤＮＡ断片が検出できた場合を「陽性」、目的の位置にＤＮＡ断片が検出できなかった場合を「陰性」とした。結果を、各菌株におけるプラスミド特異的DUF421-DUF1657遺伝子の有無と併せて下記表５に示す。 Example 5 Spore heat resistance evaluation test 3
(1) Sample preparation, PCR
The JCM strain (RIKEN Microbial Materials Development Office) (JCM2152 strain, JCM17690 strain) was used as the Bacillus cereus strain.
Each of the above strains was cultured in the same manner as in Example 1 to prepare an extracted DNA solution. Furthermore, PCR was performed in the same manner as in Example 1, and the presence or absence of DNA bands was confirmed by electrophoresis. The case where a DNA fragment could be detected at the desired position was judged as "positive", and the case where no DNA fragment could be detected at the desired position was judged as "negative". The results are shown in Table 5 below, together with the presence or absence of the plasmid-specific DUF421-DUF1657 gene in each strain.

（２）耐熱性試験（Ｄ値）
　上記バチルス・セレウスの菌株について、日本缶詰びん詰レトルト食品協会に委託し、上位実施例３と同様の方法にてＤ値を測定した。結果を、各菌株におけるプラスミド特異的DUF421-DUF1657遺伝子の有無と併せて下記表５に示す。 (2) Heat resistance test (D value)
Regarding the above Bacillus cereus strain, the D value was measured in the same manner as in Example 3 by entrusting the Japan Canned and Bottled Retort Foods Association. The results are shown in Table 5 below, together with the presence or absence of the plasmid-specific DUF421-DUF1657 gene in each strain.

Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005

　上記表５より、Multiplex PCRによって陽性と判定されたバチルス・セレウスの菌株では、Ｄ９０℃値が８２．９分であった。また、Multiplex PCRによって陰性と判定されたバチルス・セレウスの菌株では、Ｄ９０℃値が２４．３分であった。
　このように、Multiplex PCRの結果と耐熱性（Ｄ値）に相関が見られた。 From Table 5 above, the Bacillus cereus strain determined to be positive by Multiplex PCR had a D 90°C value of 82.9 minutes. Furthermore, for the Bacillus cereus strain that was determined to be negative by Multiplex PCR, the D90 °C value was 24.3 minutes.
Thus, a correlation was observed between the results of Multiplex PCR and heat resistance (D value).

実施例６　原料分離株のハイスループット解析
検体の調製
　市販及び原料メーカーから購入した原料（ＩＤ：１～３０）から単離した各種菌種に対してMicroSEQ 500 16S rDNA Sequencing Kit（Thermo Fisher Scientific社製）を用いた菌種同定を行った。バチルス・サブチルス群、バチルス・コアグランス、バチルス・セレウスと同定された菌株を解析対象とした。
　同定された各菌種を、実施例１と同様の方法で培養して抽出ＤＮＡ溶液を調製した。さらに、実施例１と同様の方法でＰＣＲを行い、電気泳動によりＤＮＡのバンドの有無を確認した。目的の位置にＤＮＡ断片が検出できた場合を「陽性」、目的の位置にＤＮＡ断片が検出できなかった場合を「陰性」とした。Multiplex PCRの結果を下記表６に示す。 Example 6 Preparation of specimens for high-throughput analysis of raw material isolates MicroSEQ 500 16S rDNA Sequencing Kit (manufactured by Thermo Fisher Scientific) was used for various bacterial species isolated from raw materials (ID: 1 to 30) commercially available and purchased from raw material manufacturers. ) was used to identify the bacterial species. Bacterial strains identified as Bacillus subtilis group, Bacillus coagulans, and Bacillus cereus were targeted for analysis.
Each of the identified bacterial species was cultured in the same manner as in Example 1 to prepare an extracted DNA solution. Furthermore, PCR was performed in the same manner as in Example 1, and the presence or absence of DNA bands was confirmed by electrophoresis. The case where a DNA fragment could be detected at the desired position was judged as "positive", and the case where no DNA fragment could be detected at the desired position was judged as "negative". The results of Multiplex PCR are shown in Table 6 below.

Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006

　表６より明らかなように、原料ＩＤ：１～２９のバチルス・サブチルス群、バチルス・コアグランス、又はバチルス・セレウスの各菌株では、いずれもMultiplex PCR法による目的のＤＮＡのバンドが検出されなかった。一方で、原料ＩＤ：３０のバチルス・サブチルス群の菌株からは、Multiplex PCR法による目的のＤＮＡバンドが検出された。よって、原料ＩＤ：３０の原料には高耐熱性を有する芽胞を形成するバチルス・サブチルス群の菌株が含まれていることが示された。
　このように本発明の方法によれば、多量のサンプルから高耐熱性を有する芽胞を形成する芽胞形成細菌を迅速に同定できることが示された。 As is clear from Table 6, no target DNA band was detected by the multiplex PCR method in any of the Bacillus subtilis group, Bacillus coagulans, or Bacillus cereus strains with raw material IDs: 1 to 29. On the other hand, the desired DNA band was detected from the strain of Bacillus subtilis group with raw material ID: 30 by multiplex PCR method. Therefore, it was shown that the raw material with raw material ID: 30 contained a strain of the Bacillus subtilis group that forms spores with high heat resistance.
As described above, it has been shown that the method of the present invention can rapidly identify spore-forming bacteria that form spores with high heat resistance from a large amount of samples.

実施例７　原料の耐熱性芽胞菌包括検査の感度と加熱処理条件の提案
（１）高耐熱性芽胞付着原料の作製
　実施例６で用いた原料ＩＤ：２８の原料１０ｇに、８０℃１０分間の加熱処理により栄養細胞を死滅させたバチルス・サブチルスJCA1403株（高耐熱性の芽胞を形成する菌株）の芽胞を、原料１ｇあたり5.0×107、5.0×105、5.0×103、5.0×101、5.0×10-1　ｓｐｏｒｅｓとなるように付着させた。対照試験として、芽胞を付着させていない原料を用いた。その後原料が十分乾燥するまで静置し、疑似的に高耐熱性芽胞が付着した原料を調製した。 Example 7 Proposal of sensitivity and heat treatment conditions for comprehensive inspection of heat-resistant spore bacteria on raw materials (1) Preparation of highly heat-resistant spore-attached raw material 10 g of raw material with raw material ID: 28 used in Example 6 was heated at 80°C for 10 minutes. The spores of Bacillus subtilis strain JCA1403 (a strain that forms highly heat-resistant spores) whose vegetative cells have been killed by heat treatment are 5.0×10 7 , 5.0×10 5 , 5.0×10 3 , 5.0×10 per gram of raw material. 1 , 5.0×10 -1 spores. As a control test, a raw material to which no spores were attached was used. Thereafter, the raw material was allowed to stand until it was sufficiently dry, to prepare a raw material to which highly heat-resistant spores were attached in a pseudo manner.

（２）菌体回収工程
　上記原料中に生理食塩水９０ｇを加え、ストマッカーを用いて粉砕・均質化処理をし、懸濁液３０ｍＬを回収した。その後１００×ｇ、５分間の遠心分離を行って原料残渣を廃棄し、さらに８０００×ｇ、２０分間の遠心分離を行い沈殿した菌体を回収した。回収した菌体を、３ｍＬの生理食塩水で再懸濁した。 (2) Bacterial body recovery step 90 g of physiological saline was added to the above raw materials, and pulverized and homogenized using a stomacher to collect 30 mL of suspension. Thereafter, centrifugation was performed at 100×g for 5 minutes to discard the raw material residue, and further centrifugation was performed at 8000×g for 20 minutes to collect precipitated bacterial cells. The collected bacterial cells were resuspended in 3 mL of physiological saline.

（３）加熱工程及び腐敗観察
　上記回収した菌懸濁液１０μＬ又は１μＬを、バチルス・サブチルスJCA1403が増殖可能な中性茶系飲料（ｐＨ：６．３５）１０ｍＬ中に接種し、１００℃、５分間の加熱処理を行った。加熱処理後、３０℃で７日間静置培養し、目視で飲料が腐敗したかどうかを観察した。３回の反復試験を行い、１つでも非増殖（飲料が腐敗しない）が認められれば死滅と判定した。なお、本加熱条件は通常の中性茶系飲料を製造する際に行う加熱処理よりも極めて緩い条件であり、食品衛生法の基準以下であるためあくまでモデル実験上の条件である。
　なお、本試験において「製品の腐敗」とは、具体的に目視による濁りが確認できる状態若しくは芽胞形成細菌の増殖が認められる状態、又は飲料１００μＬをSCD寒天培地（商品名：SCD液体培地「ダイゴ」、一般細菌検査用、富士フイルム和光純薬株式会社製）に添加し３０℃２日間の培養で目視による濁りが確認できる状態又は芽胞形成細菌の増殖が認められる状態であることを意味する。 (3) Heating process and observation of spoilage 10 μL or 1 μL of the bacterial suspension collected above was inoculated into 10 mL of a neutral tea-based beverage (pH: 6.35) in which Bacillus subtillus JCA1403 can grow, and the mixture was heated at 100°C for 50 minutes. Heat treatment was performed for 1 minute. After the heat treatment, the beverages were statically cultured at 30° C. for 7 days, and visually observed to see if the beverages had spoiled. The test was repeated three times, and if non-proliferation (beverage did not spoil) was observed in even one test, it was judged as dead. It should be noted that these heating conditions are extremely milder than the heat treatment performed when producing a normal neutral tea beverage, and are below the standards of the Food Sanitation Act, so they are only conditions for model experiments.
In this test, "product spoilage" specifically refers to a state in which turbidity can be visually confirmed or growth of spore-forming bacteria is observed, or a state in which 100 μL of the beverage is placed on SCD agar medium (product name: SCD liquid medium "Daigo"). '', for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and cultured at 30°C for 2 days.

（４）Multiplex PCR試験
　また、上記（２）で回収した菌懸濁液１０μＬ又は１μＬを、SCD液体培地(富士フイルム和光純薬株式会社製）１０ｍｌに植菌し、３０℃で４８時間培養した。培養後、菌体液を１ｍＬ回収し、実施例１と同様の方法で抽出ＤＮＡ溶液を調製した。その後実施例１と同様の方法でMultiplex PCRによりDUF421-DUF1657遺伝子の検出を行った。 (4) Multiplex PCR test In addition, 10 μL or 1 μL of the bacterial suspension collected in (2) above was inoculated into 10 ml of SCD liquid medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and cultured at 30°C for 48 hours. . After culturing, 1 mL of bacterial cell fluid was collected, and an extracted DNA solution was prepared in the same manner as in Example 1. Thereafter, the DUF421-DUF1657 gene was detected by multiplex PCR in the same manner as in Example 1.

（５）結果
　Multiplex PCRによる結果、及び加熱処理後の飲料腐敗の結果を下記表７に示す。なお、表中の「推定芽胞数(spores/ml)」は、上記加熱工程及び上記Multiplex PCR試験において、中性茶系飲料及びSCD液体培地における推定の芽胞数を示す。 (5) Results The results of Multiplex PCR and the results of beverage spoilage after heat treatment are shown in Table 7 below. The "estimated number of spores (spores/ml)" in the table indicates the estimated number of spores in the neutral tea beverage and SCD liquid medium in the heating step and the Multiplex PCR test.

Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007

　表７より明らかなように、原料１ｇあたりの芽胞数が多いサンプルでのMultiplex PCR試験で陽性と判定された場合、１００℃５分間の加熱処理を行った後に３０℃で７日間静置培養させた各対応する中性茶系飲料では、製品の腐敗が観察された。これに対し、原料１ｇあたりの芽胞数が少ないサンプルでのMultiplex PCR試験で陰性と判定された場合には、対応する中性茶系飲料では製品の腐敗が観察されなかった。
　また、原料１ｇあたりの芽胞数がやや多いサンプルでのMultiplex PCR試験で陽性と判定された場合であっても、対応する中性茶系飲料では製品の腐敗が観察されなかった。これは、１００℃５分間の加熱処理を行った後に３０℃で７日間静置培養させるという、本条件ではまだ製品が腐敗しないようなわずかな芽胞の混入であっても、本発明の方法により高感度に検出できることを示している。 As is clear from Table 7, if a sample with a large number of spores per gram of raw material is determined to be positive in the multiplex PCR test, it is subjected to heat treatment at 100°C for 5 minutes and then left to stand at 30°C for 7 days. Product spoilage was observed for each of the corresponding neutral tea-based beverages. On the other hand, when a multiplex PCR test using a sample with a low number of spores per gram of raw material was determined to be negative, no product spoilage was observed in the corresponding neutral tea beverage.
Furthermore, even when a multiplex PCR test using a sample with a slightly higher number of spores per gram of raw material was determined to be positive, no product spoilage was observed in the corresponding neutral tea beverage. This means that even if there is a small amount of spores that do not spoil under the conditions of heat treatment at 100°C for 5 minutes and then static culture at 30°C for 7 days, the method of the present invention can remove the contamination. This shows that it can be detected with high sensitivity.

　上記結果より、本発明の方法により、飲食品を被検体として高耐熱性を有する芽胞を形成する芽胞形成細菌の検出を行うことで、飲食品の腐敗を高感度に予測することができることが示された。また、本発明の方法により飲食品を被検体として高耐熱性を有する芽胞を形成する芽胞形成細菌の検出を行うことで、当該飲食品の適切な加熱処理条件を迅速かつ簡便に決定できることが示された。 The above results indicate that by using the method of the present invention to detect spore-forming bacteria that form highly heat-resistant spores using food and drink as a test subject, spoilage of food and drink can be predicted with high sensitivity. It was done. Furthermore, it has been shown that by detecting spore-forming bacteria that form highly heat-resistant spores using food and drink as a test subject using the method of the present invention, appropriate heat treatment conditions for the food and drink can be quickly and easily determined. It was done.

　上記の結果から、本発明の方法を用いることによって、中性条件で増殖可能な芽胞形成細菌におけるDUF421-DUF1657遺伝子の有無を確認することができ、また増幅産物により芽胞形成細菌を検出できる。さらに、DUF421-DUF1657遺伝子の有無を確認することで、芽胞形成細菌の芽胞耐熱性を評価することができ、芽胞形成菌が検出される原料に対し適切な加熱処理条件を決定することができる。 From the above results, by using the method of the present invention, it is possible to confirm the presence or absence of the DUF421-DUF1657 gene in spore-forming bacteria that can grow under neutral conditions, and it is also possible to detect spore-forming bacteria based on the amplification product. Furthermore, by confirming the presence or absence of the DUF421-DUF1657 gene, the spore heat resistance of spore-forming bacteria can be evaluated, and appropriate heat treatment conditions can be determined for the raw material in which spore-forming bacteria are detected.

　本発明をその実施態様とともに説明したが、我々は特に指定しない限り我々の発明を説明のどの細部においても限定しようとするものではなく、添付の請求の範囲に示した発明の精神と範囲に反することなく幅広く解釈されるべきであると考える。 Although the invention has been described in conjunction with embodiments thereof, we do not intend to limit our invention in any detail in the description unless otherwise specified and contrary to the spirit and scope of the invention as set forth in the appended claims. I believe that it should be interpreted broadly without any restrictions.

　本願は、２０２２年５月２日に日本国で特許出願された特願２０２２－０７５９４８に基づく優先権を主張するものであり、これはここに参照してその内容を本明細書の記載の一部として取り込む。 This application claims priority based on Japanese Patent Application No. 2022-075948, which was filed in Japan on May 2, 2022, and the content thereof is incorporated herein by reference. Incorporate it as a part.

Claims (16)

　下記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対、並びに
　下記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対
からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を核酸プライマーとして用いて、DUF421-DUF1657遺伝子の部分塩基配列で表される核酸を増幅し、
増幅産物の有無により中性条件で増殖可能なバチルス（Bacillus）属に属する芽胞形成細菌を検出する、
バチルス属に属する芽胞形成細菌の検出方法。
 
（ａ）配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｂ）配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｃ）配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｄ）配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｅ）配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｆ）配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｇ）配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｈ）配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｉ）配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｊ）配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｋ）配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｌ）配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。 An oligonucleotide pair consisting of the following oligonucleotides (a) and (c),
An oligonucleotide pair consisting of the following oligonucleotides (b) and (c),
An oligonucleotide pair consisting of the following oligonucleotides (d) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (e) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (f) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (g) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (h) and (j),
At least one oligonucleotide pair selected from the group consisting of an oligonucleotide pair consisting of the following oligonucleotides (i) and (j) and an oligonucleotide pair consisting of the following oligonucleotides (k) and (l) is used as a nucleic acid primer. amplify the nucleic acid represented by the partial base sequence of the DUF421-DUF1657 gene,
Detects spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions based on the presence or absence of amplification products.
A method for detecting spore-forming bacteria belonging to the genus Bacillus.

(a) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(b) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(c) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 3 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(d) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 4 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(e) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 5, or an oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 5, and growing under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(f) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 6, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(g) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 7 and grows under neutral conditions and has the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(h) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 8 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or an oligonucleotide with 80% or more identity with the base sequence represented by SEQ ID NO: 9, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(j) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 10, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(k) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 11, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 11 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions.
(l) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 12 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions. 　前記増幅産物により前記芽胞形成細菌の同定を行う、請求項１記載の方法。 The method according to claim 1, wherein the spore-forming bacteria are identified by the amplification product. 　前記増幅産物の有無により前記芽胞形成細菌の芽胞耐熱性を評価する、請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the spore heat resistance of the spore-forming bacteria is evaluated based on the presence or absence of the amplification product. 　下記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対、並びに
　下記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対
からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を核酸プライマーとして用いて、DUF421-DUF1657遺伝子の部分塩基配列で表される核酸を増幅し、
増幅産物の有無により中性条件で増殖可能なバチルス（Bacillus）属に属する芽胞形成細菌の芽胞耐熱性を評価する、
バチルス属に属する芽胞形成細菌の芽胞耐熱性の評価方法。
 
（ａ）配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｂ）配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｃ）配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｄ）配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｅ）配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｆ）配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｇ）配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｈ）配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｉ）配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｊ）配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｋ）配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｌ）配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。 An oligonucleotide pair consisting of the following oligonucleotides (a) and (c),
An oligonucleotide pair consisting of the following oligonucleotides (b) and (c),
An oligonucleotide pair consisting of the following oligonucleotides (d) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (e) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (f) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (g) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (h) and (j),
At least one oligonucleotide pair selected from the group consisting of an oligonucleotide pair consisting of the following oligonucleotides (i) and (j) and an oligonucleotide pair consisting of the following oligonucleotides (k) and (l) is used as a nucleic acid primer. amplify the nucleic acid represented by the partial base sequence of the DUF421-DUF1657 gene,
Evaluating the spore heat resistance of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions based on the presence or absence of amplification products.
A method for evaluating spore heat resistance of spore-forming bacteria belonging to the genus Bacillus.

(a) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(b) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(c) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 3 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(d) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 4 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(e) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 5, or an oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 5, and growing under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(f) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 6, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(g) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 7 and grows under neutral conditions and has the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(h) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 8 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or an oligonucleotide with 80% or more identity with the base sequence represented by SEQ ID NO: 9, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(j) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 10, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(k) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 11, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 11 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions.
(l) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 12 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions. 　請求項３又は４の方法によって評価した芽胞耐熱性に基づいて、前記芽胞形成細菌の加熱処理条件を決定することを特徴とする、バチルス属に属する芽胞形成細菌の加熱処理条件の決定方法。 A method for determining heat treatment conditions for spore-forming bacteria belonging to the genus Bacillus, the method comprising determining heat treatment conditions for the spore-forming bacteria based on the spore heat resistance evaluated by the method of claim 3 or 4. 　前記中性条件で増殖可能なバチルス属に属する芽胞形成細菌が、バチルス・サブチルス群（Bacillus subtilis group）、バチルス・コアグランス（Bacillus coagulans）、及びバチルス・セレウス（Bacillus cereus）からなる群より選ばれる少なくとも１種である、請求項１～５のいずれか１項に記載の方法。 The spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions are at least selected from the group consisting of Bacillus subtilis group, Bacillus coagulans , and Bacillus cereus . The method according to any one of claims 1 to 5, which is one type. 　前記オリゴヌクレオチド（ａ）が、配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｂ）が、配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｃ）が、配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｄ）が、配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｅ）が、配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｆ）が、配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｇ）が、配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｈ）が、配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｉ）が、配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｊ）が、配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｋ）が、配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｌ）が、配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドである、
　請求項１～６のいずれか１項に記載の方法。 The oligonucleotide (a) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 1, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (b) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 2. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (c) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 3. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (d) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 4. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (e) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 5. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (f) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 6. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (g) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 7. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (h) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 8. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (i) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 9. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene,
The oligonucleotide (j) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 10, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 10. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene,
The oligonucleotide (k) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 11, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 11. is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has a plasmid-specific DUF421-DUF1657 gene,
The oligonucleotide (l) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 12. is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has a plasmid-specific DUF421-DUF1657 gene.
The method according to any one of claims 1 to 6. 　前記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、及び前記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対をプライマー対として用いてポリメラーゼ連鎖反応法によりゲノム上のDUF421-DUF1657遺伝子の部分塩基配列で表される核酸を増幅し、
　前記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対をプライマー対として用いてポリメラーゼ連鎖反応法によりプラスミド特異的DUF421-DUF1657遺伝子の部分塩基配列で表される核酸を増幅する、
　請求項１～７のいずれか１項に記載の方法。 An oligonucleotide pair consisting of the oligonucleotides (a) and (c), an oligonucleotide pair consisting of the oligonucleotides (b) and (c), an oligonucleotide pair consisting of the oligonucleotides (d) and (j), an oligonucleotide pair consisting of the oligonucleotides (d) and (j), An oligonucleotide pair consisting of the nucleotides (e) and (j), an oligonucleotide pair consisting of the oligonucleotides (f) and (j), an oligonucleotide pair consisting of the oligonucleotides (g) and (j), an oligonucleotide pair consisting of the oligonucleotides (g) and (j), Polymerase chain reaction using at least one oligonucleotide pair selected from the group consisting of the oligonucleotide pair consisting of h) and (j) and the oligonucleotide pair consisting of the oligonucleotides (i) and (j) as a primer pair. method to amplify the nucleic acid represented by the partial base sequence of the DUF421-DUF1657 gene on the genome,
amplifying the nucleic acid represented by the partial base sequence of the plasmid-specific DUF421-DUF1657 gene by polymerase chain reaction using the oligonucleotide pair consisting of the oligonucleotides (k) and (l) as a primer pair;
The method according to any one of claims 1 to 7. 　前記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、
　前記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、並びに前記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対からなる群より選ばれる１対のオリゴヌクレオチド対、並びに
　前記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対、
を、混合して混合プライマー対として用いる、請求項８に記載の方法。 an oligonucleotide pair consisting of the oligonucleotides (a) and (c);
An oligonucleotide pair consisting of the oligonucleotides (d) and (j), an oligonucleotide pair consisting of the oligonucleotides (e) and (j), an oligonucleotide pair consisting of the oligonucleotides (f) and (j), an oligonucleotide pair consisting of the oligonucleotides (f) and (j), From the group consisting of an oligonucleotide pair consisting of nucleotides (g) and (j), an oligonucleotide pair consisting of said oligonucleotides (h) and (j), and an oligonucleotide pair consisting of said oligonucleotides (i) and (j) a selected pair of oligonucleotides, and an oligonucleotide pair consisting of the oligonucleotides (k) and (l);
The method according to claim 8, wherein the mixture is used as a mixed primer pair. 　前記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、並びに前記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を用いて増幅産物が確認されたときは、前記芽胞形成細菌をバチルス・サブチルス群と同定し、
　前記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、並びに前記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を用いて増幅産物が確認されたときは、前記芽胞形成細菌をバチルス・コアグランスと同定し、
　前記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対を用いて増幅産物が確認されたときは、前記芽胞形成細菌をバチルス・セレウスと同定する、
　請求項１～９のいずれか１項に記載の方法。 An amplification product using at least one oligonucleotide pair selected from the group consisting of the oligonucleotide pair consisting of the aforementioned oligonucleotides (a) and (c), and the oligonucleotide pair consisting of the aforementioned oligonucleotides (b) and (c). is confirmed, the spore-forming bacteria are identified as Bacillus subtilis group,
An oligonucleotide pair consisting of the oligonucleotides (d) and (j), an oligonucleotide pair consisting of the oligonucleotides (e) and (j), an oligonucleotide pair consisting of the oligonucleotides (f) and (j), an oligonucleotide pair consisting of the oligonucleotides (f) and (j), From the group consisting of an oligonucleotide pair consisting of nucleotides (g) and (j), an oligonucleotide pair consisting of said oligonucleotides (h) and (j), and an oligonucleotide pair consisting of said oligonucleotides (i) and (j) When an amplification product is confirmed using at least one selected oligonucleotide pair, identifying the spore-forming bacterium as Bacillus coagulans;
When an amplification product is confirmed using the oligonucleotide pair consisting of the oligonucleotides (k) and (l), the spore-forming bacterium is identified as Bacillus cereus;
The method according to any one of claims 1 to 9. 　下記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対、並びに
　下記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対
からなる群より選ばれる少なくとも１種のオリゴヌクレオチド対を有する、ＤＮＡキット。
 
（ａ）配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス（Bacillus）属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｂ）配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｃ）配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｄ）配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｅ）配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｆ）配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｇ）配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｈ）配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｉ）配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｊ）配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｋ）配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｌ）配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。 An oligonucleotide pair consisting of the following oligonucleotides (a) and (c),
An oligonucleotide pair consisting of the following oligonucleotides (b) and (c),
An oligonucleotide pair consisting of the following oligonucleotides (d) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (e) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (f) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (g) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (h) and (j),
A DNA kit comprising at least one oligonucleotide pair selected from the group consisting of an oligonucleotide pair consisting of the following oligonucleotides (i) and (j), and an oligonucleotide pair consisting of the following oligonucleotides (k) and (l). .

(a) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and has the DUF421-DUF1657 gene and grows under neutral conditions. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus .
(b) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(c) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 3 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(d) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 4 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(e) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 5, or an oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 5, and growing under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(f) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 6, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(g) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 7 and grows under neutral conditions and has the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(h) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 8 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or an oligonucleotide with 80% or more identity with the base sequence represented by SEQ ID NO: 9, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(j) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 10, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(k) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 11, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 11 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions.
(l) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 12 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions. 　前記バチルス属に属する菌が、バチルス・サブチルス群（Bacillus subtilis group）、バチルス・セレウス（Bacillus cereus）及びバチルス・コアグランス（Bacillus coagulans）である、
　請求項１１に記載のＤＮＡキット。 The bacteria belonging to the genus Bacillus are Bacillus subtilis group, Bacillus cereus , and Bacillus coagulans .
The DNA kit according to claim 11. 　前記オリゴヌクレオチド（ａ）が、配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｂ）が、配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｃ）が、配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｄ）が、配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｅ）が、配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｆ）が、配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｇ）が、配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｈ）が、配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｉ）が、配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｊ）が、配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｋ）が、配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｌ）が、配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドである、
　請求項１１又は１２に記載のＤＮＡキット。 The oligonucleotide (a) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 1, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (b) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 2. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (c) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 3. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (d) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 4. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (e) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 5. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (f) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 6. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (g) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 7. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (h) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 8. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (i) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 9. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (j) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 10, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 10. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (k) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 11, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 11. is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has a plasmid-specific DUF421-DUF1657 gene,
The oligonucleotide (l) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 12. is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has a plasmid-specific DUF421-DUF1657 gene.
The DNA kit according to claim 11 or 12. 　前記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、並びに前記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対がバチルス・サブチルス群の検出に使用できるオリゴヌクレオチド対であり、
　前記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、前記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、並びに前記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対がバチルス・コアグランスの検出に使用できるオリゴヌクレオチド対であり、
　前記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対がバチルス・セレウスの検出に使用できるオリゴヌクレオチド対である、
請求項１１～１３のいずれか１項に記載のＤＮＡキット。 The oligonucleotide pair consisting of the oligonucleotides (a) and (c) and the oligonucleotide pair consisting of the oligonucleotides (b) and (c) are oligonucleotide pairs that can be used for detection of Bacillus subtilis group,
An oligonucleotide pair consisting of the oligonucleotides (d) and (j), an oligonucleotide pair consisting of the oligonucleotides (e) and (j), an oligonucleotide pair consisting of the oligonucleotides (f) and (j), an oligonucleotide pair consisting of the oligonucleotides (f) and (j), The oligonucleotide pair consisting of nucleotides (g) and (j), the oligonucleotide pair consisting of the oligonucleotides (h) and (j), and the oligonucleotide pair consisting of the oligonucleotides (i) and (j) are Bacillus coagulans. is an oligonucleotide pair that can be used to detect
The oligonucleotide pair consisting of the oligonucleotides (k) and (l) is an oligonucleotide pair that can be used for detection of Bacillus cereus.
The DNA kit according to any one of claims 11 to 13. 　下記オリゴヌクレオチド（ａ）及び（ｃ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｂ）及び（ｃ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｄ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｅ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｆ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｇ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｈ）及び（ｊ）からなるオリゴヌクレオチド対、
　下記オリゴヌクレオチド（ｉ）及び（ｊ）からなるオリゴヌクレオチド対、並びに
　下記オリゴヌクレオチド（ｋ）及び（ｌ）からなるオリゴヌクレオチド対。
 
（ａ）配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス（Bacillus）属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｂ）配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｃ）配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｄ）配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｅ）配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｆ）配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｇ）配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｈ）配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｉ）配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｊ）配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列との同一性が８０％以上であり、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｋ）配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。
（ｌ）配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列との同一性が８０％以上であり、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチド。 An oligonucleotide pair consisting of the following oligonucleotides (a) and (c),
An oligonucleotide pair consisting of the following oligonucleotides (b) and (c),
An oligonucleotide pair consisting of the following oligonucleotides (d) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (e) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (f) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (g) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (h) and (j),
An oligonucleotide pair consisting of the following oligonucleotides (i) and (j), and an oligonucleotide pair consisting of the following oligonucleotides (k) and (l).

(a) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and has the DUF421-DUF1657 gene and grows under neutral conditions. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus .
(b) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(c) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 3 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(d) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 4 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(e) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 5, or an oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 5, and growing under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(f) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 6, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(g) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an oligonucleotide that has 80% or more identity with the base sequence represented by SEQ ID NO: 7 and grows under neutral conditions and has the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(h) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8, or an oligonucleotide that has 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 8 and has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or an oligonucleotide with 80% or more identity with the base sequence represented by SEQ ID NO: 9, and grown under neutral conditions that contains the DUF421-DUF1657 gene. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(j) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 10, or an oligonucleotide with 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, and which has the DUF421-DUF1657 gene and grows under neutral conditions. Oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus.
(k) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 11, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 11 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions.
(l) An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12, or a neutral oligonucleotide having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 12 and having the plasmid-specific DUF421-DUF1657 gene. An oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under certain conditions. 　前記オリゴヌクレオチド（ａ）が、配列番号１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｂ）が、配列番号２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号２で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｃ）が、配列番号３で表される塩基配列からなるオリゴヌクレオチド、又は配列番号３で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｄ）が、配列番号４で表される塩基配列からなるオリゴヌクレオチド、又は配列番号４で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｅ）が、配列番号５で表される塩基配列からなるオリゴヌクレオチド、又は配列番号５で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｆ）が、配列番号６で表される塩基配列からなるオリゴヌクレオチド、又は配列番号６で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｇ）が、配列番号７で表される塩基配列からなるオリゴヌクレオチド、又は配列番号７で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｈ）が、配列番号８で表される塩基配列からなるオリゴヌクレオチド、又は配列番号８で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｉ）が、配列番号９で表される塩基配列からなるオリゴヌクレオチド、又は配列番号９で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｊ）が、配列番号１０で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１０で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつDUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｋ）が、配列番号１１で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１１で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドであり、
　前記オリゴヌクレオチド（ｌ）が、配列番号１２で表される塩基配列からなるオリゴヌクレオチド、又は配列番号１２で表される塩基配列において１個以上４個以下の塩基が欠失、置換、挿入若しくは付加されており、かつプラスミド特異的DUF421-DUF1657遺伝子を有する中性条件で増殖可能なバチルス属に属する芽胞形成細菌の検出に使用できるオリゴヌクレオチドである、
　請求項１５に記載のオリゴヌクレオチド対。 The oligonucleotide (a) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 1, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (b) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 2. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (c) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 3. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (d) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 4. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (e) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 5. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (f) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 6. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (g) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 7. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (h) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 8. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (i) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 9. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (j) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 10, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 10. It is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has the DUF421-DUF1657 gene.
The oligonucleotide (k) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 11, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 11. is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has a plasmid-specific DUF421-DUF1657 gene,
The oligonucleotide (l) is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12, or one or more and four or less bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 12. is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and has a plasmid-specific DUF421-DUF1657 gene.
The oligonucleotide pair according to claim 15.
PCT/JP2023/017195 2022-05-02 2023-05-02 Method for detecting spore-forming bacterium Ceased WO2023214588A1 (en)

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BERENDSEN ERWIN M, BOEKHORST JOS, KUIPERS OSCAR P, WELLS-BENNIK MARJON H J: "A mobile genetic element profoundly increases heat resistance of bacterial spores", THE ISME JOURNAL, NATURE PUBLISHING GROUP UK, LONDON, vol. 10, no. 11, 1 November 2016 (2016-11-01), London, pages 2633 - 2642, XP093106218, ISSN: 1751-7362, DOI: 10.1038/ismej.2016.59 *

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