WO2023214588A1 - Procédé de détection d'une bactérie sporogène - Google Patents

Procédé de détection d'une bactérie sporogène 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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花王株式会社
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    • 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
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    • 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

L'invention concerne un procédé de détection d'une bactérie sporogène appartenant au genre Bacillus, ledit procédé comprenant l'amplification d'un acide nucléique représenté par une séquence de bases partielle du gène DUF421-DUF1657 à l'aide d'une paire d'oligonucléotides ayant des séquences spécifiques en tant qu'amorces d'acides nucléiques, et la détection d'une bactérie sporogène qui peut croître dans des conditions neutres sur la base de la présence ou de l'absence d'un produit d'amplification.
PCT/JP2023/017195 2022-05-02 2023-05-02 Procédé de détection d'une bactérie sporogène WO2023214588A1 (fr)

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WO2015126251A1 (fr) * 2014-02-20 2015-08-27 Stichting Top Institute Food And Nutrition Micro-organismes thermorésistants

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WO2015126251A1 (fr) * 2014-02-20 2015-08-27 Stichting Top Institute Food And Nutrition Micro-organismes thermorésistants

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Title
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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