Methods for detecting spore-forming bacteria

Abstract

To provide simple, quick and inexpensive methods for detecting spore-forming bacteria that form highly heat resistant spores, methods for evaluating heat resistance of spores of spore-forming bacteria, methods for determining heat treatment conditions for spore-forming bacteria and kits for detecting spore-forming bacteria having DUF421-DUF1657 gene used for the methods.SOLUTION: There is disclosed a method for detecting a spore-forming Bacillus bacterium, the method comprising amplifying a nucleic acid comprising a partial base sequence of a DUF421-DUF1657 gene by using a pair of oligonucleotides of specific sequences as a nucleic acid primer, and detecting spore-forming bacteria that can grow under neutral conditions on the basis of presence or absence of amplification products.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting spore-forming bacteria. [Background technology]

[0002] Spore-forming bacteria form spores that are highly resistant to physical treatments such as heat, drying, and ultraviolet light. Examples of spore-forming bacteria include Bacillus ( Bacillus ) Bacteria of the genus Clostridium ( Clostridium Many bacterial species, particularly those belonging to the genus *Bacteria*, are known to form spores when exposed to unsuitable environments for growth, such as nutrient deprivation, drought, or high temperatures. Spore-forming bacteria, such as those belonging to the Bacillus genus, grow widely in water and soil, posing a high risk of contamination of food ingredients and food products during the manufacturing process. While most bacteria are typically killed by heat treatment, for example, 75°C for 30 minutes, some spore-forming bacteria can withstand high temperatures around 100°C. Such bacteria may not be sufficiently sterilized by 75°C for 30 minutes. Insufficient sterilization of spore-forming bacteria can lead to their proliferation in the product, causing spoilage and deterioration. Although it is possible to sterilize such highly heat-resistant spore-forming bacteria by setting a higher heat treatment temperature, considering the deterioration of raw materials and products due to high-temperature treatment, and the cost of heat treatment, it is preferable to determine the appropriate heat treatment conditions by assessing and considering the heat resistance of the contaminated spore-forming bacteria.

[0003] Until now, the identification of spore-forming bacteria has been carried out by isolating the bacteria, culturing them in a culture medium, and then observing their morphology under a microscope. However, this method has problems such as requiring about two weeks for the characteristic morphology of each bacterial species, which serves as an indicator for identification, to form, and requiring skill to make accurate determinations, making rapid and accurate detection difficult. Furthermore, it has become clear that even among spore-forming bacteria of the same species, the heat resistance of their spores differs from strain to strain. For example, Non-Patent Literature 1 describes Bacillus subtilis, a type of spore-forming bacterium. Bacillus subtilis It has been disclosed that the spores of Bacillus subtilis exhibit diverse heat resistance. Furthermore, Non-Patent Literature 2 reports that a gene encoding a protein of unknown function, containing the DUF421 domain and the DUF1657 domain, within the transposon-derived spoVA2mob operon discovered through comparative genomic analysis of Bacillus subtilis, contributes to improved spore heat resistance.

[0004] A conventional method for evaluating the heat resistance of spores in spore-forming bacteria involves isolating and identifying the spore-forming bacteria as described above, then inducing spore formation under appropriate growth conditions, temperature, and time, and finally measuring the survival rate when exposed to various heat treatment conditions to evaluate spore heat resistance. Furthermore, for example, Patent Document 1 discloses a method for rapidly and accurately evaluating and measuring the durability of spores in real time using an atomic force microscope equipped with a cantilever. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2009-183207 [Non-patent literature]

[0006] [Non-Patent Document 1] Berendsen et al., Food Microbiol., 2015, Vol. 45, 18-25 [Non-Patent Document 2] Berendsen et al., ISME J., 2016, Vol. 10, 2633-2642 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] When evaluating the heat resistance of spores of spore-forming bacteria using conventional methods as described above, it is necessary to culture the spore-forming bacteria to form spores and then measure the heat resistance of the spores, which takes a long time. Furthermore, since it is not possible to directly evaluate the heat resistance of spores present in the raw material, it is necessary to isolate spore-forming bacteria from the raw material separately, which presents problems in terms of time and cost. Furthermore, measuring spore heat resistance using methods such as those described in Patent Document 1 requires large-scale equipment for measurement, resulting in the disadvantage of high equipment costs.

[0008] The present invention aims to provide a method for detecting spore-forming bacteria that can detect spores with high heat resistance in a simple, rapid, and low-cost manner. Furthermore, the present invention aims to provide a method for evaluating the heat resistance of spore-forming bacteria spores in a simple, rapid, and low-cost manner. Furthermore, the present invention aims to provide a method for determining heat treatment conditions for spore-forming bacteria, which ensures commercial sterility by evaluating spore heat resistance. Furthermore, the present invention aims to provide a kit or oligonucleotide pair for detecting spore-forming bacteria that form spores with high heat resistance, which can be suitably used in the above-described method. [Means for solving the problem]

[0009] In view of the above problems, the present inventors conducted intensive studies. The present inventors focused on a gene encoding a protein of unknown function having a DUF421 domain and a DUF1657 domain in the transposon-derived spoVA2mob operon, which was found to contribute to the improvement of spore heat resistance by comparative genomic analysis of Bacillus subtilis (hereinafter also referred to as the "DUF421-DUF1657 gene"), and attempted to develop a method for evaluating spore heat resistance using the gene as a DNA marker. As a result, by using an oligonucleotide pair capable of annealing to the nucleotide sequence of a specific region (hereinafter also referred to as the "specific region") of the DUF421-DUF1657 gene as a primer pair and amplifying a partial nucleotide sequence of the DUF421-DUF1657 gene, it was clarified that not only the presence or absence of the DUF421-DUF1657 gene involved in spore heat resistance can be discriminated, but also the bacterial species can be identified from the size information of the amplified nucleic acid. In addition, a correlation was found between the presence or absence of the amplification product of the DUF421-DUF1657 gene and spore heat resistance, and it was found that when an amplification product of the DUF421-DUF1657 gene can be obtained using the above primers for spore-forming bacteria, the spores formed by the spore-forming bacteria have high heat resistance. Further, it was found that by detecting the gene, the spore heat resistance of spore-forming bacteria present in the raw material can be evaluated and judged, and thereby the appropriate heat treatment conditions for the raw material can be determined. The present invention has been completed based on these findings.

[0010] The present invention relates to a method for detecting spore-forming bacteria belonging to the genus Bacillus, which uses at least one oligonucleotide pair selected from the group consisting of 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) as nucleic acid primers to amplify a nucleic acid represented by a partial base sequence of the DUF421-DUF1657 gene, and detects spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions based on the presence or absence of an amplification product. (a) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 1, or an oligonucleotide having 80% or more identity with the base sequence represented by SEQ ID NO: 1 and capable of being used for detecting spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and have the DUF421-DUF1657 gene. (b) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or an oligonucleotide having 80% or more identity with the base sequence represented by SEQ ID NO: 2 and capable of being used for detecting spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and have the DUF421-DUF1657 gene. (c) An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide having 80% or more identity with the base sequence represented by SEQ ID NO: 3 and capable of being used for detecting spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and have the DUF421-DUF1657 gene. (d) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 4, or oligonucleotides having 80% or more identity with the nucleotide sequence represented by Sequence ID No. 4 and possessing the DUF421-DUF1657 gene, which can be used for detecting spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions. (e) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 5, or oligonucleotides having 80% or more identity with the nucleotide sequence represented by Sequence ID No. 5 and possessing the DUF421-DUF1657 gene, which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions. (f) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 6, or oligonucleotides having 80% or more identity with the nucleotide sequence represented by Sequence ID No. 6 and possessing the DUF421-DUF1657 gene, which can be used for detecting spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions. (g) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 7, or oligonucleotides having 80% or more identity with the nucleotide sequence represented by Sequence ID No. 7 and possessing the DUF421-DUF1657 gene, which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions. (h) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 8, or oligonucleotides having 80% or more identity with the nucleotide sequence represented by Sequence ID No. 8 and possessing the DUF421-DUF1657 gene, which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions. (i) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 9, or oligonucleotides having 80% or more identity with the nucleotide sequence represented by Sequence ID No. 9 and possessing the DUF421-DUF1657 gene, which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions. (j) An oligonucleotide consisting of the nucleotide sequence represented by Sequence ID No. 10, or an oligonucleotide having 80% or more identity with the nucleotide sequence represented by Sequence ID No. 10 and possessing the DUF421-DUF1657 gene, which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions. (k) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 11, or oligonucleotides having 80% or more identity with the nucleotide sequence represented by Sequence ID No. 11, and possessing the plasmid-specific DUF421-DUF1657 gene, which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions. (l) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 12, or oligonucleotides having 80% or more identity with the nucleotide sequence represented by Sequence ID No. 12, and possessing the plasmid-specific DUF421-DUF1657 gene, which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions.

[0011] The present invention also relates to a method for evaluating the heat resistance of spores of spore-forming bacteria belonging to the genus Bacillus, which can grow under neutral conditions using the amplification 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 the heat treatment conditions for spore-forming bacteria belonging to the genus Bacillus based on the heat resistance of spores evaluated by the above method. Furthermore, the present invention relates to a kit and oligonucleotide pair for detecting spore-forming bacteria used in the above method. [Effects of the Invention]

[0012] The present invention provides a method for detecting spore-forming bacteria that allows for the simple, rapid, and low-cost detection of spore-forming bacteria belonging to the genus Bacillus, which possess high heat-resistant spore-forming ability. Furthermore, the present invention provides a method for evaluating the heat resistance of spores of spore-forming bacteria that allows for the simple, rapid, and low-cost evaluation of the heat resistance of spores formed by spore-forming bacteria belonging to the genus Bacillus. Moreover, the present invention provides a method for determining appropriate heat treatment conditions for spore-forming bacteria that allows for the determination of appropriate heat treatment conditions for sterilizing spore-forming bacteria. In addition, the kit and oligonucleotide pair for detecting spore-forming bacteria of the present invention can be suitably used in the above methods. [Brief explanation of the drawing]

[0013]

Figure 1

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Figure 4

[0014] The present invention provides a method for detecting spore-forming bacteria that possess the DUF421-DUF1657 gene. This method involves amplifying the DUF421-DUF1657 gene using oligonucleotide pairs that can hybridize under stringent conditions to a specific partial base sequence of the DUF421-DUF1657 gene, i.e., a region (variable region) specific to each species of spore-forming bacteria belonging to the genus Bacillus that possesses the DUF421-DUF1657 gene. The presence or absence of the amplification product is then confirmed, thereby specifically identifying and detecting spore-forming bacteria that possess the DUF421-DUF1657 gene 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 significantly between species. In this specification, "stringent conditions" refer to, for example, the method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press], which includes conditions in which the probe is incubated with a solution containing 6×SSC (composition of 1×SSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5×Denhart, and 100 mg / mL herring sperm DNA at 65°C for 8 to 16 hours to allow hybridization. Furthermore, the present invention provides a method for evaluating the heat resistance of spore-forming bacteria by checking for the presence or absence of amplified products of a partial base sequence of the DUF421-DUF1657 gene. This is based on the observation that spore-forming bacteria belonging to the genus Bacillus possessing the DUF421-DUF1657 gene exhibit improved heat resistance compared to spore-forming bacteria lacking the DUF421-DUF1657 gene. The method also provides a method for determining the heat treatment conditions for spore-forming bacteria by determining suitable heat treatment conditions based on the results of the spore heat resistance evaluation. Hereinafter, each of the methods of the present invention described above will also be referred to as "the method of the present invention."

[0015] In this invention and specification, the "DUF421-DUF1657 gene" is a gene that encodes a protein having a DUF421 domain and a DUF1657 domain, located on the spoVA2mob operon. Whether the target protein has a DUF421 domain and a DUF1657 domain can be determined by performing NCBI BLASTp analysis on the target protein. The amino acid sequence encoded by the DUF421-DUF1657 gene from Bacillus subtilis B4146 strain (GenBank: KIN50158.1) is entered as the query sequence, and the analysis is performed using blastp (protein-protein BLAST) as the Algorithm. The determination is made based on whether or not it has 90% or more coverage. In this invention and specification, a gene encoding an amino acid sequence that shows 90% or more coverage by the above method is referred to as the "DUF421-DUF1657 gene".

[0016] In this invention, "spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions" refers to bacteria belonging to the genus Bacillus that form spores and are not particularly limited as long as they can grow under aerobic conditions with a pH of 4.6 or higher and less than 8.0. Examples of such spore-forming bacteria belonging to the genus Bacillus include bacteria belonging to the genus Bacillus that can cause spoilage and deterioration in neutral foods and beverages, such as the Bacillus subtilis group, which includes Bacillus subtilis, etc. Bacillus subtilis group), Bacillus coagulans ( Bacillus coagulans ), Bacillus cereus ( Bacillus cereus ), Bacillus megatherium ( Bacillus megaterium ), Bacillus pymyrus ( Bacillus pumilus ), Bacillus simplex ( Bacillus simplex ), Bacillus anthrosis ( Bacillus anthracis ), Bacillus brevis ( Bacillusbrevis ), Bacillus lentus ( Bacillus lentus ), Bacillus mycoides ( Bacillus mycoides Examples include, etc. Among these, the spore-forming bacteria are preferably Bacillus subtilis, Bacillus coagulans, and Bacillus cereus.

[0017] <Bacillus subtilis group> In the present invention and this specification, the "Bacillus subtilis group" refers to the group of bacteria defined in the National Center for Biotechnology Information (NCBI) database (Taxonomy ID: 653685). In the method of the present invention, the Bacillus subtilis group refers to Bacillus subtilis, Bacillus licheniformis ( Bacillus licheniformis ), Bacillus amyloricephasiens ( Bacillus amyloliquefaciens ), Bacillus siamensis ( Bacillus siamensis ), Bacillus berezensis ( Bacillus velezensis ), Bacillus paralicheniformis ( Bacillus paralicheniformis ), and Bacillus sonorensis ( Bacillus sonorensis It is preferable that the group consists of ), more preferably that it consists of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloricephasiensis, Bacillus siamensis, and Bacillus berezensis, and even more preferably that it is Bacillus subtilis.

[0018] For the three species Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloricephasiensis, as described in Non-Patent Literature 2, there is a correlation between the presence or absence of the DUF421-DUF1657 gene and spore heat resistance. In other words, in the above three species of bacteria, strains possessing the DUF421-DUF1657 gene in their genome form spores with higher heat resistance compared to strains lacking the DUF421-DUF1657 gene in their genome. Furthermore, the inventors performed BLASTp analysis using the amino acid sequence encoded by the DUF421-DUF1657 gene derived from Bacillus subtilis B4146 strain, and revealed that the DUF421-DUF1657 gene is conserved in the genomes of many strains of four species: Bacillus siamensis, Bacillus berezensis, Bacillus paralicheniformis, and Bacillus sonorensis. Furthermore, the nucleotide sequences of the two DUF421-DUF1657 genes from Bacillus siamensis strain SCSIO05746 and Bacillus berezensis strain 9912D (the proteins encoded by these genes, GenBank: AUJ76361.1 and GenBank: APA02936.1) and the nucleotide sequences of the DUF421-DUF1657 genes from Bacillus subtilis strain B4146, Bacillus licheniformis strain ATCC9789, and Bacillus amyloricephasiensis strain SRCM101267 are all of over 99%. Therefore, it can be inferred that, in the four species Bacillus siamensis, Bacillus berezensis, Bacillus paralicheniformis, and Bacillus sonorensis, there is a correlation between the presence or absence of the DUF421-DUF1657 gene and spore heat resistance, similar to the three species Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloricefaciens. In other words, in the above four species, strains possessing the DUF421-DUF1657 gene are thought to form spores with higher heat resistance compared to strains lacking the DUF421-DUF1657 gene.

[0019] When the DUF421-DUF1657 gene is detected in a Bacillus subtilis strain using the method of the present invention (when the nucleic acid represented by the partial base sequence of the DUF421-DUF1657 gene is amplified using the nucleic acid primer described later, the presence of the amplification product is confirmed and judged as "positive"), the spores formed by the Bacillus subtilis strain possessing the DUF421-DUF1657 gene have high heat resistance. On the other hand, when the amplification reaction is performed using the nucleic acid primer described later but no amplification product is confirmed and judged as "negative", the test strain does not possess the DUF421-DUF1657 gene, and the heat resistance of the spores formed is relatively lower than that of the spores of the Bacillus subtilis strain possessing the DUF421-DUF1657 gene. The Decimal Reduction Value (D) of Bacillus subtilis strains possessing the DUF421-DUF1657 gene can be appropriately determined by referring to known information. For example, Berendsen et al., The ISME Journal (2016) 10, 2633-2642 describes the D value (D) at 112.5°C for Bacillus subtilis strains having at least one copy of the transposon-derived spoVA2mob operon. 112.5℃ The value has been shown to be for 1 minute or more. Since the transposon-derived spoVA2mob operon contains the DUF421-DUF1657 gene, for example, the Bacillus subtilis group D that was judged positive by the method of the present invention 112.5℃ The value can be evaluated and determined for a period of one minute or more. In this specification, for example, "D 100℃ The term "value" refers to the time required to reduce the number of target bacteria to 1 / 10 of the number before heat treatment by heating at 100°C.

[0020] <Bacillus coagulans> Furthermore, the inventors performed BLASTp analysis using the amino acid sequence encoded by the DUF421-DUF1657 gene from Bacillus subtilis B4146 strain and revealed that the DUF421-DUF1657 gene is also conserved in the genome of many strains of Bacillus coagulans. The identity between the nucleotide sequence of the DUF421-DUF1657 gene from Bacillus subtilis B4146 strain and the nucleotide sequence of the DUF421-DUF1657 gene from Bacillus coagulans DSM1=ATCC7050 strain (the same strain as NBRC12583) is approximately 74.0%.

[0021] When the DUF421-DUF1657 gene is detected in a Bacillus coagulans strain used as the test strain by the method of the present invention (when the nucleic acid represented by the partial base sequence of the DUF421-DUF1657 gene is amplified using the nucleic acid primer described later, the presence of the amplification product is confirmed and judged as "positive"), the Bacillus coagulans strain possessing the DUF421-DUF1657 gene has spores that are formed with high heat resistance. On the other hand, when the amplification reaction is performed using the nucleic acid primer described later but no amplification product is confirmed and judged as "negative", the test strain does not possess the DUF421-DUF1657 gene, and the heat resistance of the spores formed is relatively lower than that of the Bacillus coagulans strain possessing the DUF421-DUF1657 gene. The D value of the Bacillus coagulans strain having the DUF421-DUF1657 gene can also be appropriately determined by referring to the results of the examples shown below. That is, for example, from the results of the examples, the D value of Bacillus coagulans 105℃ The value can be set to 10 minutes or more. Also, for example, the D value of Bacillus subtilis at 112.5°C (D 112.5℃ Since the value can be maintained for more than 1 minute, Bacillus coagulans D, which was judged to be positive by the method of the present invention 112.5℃ Similarly, the value can also be evaluated and determined as being for more than one minute.

[0022] <Bacillus cereus> Furthermore, the inventors performed BLASTp analysis using the amino acid sequence encoded by the DUF421-DUF1657 gene derived from Bacillus subtilis B4146 strain, and revealed that the DUF421-DUF1657 gene is conserved in the genomes of almost all strains of Bacillus cereus, and that the DUF421-DUF1657 gene is additionally conserved specifically 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 DUF421-DUF1657 gene that is additionally conserved specifically in emetic toxin-producing strains will also be referred to as the "plasmid-specific DUF421-DUF1657 gene". Furthermore, the nucleotide sequence identity between the DUF421-DUF1657 gene derived from Bacillus subtilis B4146 strain and the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus AH187 strain is approximately 66.9%. In this invention and specification, unless otherwise specified, "DUF421-DUF1657 gene" means the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus, in the case of Bacillus cereus.

[0023] When the plasmid-specific DUF421-DUF1657 gene is detected using a Bacillus cereus strain as the test strain by the method of the present invention (when the nucleic acid represented by a partial base sequence of the plasmid-specific DUF421-DUF1657 gene is amplified using the nucleic acid primer described later, the presence of the amplification product is confirmed and judged as "positive"), the Bacillus cereus strain possessing the plasmid-specific DUF421-DUF1657 gene has highly heat-resistant spores. On the other hand, when the amplification reaction is performed using the nucleic acid primer described later but no amplification product is confirmed and judged as "negative", the test strain does not possess the plasmid-specific DUF421-DUF1657 gene, and the heat resistance of the spores formed is relatively lower than that of the Bacillus cereus strain possessing the plasmid-specific DUF421-DUF1657 gene. The D-value of Bacillus cereus strains possessing the plasmid-specific DUF421-DUF1657 genes can be appropriately determined by referring to known information. For example, Carlin et al., Food Microbiology (2006) 109, 132-138 states that all non-emetic toxin-producing strains have a D-value. 90℃ The value is shown to be 300 minutes or less. As described above, our analysis suggests that non-emetic toxin-producing strains do not possess the plasmid-specific DUF421-DUF1657 gene, and therefore Bacillus cereus D judged negative by the method of the present invention 90℃ The value can be evaluated and determined to be 300 minutes or less. Alternatively, the D value of Bacillus cereus can be measured in advance (for example, by measuring the D value using the method described in the examples below), and the D value of Bacillus cereus that is judged negative by the method of the present invention can be evaluated and determined to be below a specific value according to the measurement results.

[0024] The DUF421-DUF1657 gene detected by the method of the present invention is conserved in the genome or plasmid of spore-forming bacteria that form spores with high heat resistance. On the other hand, the base sequence encoding the DUF421 domain located in the upstream portion (5'-terminal side) of the DUF421-DUF1657 gene may also be conserved in the genome of spore-forming bacteria that form spores that do not have high heat resistance. Therefore, the oligonucleotide used in the method of the present invention is an oligonucleotide that satisfies all of the following conditions. In other words, 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 approximately 55 to 65°C, anneals to a region specific to the bacterial species from which the detected DUF421-DUF1657 genes originate (in the case of Bacillus cereus, further specifically to the plasmid-specific DUF421-DUF1657 genes, so as not to detect the genome-encoded DUF421-DUF1657 gene sequence), and anneals to a region that is highly conserved within the bacterial species, 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 is designed to straddle the DUF421 region (the nucleotide sequence encoding the DUF421 domain) and the DUF1657 region (the nucleotide sequence encoding the DUF1657 domain). The extents of the DUF421 and DUF1657 regions can be determined, for example, by referring to information listed in the NCBI database for each protein.

[0025] The oligonucleotide pair consisting of oligonucleotides (a) and (c) (hereinafter also referred to as "oligonucleotide pair (1)"), and the oligonucleotide pair consisting of oligonucleotides (b) and (c) (hereinafter also referred to as "oligonucleotide pair (2)") can be used to detect the DUF421-DUF1657 gene 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 that when the oligonucleotide pair is used as a primer pair, it can amplify nucleic acids consisting of a partial base sequence of the DUF421-DUF1657 gene. In the oligonucleotide(a) described above, from the viewpoint of annealing, the identity with the base sequence represented by Sequence ID No. 1 is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more. Also preferred as the oligonucleotide(a) is an oligonucleotide that has one to several bases (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 1, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably the Bacillus subtilis group) that can grow under neutral conditions and have the DUF421-DUF1657 gene. In the oligonucleotide(a) described above, "R" in the base sequence represented by Sequence ID No. 1 means a mixed base of adenine (A) and guanine (G). In the oligonucleotide(b) described above, from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 2 is 85% or more, more preferably 90% or more, and even more preferably 95% or more. Furthermore, as the oligonucleotide(b), oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus subtilis group) that can grow under neutral conditions and have the DUF421-DUF1657 gene, are also preferred, in which one to several bases (for example, one to four bases, preferably one to three bases, more preferably one or two bases, and even more preferably one base) are deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 2. In the oligonucleotide(c) described above, from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 3 is 85% or more, more preferably 90% or more, and even more preferably 95% or more. Furthermore, as the oligonucleotide(c), oligonucleotides that have one to several (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) bases deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 3, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus subtilis group) that can grow under neutral conditions and possess the DUF421-DUF1657 gene are also preferred. In this specification, the identity of the base sequence is determined using the Identity values ​​calculated by NCBI Blastn analysis. For program selection during the analysis, the Somewhat similar sequences (blastn) program was used.

[0026] The amplification products obtained by amplifying the partial base sequence of the DUF421-DUF1657 gene on the genome of Bacillus subtilis group using the oligonucleotide pair (1) and the oligonucleotide pair (2), respectively, will be described with reference to the base sequences of the DUF421-DUF1657 gene from Bacillus subtilis strain B4146 and Bacillus subtilis strain ATCC11774. The nucleotide sequence of the DUF421-DUF1657 gene from Bacillus subtilis strain B4146 is shown in Sequence ID No. 13, and the nucleotide sequence of the DUF421-DUF1657 gene from Bacillus subtilis strain ATCC11774 is shown in Sequence ID No. 14. Oligonucleotides (a) and (c) correspond to the regions from positions 364 to 381 and 695 to 712, respectively, of the nucleotide sequences described in Sequence ID No. 13 and Sequence ID No. 14. Therefore, for example, when polymerase chain reaction (PCR) is performed using oligonucleotide pair (1) and DNA containing the DUF421-DUF1657 gene from the Bacillus subtilis group as a template, the length of the amplified DNA fragment will be approximately 350 bp. Furthermore, oligonucleotides (b) and (c) correspond to the regions from positions 658 to 675 and 695 to 712, respectively, of the base sequences described in Sequence ID No. 13 and Sequence ID No. 14. Therefore, for example, when polymerase chain reaction (PCR) is performed using oligonucleotide pair (2) and DNA containing the DUF421-DUF1657 gene from the Bacillus subtilis group as a template, the length of the amplified DNA fragment will be approximately 55 bp.

[0027] The oligonucleotide pair consisting of oligonucleotides (d) and (j) (hereinafter also referred to as "oligonucleotide pair (3)"), the oligonucleotide pair consisting of oligonucleotides (e) and (j) (hereinafter also referred to as "oligonucleotide pair (4)"), the oligonucleotide pair consisting of oligonucleotides (f) and (j) (hereinafter also referred to as "oligonucleotide pair (5)"), the oligonucleotide pair consisting of oligonucleotides (g) and (j) (hereinafter also referred to as "oligonucleotide pair (6)"), the oligonucleotide pair consisting of oligonucleotides (h) and (j) (hereinafter also referred to as "oligonucleotide pair (7)"), and 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) described above, from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 4 is 85% or more, more preferably 90% or more, and even more preferably 95% or more. Furthermore, as the oligonucleotide(d), oligonucleotides that have one to several (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) bases deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 4, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus coagulans) that can grow under neutral conditions and have the DUF421-DUF1657 gene are also preferred. In the oligonucleotide(e), from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 5 is 85% or higher, more preferably 90% or higher, and even more preferably 95% or higher. Also preferred as the oligonucleotide(e) is an oligonucleotide that has one to several bases (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 5, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus coagulans) that can grow under neutral conditions and have the DUF421-DUF1657 gene. In the oligonucleotide(f), from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 6 is 85% or more, more preferably 90% or more, and even more preferably 95% or more. Furthermore, as the oligonucleotide(f), oligonucleotides that have one to several (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) bases deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 6, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus coagulans) that can grow under neutral conditions and have the DUF421-DUF1657 gene are also preferred. In the oligonucleotide(g) described above, from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 7 is 85% or more, more preferably 90% or more, and even more preferably 95% or more. Furthermore, as the oligonucleotide(g), oligonucleotides that have one to several (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) bases deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 7, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus coagulans) that can grow under neutral conditions and possess the DUF421-DUF1657 gene are also preferred. In the oligonucleotide(h), from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 8 is 85% or higher, more preferably 90% or higher, and even more preferably 95% or higher. Furthermore, as the oligonucleotide(h), oligonucleotides that have one to several (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) bases deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 8, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus coagulans) that can grow under neutral conditions and possess the DUF421-DUF1657 gene are also preferred. In the oligonucleotide(i) described above, from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 9 is 85% or more, more preferably 90% or more, and even more preferably 95% or more. Furthermore, as the oligonucleotide(i), oligonucleotides that have one to several (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) bases deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 9, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus coagulans) that can grow under neutral conditions and possess the DUF421-DUF1657 gene are also preferred. In the oligonucleotide(j) described above, from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by SEQ ID NO: 10 is 85% or more, more preferably 90% or more, and even more preferably 95% or more. Furthermore, as the oligonucleotide(j), oligonucleotides that have one to several (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) bases deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 10, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus coagulans) that can grow under neutral conditions and have the DUF421-DUF1657 gene are also preferred.

[0028] The amplification products obtained by amplifying the partial base sequence of the DUF421-DUF1657 gene on the genome of Bacillus coagulans using oligonucleotide pair (3), oligonucleotide pair (4), oligonucleotide pair (5), oligonucleotide pair (6), oligonucleotide pair (7), and oligonucleotide pair (8), respectively, will be described with reference to the base sequence of the DUF421-DUF1657 gene from Bacillus coagulans strain DSM1=ATCC7050. The nucleotide sequence of the DUF421-DUF1657 gene derived from Bacillus coagulans DSM1=ATCC7050 strain is shown in Sequence ID No. 15. Oligonucleotides (d) and (j) correspond to the regions from positions 223 to 240 and 695 to 712, respectively, of the nucleotide sequence shown in Sequence ID No. 15. Therefore, for example, when PCR is performed using oligonucleotide pair (3) and DNA containing the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be approximately 490 bp. Furthermore, oligonucleotides (e) and (j) correspond to the regions from positions 246 to 263 and 695 to 712, respectively, of the base sequence described in Sequence ID No. 15. Therefore, for example, when PCR is performed using oligonucleotide pair (4) and DNA containing the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be approximately 465 bp. Furthermore, oligonucleotides (f) and (j) correspond to the regions from positions 558 to 575 and 695 to 712, respectively, of the base sequence described in Sequence ID No. 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (5) and DNA containing the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be approximately 155 bp. Furthermore, oligonucleotides (g) and (j) correspond to the regions from positions 577 to 594 and 695 to 712, respectively, of the base sequence described in Sequence ID No. 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (6) and DNA containing the DUF421-DUF1657 gene from Bacillus coagulans as a template, the length of the amplified DNA fragment will be approximately 135 bp. Furthermore, oligonucleotides (h) and (j) correspond to the regions from positions 592 to 609 and 695 to 712, respectively, of the base sequence described in Sequence ID No. 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (7) and DNA containing the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be approximately 120 bp. Furthermore, oligonucleotides (i) and (j) correspond to the regions from positions 634 to 651 and 695 to 712, respectively, of the base sequence described in Sequence ID No. 15. Therefore, for example, when PCR is performed using the oligonucleotide pair (8) and DNA containing the DUF421-DUF1657 gene derived from Bacillus coagulans as a template, the length of the amplified DNA fragment will be approximately 80 bp.

[0029] The oligonucleotide pair consisting of oligonucleotides (k) and (l) (hereinafter also referred to as "oligonucleotide pair (9)") can be used to detect plasmid-specific DUF421-DUF1657 genes derived from Bacillus cereus. In the oligonucleotide(k), from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 11 is 85% or higher, more preferably 90% or higher, and even more preferably 95% or higher. Furthermore, as the oligonucleotide(k), oligonucleotides that have one to several (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) bases deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 11, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus cereus) that can grow under neutral conditions and possess the DUF421-DUF1657 gene are also preferred. In the oligonucleotide(l) described above, from the viewpoint of annealing, it is preferable that the identity with the base sequence represented by Sequence ID No. 12 is 85% or more, more preferably 90% or more, and even more preferably 95% or more. Furthermore, as the oligonucleotide(l), oligonucleotides that have one to several (for example, one to four, preferably one to three, more preferably one or two, even more preferably one) bases deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 12, and that can be used to detect spore-forming bacteria belonging to the genus Bacillus (preferably Bacillus cereus) that can grow under neutral conditions and have the DUF421-DUF1657 gene are also preferred.

[0030] The amplification product obtained by amplifying a partial nucleotide sequence of the plasmid-specific DUF421-DUF1657 gene of Bacillus cereus using the oligonucleotide pair (9) will be described with reference to the nucleotide sequence of the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus strain AH187. The nucleotide sequence of the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus strain AH187 is shown in Sequence ID No. 16. Oligonucleotides (k) and (l) correspond to the regions from positions 457 to 474 and 699 to 721, respectively, of the nucleotide sequence shown in Sequence ID No. 16. Therefore, for example, when PCR is performed using oligonucleotide pair (9) and DNA containing the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus as a template, the length of the amplified DNA fragment will be approximately 265 bp.

[0031] There are no particular restrictions on the method of nucleic acid amplification; conventional nucleic acid amplification methods such as PCR, real-time PCR, LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification), NASBA (Nucleic Acid Sequence-based Amplification), RCA (Rolling-circle amplification), and LAMP (Loop-mediated isothermal amplification) can be used. In this invention, PCR is preferred from the viewpoint of speed. The conditions for PCR are not particularly limited as long as the target nucleic acid (amplified DNA fragment) can be amplified to a detectable degree. A preferred example of PCR reaction conditions is, for example, when performing PCR using at least one oligonucleotide pair selected from the group consisting of oligonucleotide pair (1), oligonucleotide pair (2), oligonucleotide pair (3), oligonucleotide pair (4), oligonucleotide pair (5), oligonucleotide pair (6), oligonucleotide pair (7), oligonucleotide pair (8), and oligonucleotide pair (9) as nucleic acid primers, a thermal denaturation reaction to convert double-stranded DNA into single-stranded DNA is performed at 94-98°C, preferably 94°C, for 10-60 seconds, an annealing reaction to hybridize the primer pair to single-stranded DNA is performed at 50-62°C, preferably 58-60°C, for 30-60 seconds, and an extension reaction to act with DNA polymerase is performed at approximately 72°C for 30-60 seconds, with these being considered as one cycle, and approximately 30-35 cycles being performed. If the sample contains spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and possess the DUF421-DUF1657 gene, PCR is performed using the oligonucleotide pair of the present invention as a primer pair, and the resulting PCR product is subjected to electrophoresis, which allows for the amplification of DNA fragments of a specific size. By performing this procedure, it is possible to confirm whether the sample contains spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and possess the DUF421-DUF1657 gene, and furthermore, the bacterial species can be identified from the length of the amplified product.

[0032] In the method of the present invention, PCR may be performed using the above-mentioned 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. To enable use as oligonucleotide pairs for Multiplex PCR, each oligonucleotide pair used in the method of the present invention is designed so that the amplification product length differs for each bacterial species. Furthermore, oligonucleotide pair (1) and oligonucleotide pair (2) are oligonucleotide pairs that can specifically amplify partial nucleotide sequences of the DUF421-DUF1657 gene derived from the Bacillus subtilis group, but do not amplify partial nucleotide sequences of the DUF421-DUF1657 gene derived from Bacillus coagulans and Bacillus cereus (for Bacillus cereus, the DUF421-DUF1657 gene encoded on the genome derived from Bacillus cereus, and the plasmid-specific DUF421-DUF1657 gene). Furthermore, oligonucleotide pairs (3), (4), (5), (6), (7), and (8) are oligonucleotide pairs capable of specifically amplifying partial nucleotide sequences of the DUF421-DUF1657 gene derived from Bacillus coagulans, but do not amplify partial nucleotide sequences of the DUF421-DUF1657 gene derived from Bacillus subtilis and Bacillus cereus (for Bacillus cereus, the DUF421-DUF1657 gene encoded on the genome derived from Bacillus cereus, and the plasmid-specific DUF421-DUF1657 gene). Furthermore, the oligonucleotide pair (9) is an oligonucleotide pair that can specifically amplify a partial nucleotide sequence of the plasmid-specific DUF421-DUF1657 gene derived from Bacillus cereus, but does not amplify the DUF421-DUF1657 gene encoded on the genome of Bacillus cereus, nor the partial nucleotide sequence of the DUF421-DUF1657 gene derived from Bacillus subtilis and Bacillus coagulans.

[0033] When performing Multiplex PCR using multiple pairs of the aforementioned oligonucleotide pairs (when performing Multiplex PCR using multiple pairs of the aforementioned oligonucleotide pairs as a mixed primer pair), a preferred combination of each oligonucleotide pair is a combination of one oligonucleotide pair selected from the group consisting of oligonucleotide pair (1) and oligonucleotide pair (2) (preferably oligonucleotide pair (1)), at least one oligonucleotide pair selected from the group consisting of oligonucleotide pair (3), oligonucleotide pair (4), oligonucleotide pair (5), oligonucleotide pair (6), oligonucleotide pair (7), and oligonucleotide pair (8), and oligonucleotide pair (9).

[0034] In the present invention, the amplification of a DNA fragment can be confirmed by conventional methods. For example, methods include performing electrophoresis on the amplification product to check for the presence or absence of a band corresponding to the size of the amplified gene, measuring the amount of amplification product over time, and decoding the base sequence of the amplification product, but the present invention is not limited to these methods. In the present invention, it is preferable to perform electrophoresis after the amplification treatment of the DNA fragment to check for the presence or absence of a band corresponding to the size of the amplified DNA fragment. Furthermore, in the present invention, the amplification product can be detected by conventional methods. For example, methods include incorporating nucleotides labeled with radioactive material during the amplification reaction, using primers labeled with fluorescent material, and inserting a fluorescent material that increases fluorescence intensity by binding to DNA, such as ethidium bromide, between the two strands of amplified DNA, but the present invention is not limited to these methods. In the present invention, it is preferable to insert a fluorescent material that increases fluorescence intensity by binding to DNA between the two strands of amplified DNA.

[0035] The preparation method for the above oligonucleotides (a) to (l) used in the method of the present invention is not particularly limited. For example, they can be chemically synthesized based on the designed sequence or purchased from a reagent manufacturer. When chemically synthesized based on the designed sequence, they can be synthesized using an oligonucleotide synthesizer or the like. Alternatively, purified oligonucleotides can be used after synthesis using an adsorption column, high-performance liquid chromatography, or electrophoresis. Oligonucleotides having a base sequence in which one or more bases are substituted, deleted, inserted, or added can also be synthesized using conventional methods. Alternatively, when purchasing from reagent manufacturers, you can utilize services such as FASMAC's DNA / RNA contract synthesis service or Thermo Fisher Scientific's GeneArt artificial gene synthesis service.

[0036] There are no particular restrictions on the subjects used in the present invention; food and beverages themselves, raw materials for food and beverages, isolated bacterial cells, cultured bacterial cells, etc., can be used, and among these, neutral food and beverages and the raw materials used therein are preferred as subjects. Specific examples of such neutral food and beverages include, for example, neutral tea-based beverages such as green tea beverages, black tea beverages, and barley tea beverages, coffee beverages, dairy beverages, retort foods, and the raw materials used therein. The method for preparing DNA from the subject is not particularly limited as long as sufficient purity and quantity of DNA can be obtained for the detection of spore-forming bacteria, and it can be used in an unpurified state, but it can also be used after further pretreatment such as separation, extraction, concentration, and purification. For example, it can be purified by phenol and chloroform extraction, or by using a commercially available extraction kit to increase the purity of the nucleic acid before use. It is also possible to use DNA obtained by reverse transcription of RNA in the subject. Furthermore, the method for isolating bacteria from food or raw materials is not particularly limited, and for example, bacteria can be isolated by the method described in the examples below.

[0037] When a spore-forming bacterium having the DUF421-DUF1657 gene is detected in a raw material or a product by the method of the present invention, the spore-forming bacterium forms highly heat-resistant spores. Therefore, for a raw material or a product in which such a spore-forming bacterium is detected, the heat treatment temperature can be set to conditions (conditions under which commercial sterility of the heat-treated food is ensured) such that the spore-forming bacterium can be sufficiently sterilized. In the present specification, the "commercial sterility" of a heat-treated food means removing microorganisms that can grow in the food and viable microorganisms harmful to public health (including spores) in a non-refrigerated state during normal storage, distribution, etc. by applying heat, or removing microorganisms that can grow in the food from the food by controlling the water activity and applying heat in a normal non-refrigerated state during storage, distribution, etc.

[0038] In the present invention or in the present specification, the heat treatment conditions can be appropriately set according to the evaluation results of spore heat resistance (presence or absence of the DUF421-DUF1657 gene) and the identified bacterial species and strains. For example, when it is confirmed that all of the spore-forming bacteria belonging to the genus Bacillus detected from various raw materials of neutral foods and drinks do not have the DUF421-DUF1657 gene, or when the result of PCR for detecting the DUF421-DUF1657 gene for the DNA derived from the spore-forming bacteria belonging to the genus Bacillus extracted from the raw material is negative, it indicates that the possibility of the raw material containing highly heat-resistant spores is extremely low, and heating conditions capable of sufficiently killing the bacteria can be set such that the D value = 1 minute or less in the sterilization process. Also, in a product or an intermediate product during production that has undergone a specific sterilization process, when a "spore-forming bacterium belonging to the genus Bacillus that can grow under neutral conditions" is detected, and further when it is confirmed by the method of the present invention that the spore-forming bacterium has the DUF421-DUF1657 gene, it can be determined that the sterilization conditions were insufficient. 112.5℃ When a spore-forming bacterium having the DUF421-DUF1657 gene is detected in a raw material or a product by the method of the present invention, the spore-forming bacterium forms highly heat-resistant spores. Therefore, for a raw material or a product in which such a spore-forming bacterium is detected, the heat treatment temperature can be set to conditions (conditions under which commercial sterility of the heat-treated food is ensured) such that the spore-forming bacterium can be sufficiently sterilized. In the present specification, the "commercial sterility" of a heat-treated food means removing microorganisms that can grow in the food and viable microorganisms harmful to public health (including spores) in a non-refrigerated state during normal storage, distribution, etc. by applying heat, or removing microorganisms that can grow in the food from the food by controlling the water activity and applying heat in a normal non-refrigerated state during storage, distribution, etc. Furthermore, by evaluating the heat resistance of spores of each bacterial species and strain in advance and determining the heat treatment conditions, these conditions can be applied as is, or modified as appropriate, when the same bacterial species or strain is detected. Sufficient heat treatment conditions (temperature, time, pressure, etc.) to sterilize each bacterial species and strain can be determined, for example, based on conventional methods in this art, or by the method described in the examples.

[0039] According to the method of the present invention, the process from the preparation of the test subject to the detection of 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 test subject is a suspension inoculated with colonies of each bacterial strain, it can be carried out in about half a day, and as in Example 7, if the raw material is used as the test subject and the spore-forming bacteria contained in the raw material are detected, it can be carried out in a short time of about two days.

[0040] The kit for detecting spore-forming bacteria having the DUF421-DUF1657 gene (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 present invention. In addition to the primer pair, the kit of the present invention contains, depending on the purpose, substances commonly used for the detection of fungi, such as a labeled detection substance, a buffer solution, nucleic acid synthesis enzymes (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), and enzyme substrates (dNTPs, rNTPs, etc.). The kit of the present invention may also include a positive control to confirm that the detection reaction is possible with the detection oligonucleotide of the present invention. An example of a positive control is DNA containing the region amplified by the method of the present invention.

[0041] With respect to 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 pairs.

[0042] <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), A pair of oligonucleotides (3) consisting of the following oligonucleotides (d) and (j), A pair of oligonucleotides (4) consisting of the following oligonucleotides (e) and (j), A pair of oligonucleotides (5) consisting of the following oligonucleotides (f) and (j), A pair of oligonucleotides (6) consisting of the following oligonucleotides (g) and (j), An oligonucleotide pair (7) consisting of the following oligonucleotides (h) and (j), A pair of oligonucleotides (8) consisting of the following oligonucleotides (i) and (j), and The following oligonucleotide pair (9) consists of oligonucleotides (k) and (l). Using at least one oligonucleotide pair selected from the group consisting of the following as a nucleic acid primer, the nucleic acid represented by the partial base sequence of the DUF421-DUF1657 gene is amplified. This method 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) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 1, or oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus subtilis group, which have an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence represented by Sequence ID No. 1, and which have the DUF421-DUF1657 gene and can grow under neutral conditions. (b) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 2, or oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus subtilis group, which have an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence represented by Sequence ID No. 2, and which have the DUF421-DUF1657 gene and can grow under neutral conditions. (c) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 3, or oligonucleotides having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence represented by Sequence ID No. 3, and which can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus subtilis group, that can grow under neutral conditions and have the DUF421-DUF1657 gene. (d) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 4, or oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans, which have an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence represented by Sequence ID No. 4, and which have the DUF421-DUF1657 gene and can grow under neutral conditions. (e) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 5, or oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans, which have an identity of 80% or more, preferably 85% or more, more preferably 90% or more, with the base sequence represented by Sequence ID No. 5, and which are capable of growing under neutral conditions and possess the DUF421-DUF1657 gene. (f) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 6, or oligonucleotides having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence represented by Sequence ID No. 6, and which can be used to detect spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and possess the DUF421-DUF1657 gene, preferably Bacillus coagulans. (g) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 7, or oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans, which have an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence represented by Sequence ID No. 7, and which have the DUF421-DUF1657 gene and can grow under neutral conditions. (h) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 8, or oligonucleotides having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence represented by Sequence ID No. 8, and which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and possess the DUF421-DUF1657 gene, preferably Bacillus coagulans. (i) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 9, or oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans, which have an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence represented by Sequence ID No. 9, and which have the DUF421-DUF1657 gene and can grow under neutral conditions. (j) Oligonucleotides consisting of the base sequence represented by Sequence ID No. 10, or oligonucleotides that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably Bacillus coagulans, which have an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence represented by Sequence ID No. 10, and which have the DUF421-DUF1657 gene and can grow under neutral conditions. (k) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 11, or oligonucleotides having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the nucleotide sequence represented by Sequence ID No. 11, and which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and possess the plasmid-specific DUF421-DUF1657 gene, preferably Bacillus cereus. (l) Oligonucleotides consisting of the nucleotide sequence represented by Sequence ID No. 12, or oligonucleotides having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the nucleotide sequence represented by Sequence ID No. 12, and which can be used for the detection of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions and possess the plasmid-specific DUF421-DUF1657 gene, preferably Bacillus cereus.

[0043] <2> The amplification product is used to identify the spore-forming bacteria. <1> Methods used. <3> The heat resistance of the spores of the spore-forming bacteria is evaluated based on the presence or absence of the amplification product. <1> or <2> Methods used.

[0044] <4> Using at least one oligonucleotide pair selected from the group consisting of oligonucleotide pair (1), oligonucleotide pair (2), oligonucleotide pair (3), oligonucleotide pair (4), oligonucleotide pair (5), oligonucleotide pair (6), oligonucleotide pair (7), oligonucleotide pair (8), and oligonucleotide pair (9) as a nucleic acid primer, the nucleic acid represented by the partial nucleotide sequence of the DUF421-DUF1657 gene is amplified. The heat resistance of spores of spore-forming bacteria belonging to the genus Bacillus, which can grow under neutral conditions, is evaluated based on the presence or absence of amplification products. A method for evaluating the heat resistance of spores of spore-forming bacteria belonging to the genus Bacillus. <5> The aforementioned <3> or <4> A method for determining heat treatment conditions for spore-forming bacteria belonging to the genus Bacillus, characterized by determining the heat treatment conditions for the spore-forming bacteria based on the heat resistance of the spores evaluated by the method described above.

[0045] <6> The spore-forming bacteria belonging to the genus Bacillus that can grow under the aforementioned neutral conditions are spore-forming bacteria that can grow under aerobic conditions with a pH of 4.6 or higher and less than 8.0, <1> ~ <5> The method described in any one of the items. <7> The spore-forming bacteria belonging to the genus Bacillus that can grow under the aforementioned neutral conditions are at least one selected from the group consisting of Bacillus subtilis, Bacillus coagulans, Bacillus cereus, Bacillus megatherium, Bacillus pymirus, Bacillus simplex, Bacillus anthrosis, Bacillus brevis, Bacillus lentus, and Bacillus mycoides, preferably at least one selected from the group consisting of Bacillus subtilis, Bacillus coagulans, and Bacillus cereus. <1> ~ <6> The method described in any one of the items. <8> The Bacillus subtilis group is at least one selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloricephasiensis, Bacillus siamensis, Bacillus berezensis, Bacillus paralicheniformis, and Bacillus sonorensis, preferably at least one selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloricephasiensis, Bacillus siamensis, and Bacillus berezensis, more preferably Bacillus subtilis, <7> Methods used.

[0046] <9> The oligonucleotide (a) is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably the Bacillus subtilis group, which have the DUF421-DUF1657 gene and can grow under neutral conditions, and which have one to several, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, deleted, substituted, inserted, or added to the base sequence represented by Sequence ID No. 1. <1> ~ <8> The method described in any one of the items. <10> The oligonucleotide (b) is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably the Bacillus subtilis group, which have the DUF421-DUF1657 gene and can grow under neutral conditions, and which have one to several, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, deleted, substituted, inserted, or added to the base sequence represented by Sequence ID No. 2. <1> ~ <9> The method described in any one of the items. <11> The oligonucleotide (c) is an oligonucleotide that can be used to detect spore-forming bacteria belonging to the genus Bacillus, preferably the Bacillus subtilis group, which have the DUF421-DUF1657 gene and can grow under neutral conditions, and which have one to several, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, deleted, substituted, inserted, or added bases in the base sequence represented by Sequence ID No. 3. <1> ~ <10> The method described in any one of the items. <12> The oligonucleotide (d) is an oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which has the DUF421-DUF1657 gene and can grow under neutral conditions, wherein the base sequence represented by Sequence ID No. 4 has one to several bases, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, deleted, substituted, inserted, or added, and which can grow under neutral conditions. <1> ~ <11> The method described in any one of the items. <13> The oligonucleotide (e) is an oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which has the DUF421-DUF1657 gene and can grow under neutral conditions, wherein the base sequence represented by Sequence ID No. 5 has one to several, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, bases deleted, substituted, inserted, or added, and which can grow under neutral conditions. <1> ~ <12> The method described in any one of the items. <14> The oligonucleotide (f) is an oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which has the DUF421-DUF1657 gene and can grow under neutral conditions, wherein the base sequence represented by Sequence ID No. 6 has one to several, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, bases deleted, substituted, inserted, or added, and which can grow under neutral conditions. <1> ~ <13> The method described in any one of the items. <15> The oligonucleotide (g) is an oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which has the DUF421-DUF1657 gene and can grow under neutral conditions, and in which one to several bases, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, are deleted, substituted, inserted, or added in the base sequence represented by Sequence ID No. 7. <1> ~ <14> The method described in any one of the items. <16> The oligonucleotide (h) is an oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which has the DUF421-DUF1657 gene and can grow under neutral conditions, wherein the base sequence represented by Sequence ID No. 8 has one to several, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, bases deleted, substituted, inserted, or added, and which can grow under neutral conditions. <1> ~ <15> The method described in any one of the items. <17> The oligonucleotide (i) is an oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which has the DUF421-DUF1657 gene and can grow under neutral conditions, wherein the base sequence represented by Sequence ID No. 9 has one to several, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, bases deleted, substituted, inserted, or added, and which can grow under neutral conditions. <1> ~ <16> The method described in any one of the items. <18> The oligonucleotide (j) is an oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus coagulans, which has the DUF421-DUF1657 gene and can grow under neutral conditions, wherein the base sequence represented by Sequence ID No. 10 has one to several bases, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, deleted, substituted, inserted, or added. <1> ~ <17> The method described in any one of the items. <19> The oligonucleotide (k) is an oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus cereus, which can grow under neutral conditions and has the plasmid-specific DUF421-DUF1657 gene, and in which the base sequence represented by Sequence ID No. 11 has one to several, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, deleted, substituted, inserted, or added bases. <1> ~ <18> The method described in any one of the items. <20> The oligonucleotide (l) is an oligonucleotide that can be used to detect a spore-forming bacterium belonging to the genus Bacillus, preferably Bacillus cereus, which can grow under neutral conditions and has the plasmid-specific DUF421-DUF1657 gene, and in which the base sequence represented by Sequence ID No. 12 has one to several, preferably one to four, more preferably one to three, more preferably one or two, and even more preferably one, deleted, substituted, inserted, or added bases. <1> ~ <19> The method described in any one of the items.

[0047] <21> At least one oligonucleotide pair selected from the group consisting of oligonucleotide pair (1), oligonucleotide pair (2), oligonucleotide pair (3), oligonucleotide pair (4), oligonucleotide pair (5), oligonucleotide pair (6), oligonucleotide pair (7), and oligonucleotide pair (8) is used as a primer pair to amplify the nucleic acid represented by the partial nucleotide sequence of the DUF421-DUF1657 gene on the genome by polymerase chain reaction. The nucleic acid represented by the plasmid-specific DUF421-DUF1657 gene is amplified by polymerase chain reaction using the oligonucleotide pair (9) as a primer pair. The aforementioned <1> ~ <20> The method described in any one of the items. <22> A pair of oligonucleotides selected from the group consisting of oligonucleotide pair (1) and oligonucleotide pair (2), preferably oligonucleotide pair (1), At least one pair of oligonucleotide pairs selected from the group consisting of oligonucleotide pair (3), oligonucleotide pair (4), oligonucleotide pair (5), oligonucleotide pair (6), oligonucleotide pair (7), and oligonucleotide pair (8), and The aforementioned oligonucleotide pair (9), The above is used as a mixed primer pair by mixing the following: <21> Methods used. <23> When an amplification product is confirmed using at least one oligonucleotide pair selected from the group consisting of oligonucleotide pair (1) and oligonucleotide pair (2), the spore-forming bacteria contained in the sample are identified as Bacillus subtilis. When an amplification product is confirmed using at least one oligonucleotide pair selected from the group consisting of oligonucleotide pair (3), oligonucleotide pair (4), oligonucleotide pair (5), oligonucleotide pair (6), oligonucleotide pair (7), and oligonucleotide pair (8), the spore-forming bacteria contained in the sample are identified as Bacillus coagulans. When the amplification product is confirmed using the oligonucleotide pair (9), the spore-forming bacteria contained in the sample are identified as Bacillus cereus. The aforementioned <1> ~ <22> The method described in any one of the items. <24> The subject is a neutral food and beverage and the raw materials used therein, <1> ~ <23> The method described in any one of the items.

[0048] <25> A DNA kit comprising at least one oligonucleotide pair selected from the group consisting of oligonucleotide pair (1) consisting of oligonucleotide (a) and (c), oligonucleotide pair (2) consisting of oligonucleotide (b) and (c), oligonucleotide pair (3) consisting of oligonucleotide (d) and (j), oligonucleotide pair (4) consisting of oligonucleotide (e) and (j), oligonucleotide pair (5) consisting of oligonucleotide (f) and (j), oligonucleotide pair (6) consisting of oligonucleotide (g) and (j), oligonucleotide pair (7) consisting of oligonucleotide (h) and (j), oligonucleotide pair (8) consisting of oligonucleotide (i) and (j), and oligonucleotide pair (9) consisting of oligonucleotide (k) and (l). <26> An oligonucleotide pair (1) consisting of oligonucleotides (a) and (c), an oligonucleotide pair (2) consisting of oligonucleotides (b) and (c), an oligonucleotide pair (3) consisting of oligonucleotides (d) and (j), an oligonucleotide pair (4) consisting of oligonucleotides (e) and (j), an oligonucleotide pair (5) consisting of oligonucleotides (f) and (j), an oligonucleotide pair (6) consisting of oligonucleotides (g) and (j), an oligonucleotide pair (7) consisting of oligonucleotides (h) and (j), an oligonucleotide pair (8) consisting of oligonucleotides (i) and (j), and an oligonucleotide pair (9) consisting of oligonucleotides (k) and (l). <27> The oligonucleotide (a) is the <9> The oligonucleotide described above, The oligonucleotide (b) is the <10> The oligonucleotide described above, The oligonucleotide (c) is the <11> The oligonucleotide described above, The oligonucleotide (d) is the <12> The oligonucleotide described above, The oligonucleotide (e) is the <13> The oligonucleotide described above, The oligonucleotide (f) is the <14> The oligonucleotide described above, The oligonucleotide (g) is the <15> The oligonucleotide described above, The oligonucleotide (h) is the <16> The oligonucleotide described above, The oligonucleotide (i) is the <17> The oligonucleotide described above, The oligonucleotide (j) is the <18> The oligonucleotide described above, The oligonucleotide (k) is the <19> The oligonucleotide described above, The oligonucleotide (l) is the <20> The oligonucleotide described above, The aforementioned <25> or <26> The DNA kit or oligonucleotide pair described above. <28> The oligonucleotide pair (1) and the oligonucleotide pair (2) are oligonucleotide pairs that can be used for the detection of Bacillus subtilis group. The oligonucleotide pairs (3), (4), (5), (6), (7), and (8) are oligonucleotide pairs that can be used for the detection of Bacillus coagulans. The oligonucleotide pair (9) is an oligonucleotide pair that can be used for the detection of Bacillus cereus. The aforementioned <25> ~ <27> An oligonucleotide pair as described in any one of the items. [Examples]

[0049] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0050] Example 1: Multiplex PCR of 3 bacterial species (1) Primer design Based on the nucleotide sequence information of the DUF421-DUF1657 gene (SEQ ID NO: 13) from Bacillus subtilis B4146 strain, we designed primers consisting of oligonucleotides represented by the nucleotide sequence of SEQ ID NO: 1 (Primer 1) and primers represented by the nucleotide sequence of SEQ ID NO: 3 (Primer 3). In addition, based on the nucleotide sequence of the DUF421-DUF1657 gene (SEQ ID NO: 15) from Bacillus coagulans DSM1=ATCC7050 strain, we designed primers consisting of oligonucleotides represented by the nucleotide sequence of SEQ ID NO: 5 (Primer 5) and primers represented by the nucleotide sequence of SEQ ID NO: 10 (Primer 10). Furthermore, based on the nucleotide sequence of the DUF421-DUF1657 gene (SEQ ID NO: 16) from Bacillus cereus AH187 strain, we designed primers consisting of oligonucleotides represented by the nucleotide sequence of SEQ ID NO: 11 (Primer 11) and primers represented by the nucleotide sequence of SEQ ID NO: 12 (Primer 12). Based on the information of each designed primer, we obtained purified reverse-phase column-processed primers from FASMAC's DNA / RNA contract synthesis service. The nucleotide sequence information for the DUF421-DUF1657 genes of each bacterial species was obtained from the NCBI (National Center for Biotechnology Information). If each strain of the bacterial species possesses the DUF421-DUF1657 gene, the length of the DNA fragment amplified by the primer pair of primer 1 and primer 3 is approximately 350 bp, the length of the DNA fragment amplified by the primer pair of primer 5 and primer 10 is approximately 465 bp, and the length of the DNA fragment amplified by the primer pair of primer 11 and primer 12 is approximately 265 bp. (2) Preparation of the subject For the evaluation of each primer prepared as described above, we used the following strains of Bacillus subtilis: JCA1402, JCA1403, and JCM1465; Bacillus coagulans: NBRC12583, JCA1108, and DSM2314; and Bacillus cereus: JCM2152 and JCM17690, as listed in Table 1 below. The JCA strains were obtained from the Japan Canned, Bottled and Retort Foods Association; the JCM strains from the Microbial Materials Development Laboratory, RIKEN; the NBRC strains from the National Institute of Technology and Evaluation; and the DSM strains from the German Microbial Strain Preservation Institute, Leibniz Institute. Furthermore, Bacillus subtilis strain JCA1403, Bacillus coagulans strain NBRC12583, and Bacillus coagulans strain JCA1108 each possess the DUF421-DUF1657 gene, which is involved in improving spore heat resistance, on their genomes, while Bacillus cereus strain JCM17690 possesses it on its plasmid. Each bacterial strain was seeded on SCD agar medium (product name: SCD Medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the plates were left standing for 1-2 days to culture Bacillus subtilis and Bacillus cereus at 30°C, and Bacillus coagulans at 45°C. After culturing on agar medium, each colony was scraped off with a platinum loop and inoculated onto SCD liquid medium (product name: SCD Liquid Medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and cultured for 2-3 days under the same temperature conditions.

[0051] (3) Preparation of genomic DNA After culturing, 1.0 mL of suspension was collected from each SCD liquid medium. DNA was extracted from the cells using a genomic DNA preparation kit (product name: NucleoSpin Tissue, manufactured by Takara Bio Inc.), following the protocol provided with the kit, except that lysozyme lysis was used during cell lysis. Since this kit allows for the extraction of DNA including plasmids, the extracted DNA will be referred to as "extracted DNA" below. The concentration of the obtained extracted DNA solution was adjusted to 50 ng / μL.

[0052] (4) Multiplex PCR reaction Using the extracted DNA solution obtained above as a DNA template, a Multiplex PCR solution was prepared using the Multiplex PCR Assay Kit Ver.2 (product name, manufactured by Takara Bio Inc.). 0.25 μL of Multiplex PCR Enzyme Mix and 2x Multiplex PCR Buffer (Mg 2+ 25 μL of dNTP plus, 1 μL of extracted DNA solution (equivalent to 50 ng of extracted DNA), and primers 1, 3, 5, 10, 11, and 12 were added to a final concentration of 0.2 μM each. Sterile distilled water was then added to bring the total volume of the Multiplex PCR solution to 50 μL. A mixture of extracted DNA solutions from 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 (Thermo Fisher Scientific) was used as the thermal cycler. The reaction cycle consisted of (i) a thermal denaturation reaction at 94°C for 60 seconds, (ii) a thermal denaturation reaction at 94°C for 30 seconds, (iii) an annealing reaction at 60°C for 30 seconds, (iv) an extension reaction at 72°C for 30 seconds, and (v) an extension reaction at 72°C for 300 seconds. Cycles (ii) through (iv) constituted one cycle, and this was repeated 30 times.

[0053] (5) Electrophoresis and imaging After PCR, 2 μL of the PCR solution was taken and thoroughly mixed with loading buffer. Electrophoresis (100V, 30 minutes) was performed on a 2% agarose gel using Mupid-exU (Takara Bio Inc.). A 100 bp DNA Step Ladder (Promega) was used as the DNA marker. After electrophoresis, the agarose gel was stained with SYBR Safe (Thermo Fisher Scientific) for 30 minutes. The gel was then irradiated with 254 nm ultraviolet light using BioDoc-It Imaging Systems (UVP) with an exposure time of 3-5 seconds to check for the presence of amplified DNA fragments. The images of the agarose gel are shown in Figure 1. Note that numbers 1 to 10 in Figure 1 indicate that the reaction was performed using DNA extracted from the sample corresponding to the sample number listed in Table 1 below. The correspondence between each lane number in Figure 1 and the applied sample is shown in Table 1 below.

[0054] [Table 1]

[0055] As shown in Figure 1, when PCR was performed using Bacillus subtilis JCA1403, Bacillus coagulans NBRC12583 and JCA1108, and Bacillus cereus JCM17690, all possessing the DUF421-DUF1657 gene, DNA fragments of a size specific to each bacterial species were detected. Furthermore, even when a mixture of extracted DNA solutions from Bacillus subtilis JCA1403, Bacillus coagulans NBRC12583, and Bacillus cereus JCM17690 was used as a template, DNA fragments of a size specific to each bacterial species were detected. In contrast, when PCR was performed using Bacillus subtilis strains JCA1402, JCM1465, Bacillus coagulans strain DSM2314, and Bacillus cereus strain JCM2152 as subjects, no DNA fragments were detected because these strains do not possess the DUF421-DUF1657 gene. From the above, it has been shown that the method of the present invention can detect the DUF421-DUF1657 gene in a species-specific manner, even when the sample is a mixture of different bacterial species.

[0056] Example 2: Multiplex PCR of 3 bacterial species (1) Primer design Based on the nucleotide sequence information of the DUF421-DUF1657 gene (SEQ ID NO: 13) derived from Bacillus subtilis B4146 strain, a primer (Primer 2) consisting of oligonucleotides represented by the nucleotide sequence of SEQ ID NO: 2 was designed. If the Bacillus subtilis B4146 strain possesses 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 approximately 55 bp.

[0057] Furthermore, based on the nucleotide sequence of the DUF421-DUF1657 gene (SEQ ID NO: 15) derived from Bacillus coagulans DSM1=ATCC7050 strain, primers were designed to pair with primer 10, which is represented by the nucleotide sequence of SEQ ID NO: 10. These primers consist of oligonucleotides represented by the nucleotide sequence of SEQ ID NO: 4 (Primer 4), SEQ ID NO: 6 (Primer 6), SEQ ID NO: 7 (Primer 7), SEQ ID NO: 8 (Primer 8), and SEQ ID NO: 9 (Primer 9). Based on the information of each designed primer, primers were obtained as reverse-phase column purified products from FASMAC's DNA / RNA contract synthesis service. When Bacillus coagulans DSM1=ATCC7050 strain possesses the DUF421-DUF1657 gene, the length of the DNA fragment amplified by the primer pair of primer 4 and primer 10 is approximately 490 bp, the length of the DNA fragment amplified by the primer pair of primer 6 and primer 10 is approximately 155 bp, the length of the DNA fragment amplified by the primer pair of primer 7 and primer 10 is approximately 135 bp, the length of the DNA fragment amplified by the primer pair of primer 8 and primer 10 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.

[0058] (2) Multiplex PCR reaction, electrophoresis and imaging For the Multiplex PCR, the extracted DNA solutions obtained in Example 1—from Bacillus subtilis JCA1403 strain, Bacillus coagulans NBRC12583 strain, and Bacillus cereus JCM17690 strain—were used as templates. Multiplex PCR was performed using the primer pair combinations shown in Table 2 below, under the conditions described in Example 1, and images of the agarose gel were obtained after electrophoresis. The images are shown in Figures 2 and 3. Note that lanes 11 and 18 are marker 100bp DNA Step Ladder (Promega).

[0059] [Table 2]

[0060] In Figures 2 and 3, left-pointing black triangles indicate amplification products specific to the DUF421-DUF1657 gene from the Bacillus subtilis group, asterisks indicate amplification products specific to the DUF421-DUF1657 gene from Bacillus coagulans, and left-pointing arrowheads indicate plasmid-specific amplification products specific to the DUF421-DUF1657 gene from Bacillus cereus. As is clear from Figures 2 and 3, it was shown that regardless of the primer pair combination, the DUF421-DUF1657 gene could be detected in a species-specific manner in a sample containing mixed DNA from different bacterial species.

[0061] Example 3: Spore heat resistance evaluation test 1 (1) Sample preparation, PCR The following strains of Bacillus subtilis were used: JCA strains (Japan Canned, Bottled and Retort Foods Association) (JCA1402, JCA1403, JCA1404, JCA1405, JCA1407, JCA1408, JCA1409, JCA1410, JCA1411), JCM strains (RIKEN Microbial Materials Development Laboratory) (JCM1465), KM strains (environmentally isolated strains) (KM166, KM167), and 168. Each of the above bacterial 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. A result was defined as "positive" if a DNA fragment was detected at the target location, and as "negative" if a DNA fragment was not detected at the target location. The results, along with the presence or absence of the DUF421-DUF1657 gene in each bacterial strain, are shown in Table 3 below.

[0062] (2) Simple heat resistance test The above bacterial strains were cultured for 1 week at an appropriate temperature on 1 L of DSM agar medium (prepared with 400 mL of 0.8% Nutrient Broth (Difco), 0.1% KCl, 0.012% MgSO4·7H2O, and 5% Agar, with 200 μL of 1N NaOH added to adjust the pH to 7.0. After autoclaving, 0.4 mL of 1 M Ca(NO3)2, 0.4 mL of 0.01 MnCl2, and 0.4 mL of 1 mM FeSO4 were added). Spore formation was confirmed using a phase-contrast microscope, and the spores were collected with ice-cold sterile water. The collected material was centrifuged at 8000 × g, 4°C, and for 20 minutes, and the supernatant was removed and washed again with ice-cold sterile water a total of 5 times. The recovered material (precipitate) after washing was suspended in ice-cold sterile water to a volume of approximately 3 mL per culture plate, then dispensed into smaller portions and stored frozen at -20°C until use. The number of spores (number of spores per 1 mL of suspension) was measured by heating the spores at 80°C for 10 minutes, followed by serial dilution of the spore solution, which was then spread onto SCD agar medium (product name: SCD Agar Medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The obtained spores are placed in 10 mL of SCD liquid medium (product name: SCD liquid medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and filled to 106 ~10 7 The solution was added to a concentration of spores / mL, heated at 90°C, 95°C, or 100°C for 30 minutes, or at 105°C for 10 minutes, and then incubated at 30°C for 7 days. Heat resistance was determined by observing the growth of the bacterial strain after incubation, based on the evaluation criteria below. This test was repeated three times for each bacterial strain, and if death was observed in even one test, it was determined that the strain had "died upon heating." Whether or not a bacterial strain had died was determined as follows: if no turbidity was observed visually after incubation, it was determined that the strain had "died upon heating," and if turbidity was observed visually, it was determined that the strain "did not die upon heating." 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, but killed by heating at 100°C for 30 minutes. Level 4: Not killed by heating at 100°C for 30 minutes, but killed by heating at 105°C for 10 minutes. Level 5: Not killed even after heating at 105°C for 10 minutes.

[0063] (3) Heat resistance test (D value) The above-mentioned strains of Bacillus subtilis were entrusted to the Japan Canned, Bottled and Retort Foods Association, and the D-value (112.5°C) was measured using the following method. The heat resistance of each test strain's spores in M / 15 phosphate buffer (pH 7.0) was measured. The test method involved serially diluting the spore solution of each test strain with sterile 0.1% peptone water, and then adding approximately 10 units of the solution to M / 15 phosphate buffer and each test solution. 5 The solution was suspended to a CFU / mL concentration, mixed, and then dispensed into TDT tubes and sealed. These TDT tubes were heat-treated under specified conditions. After heating, the TDT tubes were opened, the number of spores was measured, and the D value was calculated. The results are shown in Table 3 below.

[0064] [Table 3]

[0065] As is clear from Table 3, the Bacillus subtilis strains that possess the DUF421-DUF1657 gene and were determined to be "positive" by Multiplex PCR all had a heat resistance level of level 5 in the simplified heat resistance test, and the D of the Bacillus subtilis JCA1403 strain that was determined to be "positive" 112.5℃ The value was 1.18 min. In contrast, Bacillus subtilis strains that did not possess the DUF421-DUF1657 gene and were judged "negative" by Multiplex PCR all had a heat resistance level of level 2 in the simplified heat resistance test, and the D of Bacillus subtilis strains JCA1402 and JCM1465, which were judged "negative", was also 2. 112.5℃ The values ​​were 0.09 minutes and 0.08 minutes, respectively. Therefore, it became clear that there is a correlation between the results of Multiplex PCR and heat resistance. Furthermore, based on the above results, if the Multiplex PCR result is positive, heating at 105°C for 10 minutes is sufficient (10 6 ~10 7 If the spores (10 / mL) are not killed and the Multiplex PCR result is negative, heating at 95°C for 30 minutes is sufficient. 6 ~10 7 It was shown that it can kill spores (spores / mL).

[0066] Example 4: Spore heat resistance evaluation test 2 (1) Sample preparation, PCR The following strains of Bacillus coagulans were used: NBRC strain (National Institute of Technology and Evaluation) (NBRC12583), JCA strain (Japan Canned, Bottled and Retort Foods Association) (JCA1108, JCA1109, JCA1116, JCA1117, JCA1120, JCA1122, JCA1158, JCA1174, JCA1180, JCA1182), and DSM strain (Leibniz Institute German Microbial Strain Preservation Service) (DSM2308, DSM2311, DSM2312, DSM2314, DSM2350, DSM2356, DSM2383, DSM2384, DSM2385). Each of the above bacterial 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. A result was defined as "positive" if a DNA fragment was detected at the target location, and as "negative" if a DNA fragment was not detected at the target location. The results, along with the presence or absence of the DUF421-DUF1657 gene in each bacterial strain, are shown in Table 4 below.

[0067] (2) Heat resistance test (D value) The above-mentioned Bacillus coagulans strains were commissioned to the Japan Canned, Bottled and Retort Food Association, and the D-value was measured using the same method as in Example 3. The results are shown in Table 4 below. Furthermore, Figure 4 shows a box plot of the results from Table 4 below.

[0068] [Table 4]

[0069] From Table 4 and Figure 4 above, all Bacillus coagulans strains possessing the DUF421-DUF1657 gene and determined to be "positive" by Multiplex PCR were classified as D 105℃ The value was 10 minutes or more. Also, in Bacillus coagulans strains that did not possess the DUF421-DUF1657 gene and were judged "negative" by Multiplex PCR, all were D 105℃ The value was less than 10 minutes. Thus, a correlation was observed between the results of Multiplex PCR and heat resistance (D value), and if the Multiplex PCR result was positive, D 105℃ If the value remains negative for 10 minutes or more, the result is D. 105℃ It was shown that the value could be evaluated as less than 10 minutes.

[0070] Example 5: Spore heat resistance evaluation test 3 (1) Sample preparation, PCR The Bacillus cereus strains used were JCM strains (Microbial Materials Development Laboratory, RIKEN) (JCM2152 strain, JCM17690 strain). Each of the above bacterial 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. A result was defined as "positive" if a DNA fragment was detected at the target location, and as "negative" if a DNA fragment was not detected at the target location. The results, along with the presence or absence of plasmid-specific DUF421-DUF1657 genes in each bacterial strain, are shown in Table 5 below.

[0071] (2) Heat resistance test (D value) The above Bacillus cereus strains were commissioned to the Japan Canned, Bottled and Retort Food Association, and the D-value was measured using the same method as in Example 3. The results, along with the presence or absence of plasmid-specific DUF421-DUF1657 genes in each strain, are shown in Table 5 below.

[0072] [Table 5]

[0073] From Table 5 above, among the Bacillus cereus strains that tested positive by Multiplex PCR, D 90℃ The value was 82.9 minutes. In addition, in Bacillus cereus strains that were determined to be negative by Multiplex PCR, D 90℃ The value was 24.3 minutes. Thus, a correlation was observed between the results of Multiplex PCR and heat resistance (D value).

[0074] Example 6: High-throughput analysis of raw material isolates Sample preparation Species identification was performed on various bacterial species isolated from commercially available and raw materials purchased from raw material manufacturers (ID: 1-30) using the MicroSEQ 500 16S rDNA Sequencing Kit (Thermo Fisher Scientific). Strains identified as Bacillus subtilis, Bacillus coagulans, and Bacillus cereus were included in the analysis. Each 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. A result was defined as "positive" if a DNA fragment was detected at the target location, and "negative" if a DNA fragment was not detected at the target location. The results of the Multiplex PCR are shown in Table 6 below.

[0075] [Table 6]

[0076] As is clear from Table 6, the target DNA band was not detected by Multiplex PCR in any of the Bacillus subtilis, Bacillus coagulans, or Bacillus cereus strains with raw material IDs 1-29. On the other hand, the target DNA band was detected by Multiplex PCR in the Bacillus subtilis strain with raw material ID 30. Therefore, it was shown that the raw material with raw material ID 30 contains a Bacillus subtilis strain that forms highly heat-resistant spores. Thus, the present invention demonstrates that spore-forming bacteria that form highly heat-resistant spores can be rapidly identified from a large amount of sample.

[0077] Example 7: Proposed sensitivity and heat treatment conditions for comprehensive testing of heat-resistant spore-forming bacteria in raw materials. (1) Preparation of high heat-resistant spore-attached raw materials To 10g of raw material ID:28 used in Example 6, spores of Bacillus subtilis JCA1403 strain (a strain that forms highly heat-resistant spores), whose vegetative cells were killed by heat treatment at 80°C for 10 minutes, were added at a rate of 5.0 × 10 per gram of raw material. 7 , 5.0×10 5 , 5.0×10 3 , 5.0×10 1 , 5.0×10 -1 The spores were attached to the raw material to form spores. As a control, raw material without attached spores was used. The raw material was then left to stand until it was completely dry to prepare raw material with artificially attached high-heat-resistant spores.

[0078] (2) Bacterial body collection process 90 g of physiological saline was added to the above raw material, and the mixture was crushed and homogenized using a stomacher. A 30 mL suspension was collected. Then, the raw material residue was discarded after centrifugation at 100 × g for 5 minutes, and the precipitated bacterial cells were collected after further centrifugation at 8000 × g for 20 minutes. The collected bacterial cells were resuspended in 3 mL of physiological saline.

[0079] (3) Heating process and observation of spoilage 10 μL or 1 μL of the recovered bacterial suspension was inoculated into 10 mL of a neutral tea-based beverage (pH: 6.35) in which Bacillus subtilis JCA1403 can grow, and heat-treated at 100°C for 5 minutes. After heat treatment, the beverage was incubated at 30°C for 7 days, and the beverage was visually observed to determine if it had spoiled. Three repeated tests were performed, and if even one non-growth (no spoilage of the beverage) was observed, the bacteria were considered dead. Note that these heating conditions are extremely mild compared to the heat treatment used when manufacturing normal neutral tea-based beverages and are below the standards of the Food Sanitation Act, therefore they are merely conditions for a model experiment. In this test, "product spoilage" specifically refers to a state in which turbidity can be visually confirmed or growth of spore-forming bacteria can be observed, or a state in which turbidity can be visually confirmed or growth of spore-forming bacteria can be observed after adding 100 μL of the beverage to SCD agar medium (product name: SCD Liquid Medium "Daigo", for general bacterial testing, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and culturing at 30°C for 2 days.

[0080] (4) Multiplex PCR test Furthermore, 10 μL or 1 μL of the bacterial suspension recovered 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 saturation was collected, and an extracted DNA solution was prepared in the same manner as in Example 1. Subsequently, the DUF421-DUF1657 gene was detected by Multiplex PCR in the same manner as in Example 1.

[0081] (5) Results The results of Multiplex PCR and the spoilage of the beverage after heat treatment are shown in Table 7 below. In the table, "Estimated spore count (spores / ml)" indicates the estimated spore count in the neutral tea beverage and SCD liquid medium in the above heating process and Multiplex PCR test.

[0082] [Table 7]

[0083] As is clear from Table 7, when a Multiplex PCR test was positive for samples with a high number of spores per gram of raw material, spoilage was observed in the corresponding neutral tea beverages that were heat-treated at 100°C for 5 minutes and then incubated at 30°C for 7 days. In contrast, when a Multiplex PCR test was negative for samples with a low number of spores per gram of raw material, no spoilage was observed in the corresponding neutral tea beverages. Furthermore, even when a Multiplex PCR test was positive for a sample with a slightly higher spore count per gram of raw material, no spoilage was observed in the corresponding neutral tea-based beverage. This demonstrates that even a small amount of spore contamination, which would not cause product spoilage under these conditions (heat treatment at 100°C for 5 minutes followed by static incubation at 30°C for 7 days), can be detected with high sensitivity using the method of the present invention.

[0084] The results above demonstrate that the method of the present invention allows for highly sensitive prediction of spoilage of food and beverages by detecting spore-forming bacteria that form highly heat-resistant spores in food and beverages. Furthermore, the method of the present invention allows for the rapid and simple determination of appropriate heat treatment conditions for food and beverages by detecting spore-forming bacteria that form highly heat-resistant spores in food and beverages.

[0085] From the results above, it is possible to confirm the presence or absence of the DUF421-DUF1657 gene in spore-forming bacteria that can grow under neutral conditions by using the method of the present invention, and to detect spore-forming bacteria using the amplification product. Furthermore, by confirming the presence or absence of the DUF421-DUF1657 gene, the heat resistance of spore-forming bacteria can be evaluated, and appropriate heat treatment conditions can be determined for raw materials in which spore-forming bacteria are detected.

Claims

1. 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); An oligonucleotide pair 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; Based on the presence or absence of an amplification product, spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions are detected. 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 oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:1 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (b) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 2 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (c) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 3 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (d) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 4 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (e) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:5, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:5 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (f) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:6, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:6 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (g) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 7 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (h) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 8 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:9, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:9 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (j) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:10, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:10 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (k) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:11, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:11 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has a plasmid-specific DUF421-DUF1657 gene. (l) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:12, or an oligonucleotide which has an identity of 80% or more with the base sequence represented by SEQ ID NO:12 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has a plasmid-specific DUF421-DUF1657 gene.

2. The method of claim 1 , wherein the amplification product identifies the spore-forming bacterium.

3. The method according to claim 1 , wherein the spore heat resistance of the spore-forming bacteria is evaluated based on the presence or absence of the amplification product.

4. 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); An oligonucleotide pair 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; Based on the presence or absence of amplification products, the spore heat resistance of spore-forming bacteria belonging to the genus Bacillus that can grow under neutral conditions is evaluated. A method for evaluating the heat resistance of spores of spore-forming bacteria belonging to the genus Bacillus. (a) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:1, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:1 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (b) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 2 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (c) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 3 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (d) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 4 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (e) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:5, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:5 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (f) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:6, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:6 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (g) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 7 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (h) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 8 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:9, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:9 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (j) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:10, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:10 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (k) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:11, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:11 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has a plasmid-specific DUF421-DUF1657 gene. (l) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:12, or an oligonucleotide which has an identity of 80% or more with the base sequence represented by SEQ ID NO:12 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has a plasmid-specific DUF421-DUF1657 gene.

5. A method for determining heat treatment conditions for a spore-forming bacterium belonging to the genus Bacillus, comprising determining heat treatment conditions for the spore-forming bacterium based on the spore heat resistance evaluated by the method of claim 3 or 4.

6. 2. The method according to claim 1, wherein the spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions is at least one species selected from the group consisting of Bacillus subtilis group, Bacillus coagulans, and Bacillus cereus.

7. amplifying a nucleic acid represented by a partial base sequence of DUF421-DUF1657 gene on a genome by a polymerase chain reaction using at least one oligonucleotide pair selected from the group consisting of 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 (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 (h) and (j), and an oligonucleotide pair consisting of the oligonucleotides (i) and (j), amplifying a nucleic acid represented by a partial base sequence of the plasmid-specific DUF421-DUF1657 gene by a polymerase chain reaction method using an oligonucleotide pair consisting of the oligonucleotides (k) and (l) as a primer pair; The method of claim 1.

8. an oligonucleotide pair consisting of the oligonucleotides (a) and (c); An oligonucleotide pair selected from the group consisting of 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 (g) and (j), an oligonucleotide pair consisting of the oligonucleotides (h) and (j), and an oligonucleotide pair consisting of the oligonucleotides (i) and (j), An oligonucleotide pair consisting of the oligonucleotides (k) and (l); The method according to claim 7, wherein the above primers are mixed and used as a mixed primer pair.

9. When an amplification product is confirmed using at least one oligonucleotide pair selected from the group consisting of the oligonucleotide pair consisting of the oligonucleotide pair (a) and (c) and the oligonucleotide pair consisting of the oligonucleotide pair (b) and (c), the spore-forming bacterium is identified as a Bacillus subtilis group bacterium; When an amplification product is confirmed using at least one oligonucleotide pair selected from the group consisting of the oligonucleotide pair consisting of the oligonucleotide pair (d) and (j), the oligonucleotide pair consisting of the oligonucleotide pair (e) and (j), the oligonucleotide pair consisting of the oligonucleotide pair (f) and (j), the oligonucleotide pair consisting of the oligonucleotide pair (g) and (j), the oligonucleotide pair consisting of the oligonucleotide pair (h) and (j), and the oligonucleotide pair consisting of the oligonucleotide pair (i) and (j), the spore-forming bacterium is identified 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 of claim 1.

10. 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); An oligonucleotide pair consisting of the following oligonucleotides (k) and (l): A DNA kit comprising at least one oligonucleotide pair selected from the group consisting of: (a) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:1, or an oligonucleotide which has an identity of 80% or more with the base sequence represented by SEQ ID NO:1 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (b) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 2 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (c) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 3 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (d) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 4 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (e) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:5, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:5 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (f) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:6, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:6 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (g) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 7 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (h) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 8 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:9, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:9 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (j) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:10, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:10 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (k) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:11, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:11 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has a plasmid-specific DUF421-DUF1657 gene. (l) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:12, or an oligonucleotide which has an identity of 80% or more with the base sequence represented by SEQ ID NO:12 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has a plasmid-specific DUF421-DUF1657 gene.

11. The bacterium belonging to the genus Bacillus is selected from the group consisting of Bacillus subtilis, Bacillus cereus, and Bacillus coagulans. The DNA kit according to claim 10.

12. the oligonucleotide pair consisting of the oligonucleotides (a) and (c) and the oligonucleotide pair consisting of the oligonucleotides (b) and (c) are oligonucleotide pairs usable for detecting Bacillus subtilis group; the oligonucleotide pair consisting of the oligonucleotides (d) and (j), the oligonucleotide pair consisting of the oligonucleotides (e) and (j), the oligonucleotide pair consisting of the oligonucleotides (f) and (j), the oligonucleotide pair consisting of the oligonucleotides (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 oligonucleotide pairs usable for detecting Bacillus coagulans; The oligonucleotide pair consisting of the oligonucleotides (k) and (l) is an oligonucleotide pair that can be used for detecting Bacillus cereus. The DNA kit according to claim 10 or 11.

13. 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); An oligonucleotide pair consisting of the following oligonucleotides (k) and (l): (a) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:1, or an oligonucleotide which has an identity of 80% or more with the base sequence represented by SEQ ID NO:1 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (b) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 2 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (c) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 3 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (d) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 4 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (e) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:5, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:5 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (f) an oligonucleotide consisting of the base sequence represented by SEQ ID NO:6, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:6 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (g) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 7 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (h) an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO: 8 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (i) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:9, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:9 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions, which has the DUF421-DUF1657 genes. (j) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:10, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:10 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has the DUF421-DUF1657 genes. (k) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:11, or an oligonucleotide which has an identity of 80% or more to the base sequence represented by SEQ ID NO:11 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has a plasmid-specific DUF421-DUF1657 gene. (l) An oligonucleotide consisting of the base sequence represented by SEQ ID NO:12, or an oligonucleotide which has an identity of 80% or more with the base sequence represented by SEQ ID NO:12 and can be used for detecting a spore-forming bacterium belonging to the genus Bacillus capable of growing under neutral conditions and which has a plasmid-specific DUF421-DUF1657 gene.

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