JP7685202B2 - Oligonucleotides for detecting target nucleic acids and uses thereof - Google Patents

Description

The present invention relates to an oligonucleotide for detecting a target nucleic acid and its use.

The causes of spoilage and deterioration of food during long-term storage are microorganisms such as bacteria and fungi that remain in food. Food processing technologies, mainly heat treatment, have been developed with the aim of inactivating these microorganisms. Furthermore, in recent years, medium-high pressure processing technology and vacuum packaging technology have been developed, which have made it possible to significantly extend food storage periods. However, microbial contamination has also been reported in processed foods using these technologies, and microorganisms collectively known as heat-resistant bacteria are believed to be the cause of contamination. Among heat-resistant bacteria, the genera Byssochlamys, Neosartoria, and Eurotium are types of fungi known as heat-resistant fungi, and cases of contamination have been reported both domestically and overseas.

As a method for detecting heat-resistant fungi, a method has been proposed that uses primers specific to the target organism, designed from the β-tubulin gene and ITS region of each fungus (e.g., Patent Documents 1 to 6).

JP 2010-4880 A JP 2010-4876 A JP 2013-34447 A JP 2010-4879 A JP 2009-284832 A International Publication No. 2012/169099

Conventional detection methods mainly use the coding region of the β-tubulin gene and the ITS region. Sequence information of the gene region is registered for many fungi, so it is suitable for comprehensive detection of fungi. On the other hand, the gene is conserved in a wide range of biological species and has a highly conserved sequence. Therefore, strict primer design is required to specifically detect microorganisms classified into the same genus. In general, differences of about one base may be ignored in the formation of complementary strands of DNA, so when performing the PCR method, the amplification reaction may proceed by complementing a sequence similar to the base sequence of the primer set. In order to suppress such non-specific amplification reactions, a region in which a continuous base sequence specific to the organism to be detected is usually set as a primer. In this case, although the detection accuracy is improved, comprehensiveness by genus is lost and the detection ability for unknown samples is reduced. In conventional techniques, primer sets made only from normal DNA are used, and it was difficult to design primers that combine specificity and comprehensiveness. In addition, conventional techniques may not detect some heat-resistant fungi isolated in Japan, and there is a risk that the occurrence of contamination by heat-resistant fungi cannot be sufficiently suppressed. Thus, currently, methods for detecting heat-resistant fungi in foods, such as long-term storage foods, have not been established or disseminated, and measures are taken after the fact when heat-resistant fungal contamination occurs. Therefore, in order to prevent food contamination with heat-resistant fungi after processing and distribution, there has been a demand for the establishment of testing technology that can detect the causative bacteria in advance.

The object of the present invention is to provide a means for specifically detecting a target nucleic acid.

The present invention provides the following:
[1] A nucleic acid having a base sequence complementary to a portion of a target nucleic acid that contains at least two different bases compared to a corresponding sequence of a non-target nucleic acid;
having at least one modified base,
Oligonucleotides for detecting target nucleic acids.
[2] The oligonucleotide according to [1], wherein the distance between the base complementary to the at least two different bases and the modified base is 0 to 15 bases.
[3] The oligonucleotide according to [1] or [2], wherein the distance between the 3'-terminal base of the oligonucleotide and the modified base is 0 to 10 bases.
[4] The oligonucleotide according to any one of [1] to [3], wherein the modified base is LNA or a BNA other than LNA.
[5] The oligonucleotide according to any one of [1] to [4], wherein the modified base is a modified adenine or guanine base.
[6] The oligonucleotide according to any one of [1] to [5], wherein the target nucleic acid is at least a partial base sequence of a gene of a fungus belonging to a single genus of thermotolerant fungi.
[7] The oligonucleotide according to any one of [1] to [6], wherein the target nucleic acid is at least a part of a gene of at least one bacterium selected from the genera Byssoclamis, Neosartoria, and Eurotium.
[8] The oligonucleotide according to any one of [1] to [7], wherein the target nucleic acid is a nucleic acid containing at least a part of a nucleotide sequence of a thermotolerant fungus gene, which corresponds to the portion of nucleotide numbers 352 to 569 or 352 to 703 of the nucleotide sequence of SEQ ID NO: 1, or the portion of nucleotide numbers 52 to 545 of the nucleotide sequence of SEQ ID NO: 26.
[9] The target nucleic acid is at least a part of a β-tubulin gene of a thermotolerant fungus;
When PCR is performed on a thermotolerant fungus using an oligonucleotide containing the base sequence of SEQ ID NO: 20 as a primer, an amplification product of 100 to 600 bases is obtained.
The oligonucleotide according to any one of [1] to [8].
[10] The oligonucleotide according to any one of [1] to [9], wherein the oligonucleotide is any one of the following:
(1) An oligonucleotide in which the base at base number 18 in the base sequence of SEQ ID NO: 2 is a modified base and which contains at least the base portion of base numbers 6 to 18;
(2) An oligonucleotide in which the base at base number 25 in the base sequence of SEQ ID NO: 9 is a modified base and which contains at least the base portion of base numbers 20 to 25;
(3) An oligonucleotide in which the base at base number 24 in the base sequence of SEQ ID NO:14 is a modified base and which contains at least the base portion of base numbers 20 to 24.
[11] The target nucleic acid is at least a part of an ITS region of a thermotolerant fungus;
When PCR is performed on a thermotolerant fungus using an oligonucleotide containing the base sequence of SEQ ID NO: 28 as a primer, an amplification product of 100 to 600 bases is obtained.
The oligonucleotide according to any one of [1] to [8].
[12] The oligonucleotide according to any one of [1] to [11], wherein the oligonucleotide is an oligonucleotide in which the base at base number 16 in the base sequence of SEQ ID NO:27 is a modified base and which contains at least base numbers 13 to 19.
[13] The oligonucleotide according to any one of [1] to [12], which is a primer for amplifying a target nucleic acid.
[14] The oligonucleotide according to [13], which is any one of the oligonucleotides (1) to (3) and is a reverse primer.
[15] The oligonucleotide according to [13], which is the oligonucleotide (4) and is a forward primer.
[16] A primer set for detecting a target nucleic acid, comprising the oligonucleotide according to any one of [13] to [15].
[17] The primer set according to [16], comprising the oligonucleotide according to [14] and a forward primer, wherein the forward primer is an oligonucleotide having at least a portion of base portion 1 to 544 of the base sequence of SEQ ID NO: 1 or a sequence having 90% or more identity thereto.
[18] The primer set according to [17], wherein the forward primer is an oligonucleotide comprising at least the base sequence of SEQ ID NO: 20.
[19] The primer set according to [16], comprising the oligonucleotide according to [15] and a reverse primer, wherein the reverse primer is an oligonucleotide having a sequence complementary to at least a portion of the portion of base numbers 73 to 1108 of the base sequence of SEQ ID NO:26 or a sequence having 90% or more identity thereto.
[20] The primer set according to [19], wherein the reverse primer is an oligonucleotide comprising at least the base sequence of SEQ ID NO:28.
[21] A method for detecting a target nucleic acid that may be contained in a sample, using the oligonucleotide according to any one of [1] to [15], or the primer set according to any one of [16] to [19].
[22] The detection method according to [21], comprising a step of amplifying a target nucleic acid in a sample by PCR using the oligonucleotide according to any one of [1] to [15], or the primer set according to any one of [16] to [19].
[23] A method for detecting a heat-resistant fungus that may be contained in a sample, comprising using the oligonucleotide according to any one of [1] to [15], or the primer set according to any one of [16] to [19].
[24] The detection method described in [23], wherein the heat-resistant fungus is at least one fungus selected from the genera Byssoclamis, Neosartoria, and Eurotium.
[25] The detection method described in [23] or [24], wherein the sample is a long-term storage food that has undergone at least one of heat treatment, high pressure treatment, and vacuum packaging treatment during the manufacturing process.
[26] The detection method according to any one of [23] to [25], wherein the sample is a canned, bottled, or retort food.
[27] The detection method according to any one of [23] to [26], wherein the detection is carried out immediately after production.
[28] A manufacturing process for long-term storage foods that involves at least one of heat treatment, high pressure treatment, and vacuum packaging treatment; and
A method for producing a long-term storage food, comprising a detection step of detecting a heat-resistant fungus using the detection method according to any one of [23] to [27] after the completion of the production process and before shipping.

The present invention provides oligonucleotides capable of specifically detecting target nucleic acids and uses thereof. The present invention can be widely used in the fields of food, medicine, and research. In particular, the use of the present invention enables simple and highly sensitive detection of target nucleic acids, particularly heat-resistant fungi. By carrying out tests based on the detection method of the present invention during food production, it is expected that food contamination by heat-resistant fungi after processing and distribution can be prevented in advance.

FIG. 1 shows the difference between LNA and DNA. 2 shows an alignment of the nucleotide sequence amplified with oligonucleotide (1) with the corresponding portions of the nucleotide sequences of the β-tubulin genes of Byssochlamys sp. and P. variotii. 3 shows an alignment of the nucleotide sequence amplified with oligonucleotide (2) with the corresponding portions of the nucleotide sequences of the β-tubulin genes of Neosartoria sp. and A. fumigatus. 4 shows an alignment of the nucleotide sequence amplified with oligonucleotide (3) with the corresponding portions of the nucleotide sequences of the β-tubulin genes of Neosartoria sp. and A. fumigatus. 5 is an electrophoretic diagram showing the results of Example 1. The upper, middle and lower rows show the results for primer sets 1, 2 and 3, respectively. 6 is an electrophoretic diagram showing the results of Example 2(2). The upper, middle, and lower rows in the left column show the results for primer sets 1, 2, and 3, and the upper and lower rows in the right column show the results for primer sets 4 and 5. 7 is an electrophoretic diagram showing the results of Example 2(3). The upper, middle, and lower rows show the results for primer sets 1, 4, and 5, respectively. 8 is an explanatory diagram showing the correspondence between the base sequence of SEQ ID NO:1 and preferred embodiments of the oligonucleotides of the present invention. The boxed areas indicate the design positions of primers Bt2a (corresponding to bases 352-375 of SEQ ID NO:1) and Bt2b (corresponding to bases 825-848 of SEQ ID NO:1). The italicized areas (corresponding to bases 545-569 of SEQ ID NO:1) and the shaded areas (corresponding to bases 681-703 of SEQ ID NO:1) indicate the design positions of the oligonucleotides (for Neosartoria and Bysochlamys), respectively. The double underlines indicate the amino acid coding regions (ORFs). FIG. 9 shows an alignment of the nucleotide sequence of oligonucleotide (4) with the corresponding portion of the ITS region of Eurotium sp. FIG. 10 is an electrophoretic diagram showing the results of Example 3(2). 11 is an electrophoretic diagram showing the results of Example 3(3). The upper row shows the results when the Eur-F1 primer was used, and the lower row shows the results when the Eur-F1L primer was used. 12 is an explanatory diagram showing the correspondence between the base sequence of SEQ ID NO:25 and a preferred embodiment of the oligonucleotide of the present invention. The boxed portion is the portion on which primer Eur-FIL (SEQ ID NO:27) was designed (positions 2396-2417 of SEQ ID NO:25). The underlined portion is the portion corresponding to primer ITS4 (positions 2517-2539 of SEQ ID NO:25). 13 is an explanatory diagram showing the correspondence between the base sequence of SEQ ID NO:26 and a preferred embodiment of the oligonucleotide of the present invention. The boxed portion is the portion on which primer Eur-FIL (SEQ ID NO:27) was designed (bases 52-72 of SEQ ID NO:26). The underlined portion is the portion corresponding to primer ITS4 (corresponding to the complementary sequence of bases 526-545 of SEQ ID NO:26).

[Oligonucleotides for detecting target nucleic acids]
The oligonucleotide for detecting a target nucleic acid is an oligonucleotide having a base sequence complementary to a portion of the base sequence of a target nucleic acid and having at least one modified base.

(Target nucleic acid, heat-resistant fungus)
The target nucleic acid that can be detected by the oligonucleotide is not particularly limited, but is preferably a base sequence of a gene of a group of bacteria belonging to a single genus, and more preferably a base sequence of a gene of a group of bacteria belonging to a single genus selected from fungi, especially thermotolerant fungi. This allows heat-resistant fungi that have high homology between genera and have been difficult to classify to each genus. In this specification, a heat-resistant fungus is a fungus that forms spores such as ascospores and has resistance to heat treatment and high pressure treatment. According to a general classification system, heat-resistant fungi are said to belong to a taxonomic group called ascomycetes, but are not limited to this. When it is confirmed that a sexual spore called an ascospore is produced, it is named as a perfect generation, and heat-resistant fungi can be classified based on the presence or absence of a perfect generation and differences in phenotype, etc. However, this classification system was established before the development of genetic analysis technology, and there are taxonomic groups that do not have a perfect generation (sexual generation): improper generation (asexual generation) = 1:1 relationship, so although it is a guideline for the definition of heat-resistant fungi, it is not limited to this classification.
Heat-resistant fungi include bacteria that cause contamination of long-term storage foods that undergo at least one of heat treatment, high-pressure treatment, and vacuum packaging during the manufacturing process, and are generally considered to be different from normal fungi and not inactivated by heat treatment (e.g., 70°C, 10 minutes) (surviving even after heat treatment). Examples of the aforementioned long-term storage foods include canned foods, bottled foods, and retort foods. Examples of heat-resistant fungi include the genus Byssochlamys, Neosartoria, Aspergillus, Paecilomyces, Talaromyces, Hamigera, Thermoascus, Eupenicillium, and Eurotium. Byssochlamys and Thermoascus are one of the groups of Paecilomyces that have a perfect stage. Some perfect stages of Paecilomyces are also classified as Talaromyces. Neosartoria is one of the groups of Aspergillus that have a teleomorph. Eurotium is also a teleomorph of Aspergillus.

Examples of the Byssochlamys genus include Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys zollerniae, Byssochlamys lagunculariae, Byssochlamys nivea, and Byssochlamys sp. Examples of the Neosartoria genus include Neosartoria fennelliae and Neosartoria fischeri var. Examples of the fungi of the genus Aspergillus include Aspergillus fumigatus. Examples of the fungi of the genus Paecilomyces include Paecilomyces variotii. Examples of the fungi of the genus Aspergillus include Neosartoria fischeri, Neosartoria hiratsukae, Neosartoria pseudofischeri, Neosartoria udagawae, Neosartoria primulina, Neosartoria quadricincta, and Neosartoria stramenia. Examples of the fungi of the genus Aspergillus include Aspergillus fumigatus. Examples of the fungi of the genus Paecilomyces include Paecilomyces variotii. Examples of the genus Talaromyces include Talaromyces cejpii, Talaromyces apiculatus, Talaromyces flavus, Talaromyces luteus, Talaromyces trachyspermus, Talaromyces wortmannii, Talaromyces bacillisporus, and Talaromyces macrosporus. Examples of the genus Hamigera include Hamigera insecticola, Hamigera striata, and Hamigera avellanea. Examples of the Thermoascus genus include Thermoascus thermophilus and Thermoascus aegyptiacus. Examples of the Eupenicillium genus include Eupenicillium brefeldianum and Eupenicillium cinnamopurpureum. Examples of the Eurotium genus include Eurotium rubrum, Eurotium chevalieri, Eurotium echinulatum, and Eurotium repens.

(Base sequence of target nucleic acid)
The oligonucleotide has a base sequence complementary to a portion of the base sequence of the target nucleic acid that contains at least two different bases compared to the corresponding sequence of the non-target nucleic acid. Examples of the base sequence of the target nucleic acid include, but are not limited to, at least a portion of a gene of a fungus group belonging to a single genus of thermotolerant fungi (e.g., β-tubulin gene, DNA encoding ribosomal RNA and an ITS (Internal Transcribed Spacer) region present in the gap therebetween). Examples of at least a portion of the β-tubulin gene of a thermotolerant fungus include a base portion corresponding to base numbers 352 to 569 or 352 to 703 of the base sequence of SEQ ID NO: 1 in the β-tubulin gene of a thermotolerant fungus. SEQ ID NO: 1 is a partial sequence from the 202nd base A, which is the translation initiation point of the gene coding region, to the 2213th base A in the base sequence encoding the β-tubulin gene of Neurospora crassa, a type of fungus (NC_026506.1: available in the NCBI database (https://www.ncbi.nlm.nih.gov/nuccore/759001909)). The correspondence between the β-tubulin gene of a thermotolerant fungus and SEQ ID NO: 1 can be confirmed by aligning the two to confirm the correspondence (Glass and Donaldson (1995) Applied and Environmental Microbiology, p. 1323-1330). An example of at least a portion of the ITS region of a thermotolerant fungus is a nucleotide portion corresponding to nucleotides 52 to 545 of the nucleotide sequence of SEQ ID NO:26 in the ITS region of a thermotolerant fungus.

In this specification, the identity of the base sequence is, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The identity can be determined by appropriately setting search conditions using an algorithm such as BLAST or FASTA.

The base length of the portion of the base sequence of the target nucleic acid that contains at least two different bases compared to the corresponding sequence of the non-target nucleic acid is not particularly limited, but may be, for example, 5 or more bases, 6 or more bases, 7 or more bases, 8 or more bases, 9 or more bases, or 10 or more bases. The upper limit may be 50 or less bases, 45 or less bases, 40 or less bases, 35 or less bases, 30 or less, or 25 or less bases. The number of bases that differ compared to the corresponding sequence of the non-target nucleic acid may be at least two. The upper limit is not particularly limited, but may be, for example, 5 or less, 4 or less, or 3 or less.

The portion containing at least two different bases compared to the corresponding sequence of the non-target nucleic acid can be appropriately selected from the base sequence of the target nucleic acid, but preferably contains a base sequence corresponding to at least a part of base numbers 545 to 569 and 681 to 702 of the base sequence of SEQ ID NO: 1, and preferably a base sequence corresponding to a portion containing base numbers 545 and 546 (e.g., base numbers 545 to 546), a portion containing base numbers 545 and 550 (e.g., base numbers 545 to 550), or a portion containing base numbers 683 and 686 (e.g., base numbers 683 to 686). Base numbers 545, 546, and 550 of SEQ ID NO: 1 differ between the corresponding sequences of Neosartoria (SEQ ID NOs: 10 to 12, 15 to 19) and Aspergillus fumigatus (SEQ ID NO: 13) (Figures 3 and 4). Base numbers 683, 686, and 698 in SEQ ID NO:1 differ between Byssochlamys genus (SEQ ID NO:3-6) and Paecilomyces variotii (SEQ ID NO:7-8) (Figure 2). It is also preferable to include a base sequence corresponding to at least a portion of base numbers 52-72 (preferably base number 27) of the base sequence of SEQ ID NO:26. This base sequence portion (particularly the above base portion of SEQ ID NO:26) is common to Eurotium genus bacteria and differs from the primer portion described in Patent Document 6 (Figures 9 and 13). The primer used in Patent Document 6 corresponds to the portion from base 2517 to base 2539 in the base sequence (SEQ ID NO:25) of the rDNA of Aspergillus oryzae RIB40 rDNA (ACCESSION No.AP007173) shown in Figure 12, and the base sequence of SEQ ID NO:27 corresponds to bases 2396 to 2417 (Figure 12).

(Modified bases)
The modified base contained in the oligonucleotide may be any modified base capable of stabilizing the binding of nucleic acid, and examples thereof include LNA (locked nucleic acid: FIG. 1), BNA (bridged nucleic acid) other than LNA, 2'-O-methoxyethyl nucleic acid, 2'-O-methyl nucleic acid, and 2'-fluoro nucleic acid. The modified base may further have one or more substituents (e.g., methyl group, etc.) within a range that does not interfere with the stabilization of the binding of nucleic acid. The number of modified bases may be at least one, and the upper limit is not particularly limited, and is usually 5 or less, 4 or less, 3 or less, or 2 or less. When two or more modified bases are contained, each modified base may be different.

The modified base is preferably the same as the base before modification. For example, if the base before modification is adenine, the modified base is preferably a modified adenine base (e.g., an LNA of adenine). The modified base is preferably a modified adenine or guanine base.

The position of the modified base in the oligonucleotide is not particularly limited, but the distance between the base complementary to the base different from the corresponding sequence of the non-target nucleic acid and the modified base is preferably, for example, 0 to 15 bases, 0 to 14 bases, 0 to 13 bases, or 0 to 12 bases. Note that a distance of 0 bases means that the base complementary to the base different from the corresponding sequence of the non-target nucleic acid is a modified base. It is also preferable that the distance between the base at the 3' end of the oligonucleotide and the modified base is closer than the distance between the base at the 5' end and the modified base. This can cause a structural change in the base sequence on the 3' side, which has a significant effect on the amplification reaction. The distance between the base at the 3' end of the oligonucleotide and the modified base is preferably, for example, 0 to 10 bases, 0 to 9 bases, 0 to 8 bases, 0 to 7 bases, or 0 to 6 bases. Note that a distance of 0 bases means that the base at the 3' end of the oligonucleotide is a modified base.

(Examples of Oligonucleotides)
The length of the oligonucleotide is not particularly limited, but typically has 15 or more or 20 or more bases, preferably 21 or more, 22 or more, or 23 or more bases. The upper limit is also not particularly limited, but typically has 50 or less or 45 or less, preferably 40 or less, 35 or less, or 30 or less.

As the oligonucleotide for detecting a target nucleic acid, an oligonucleotide that can be used as a primer capable of amplifying a gene of a heat-resistant fungus together with an oligonucleotide containing a part of the base sequence of SEQ ID NO: 1 or 26 is preferred. In particular, it is preferred that the size of the amplified product is, for example, 100 bases or more, 120 bases or more, 140 bases or more, or 150 bases or more, with an upper limit of, for example, 600 bases or less, 500 bases or less, 450 bases or less, 400 bases or less, or 350 bases or less.

Examples of oligonucleotides for detecting target nucleic acids include the following:
(1) an oligonucleotide in which the base at base number 18 in the base sequence of SEQ ID NO:2 is a modified base and which contains at least the base portion of base numbers 6 to 18;
(2) an oligonucleotide in which the base at base number 25 in the base sequence of SEQ ID NO:9 is a modified base and which contains at least a portion of base numbers 20 to 25; and (3) an oligonucleotide in which the base at base number 24 in the base sequence of SEQ ID NO:14 is a modified base and which contains at least a portion of base numbers 20 to 24.

SEQ ID NO:2 is complementary to the base sequence of the β-tubulin gene of a thermotolerant fungus, which corresponds to the portion of nucleotides 681 to 702 of SEQ ID NO:1. SEQ ID NOs:9 and 14 are complementary to the base sequence of the β-tubulin gene of a thermotolerant fungus, which corresponds to the portion of nucleotides 545 to 569 of SEQ ID NO:1 (Figure 8).

Examples of the oligonucleotide for detecting a target nucleic acid include: (4) an oligonucleotide in which the base at base number 16 in the base sequence of SEQ ID NO:27 is a modified base and which contains at least the base portion of base numbers 13 to 19;
Oligonucleotides (1) to (4) preferably contain, and more preferably consist of, the full-length base sequences of SEQ ID NOs: 2, 9, 14, and 27, respectively.

Figure 2 shows an example of a base sequence amplified by oligonucleotide (1) (complementary sequence of oligonucleotide (1): SEQ ID NO: 2) and an alignment of the corresponding portions of the base sequences of the β-tubulin genes of Byssochlamys (SEQ ID NOs: 3-6) and P. variotii (SEQ ID NOs: 7-8). The bases corresponding to thymine (T) at base number 3, cytosine (C) at base number 6, and thymine (T) at base number 18 in the base sequence amplified by oligonucleotide (1) are cytosine (C), thymine (T), and cytosine (C) in P. variotii, respectively, and differ at three positions. On the other hand, in Byssochlamys, these bases are thymine (T), thymine (T) or cytosine (C), thymine (T), and only base number 6 may differ.

Figure 3 shows an example of a base sequence amplified by oligonucleotide (2) (complementary sequence of oligonucleotide (2): SEQ ID NO: 9) and an alignment of the corresponding portions of the base sequences of the β-tubulin genes of Neosartoria (N. primulina, N. quadricincta, N. stramenia: SEQ ID NOs: 10-12, respectively) and A. fumigatus (SEQ ID NO: 13). The bases corresponding to thymine (T) at base number 1 and cytosine (C) at base number 6 of the base sequence amplified by oligonucleotide (2) (the base numbers in SEQ ID NO: 1 are adenine (A) and thymine (T), respectively, in A. fumigatus, which differ at two positions. On the other hand, in Neosartoria, these bases are thymine (T) and cytosine (C), which are the same.

Figure 4 shows an example of a base sequence amplified by oligonucleotide (3) (complementary sequence of oligonucleotide (3): SEQ ID NO: 14) and an alignment of the corresponding portions of the base sequences of the β-tubulin genes of Neosartoria sp. (N. fennelliae, N. fischeri, N. hiratsukae, N. pseudofischeri, N. udagawae: SEQ ID NO: 15-19) and A. fumigatus (SEQ ID NO: 13). In A. fumigatus, the bases corresponding to guanine (G) at base number 1 and thymine (T) at base number 2 in the base sequence amplified by oligonucleotide (3) are adenine (A) and adenine (A), respectively, and differ at two positions. On the other hand, in five species of Neosartoria sp., these bases are guanine (G), adenine (A), or thymine (T), and only the base at base number 2 may differ.

Figure 9 shows an example of a base sequence amplified by oligonucleotide (4) (sequence of oligonucleotide (4): SEQ ID NO: 27) and an alignment of the corresponding portions of the base sequences of the ITS regions of Eurotium genus bacteria (E. echinulatum, E. rubrum, E. rubrum, E. chevalieri: SEQ ID NOs: 29 to 38). The base portion amplified by oligonucleotide (4) is different from the base portion amplified by the primers shown in Patent Document 6 and is common to each bacteria.

The upper part of Figure 9 shows an alignment of the corresponding parts of the ITS region of Eurotium bacteria (E. echinulatum, E. rubrum, E. chevalieri: SEQ ID NOs: 29 to 33) amplified with oligonucleotide (4). The lower part of Figure 9 shows an alignment of the corresponding parts (SEQ ID NOs: 34 to 38) of each bacterium in the upper part amplified with the primers described in Patent Document 6. The region amplified by oligonucleotide (4) is common to 27 strains of Eurotium bacteria, including each bacterium. On the other hand, the region amplified by the primers described in Patent Document 6 differs by one base in E. chevalieri (E in the figure), and therefore the detection accuracy is thought to be lower than that of the present invention.

In addition, as long as the effect of the present invention is not impaired, oligonucleotides (1) to (4) may have modified base sequences within a range of 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, or 98% or more identity, and may have modified bases in other than the specified positions.

(Detection of target nucleic acid)
The oligonucleotide is used for detecting a target nucleic acid, preferably for detecting the target nucleic acid by PCR. The oligonucleotide is more preferably used as a PCR primer for detecting the target nucleic acid, and even more preferably as a reverse primer. When the target nucleic acid is a gene or a part thereof of a fungus group belonging to a single genus of thermotolerant fungi, it is used for detecting the thermotolerant fungus. In this specification, detection of a target nucleic acid includes confirmation of the target nucleic acid and identification of the target nucleic acid.

[Primer set]
The above oligonucleotides can be used as primers for detecting a target nucleic acid together with an oligonucleotide as a predetermined primer to form a primer set for detecting a target nucleic acid. Among them, it is preferable to use oligonucleotides (1) to (3) together with a predetermined forward primer, and oligonucleotide (4) together with a predetermined reverse primer.

(Forward primer in combination with oligonucleotides (1) to (3))
The oligonucleotide as a forward primer may be designed to amplify at least a portion of the target nucleic acid (e.g., 100 bases or more, 120 bases or more, 140 bases or more, or 150 bases or more, with an upper limit of, for example, 600 bases or less, 500 bases or less, 450 bases or less, 400 bases or less, or 350 bases or less). The length of the oligonucleotide as a forward primer is not particularly limited, but is usually 15 or more or 20 or more bases, preferably 21 or more, 22 or more, or 23 or more. The upper limit is also not particularly limited, but is usually 50 or less or 45 or less, preferably 40 or less, 35 or less, or 30 or less. Examples of oligonucleotides as forward primers include oligonucleotides having at least a portion of the base portion 1 to 544 of the base sequence of SEQ ID NO: 1 or a sequence having 90% or more identity thereto, and preferably an oligonucleotide having a base sequence represented by SEQ ID NO: 20.

(Reverse primer to be combined with oligonucleotide (4))
The oligonucleotide as a reverse primer may be designed to amplify at least a portion of the target nucleic acid (e.g., 100 bases or more, 120 bases or more, 140 bases or more, or 150 bases or more, with an upper limit of, for example, 600 bases or less, 500 bases or less, 450 bases or less, 400 bases or less, or 350 bases or less). The length of the oligonucleotide as a reverse primer is not particularly limited, but is usually 15 or more or 17 or more bases, preferably 20 or more. The upper limit is also not particularly limited, but is usually 50 or less or 45 or less, preferably 40 or less, 35 or less, or 30 or less. Examples of oligonucleotides as reverse primers include oligonucleotides having a sequence complementary to at least a portion of the base portion 73 to 1108 of the base sequence of SEQ ID NO: 26 or a sequence having 90% or more identity thereto, and preferably an oligonucleotide having the base sequence represented by SEQ ID NO: 28 (base numbers 526 to 545 of the base sequence of SEQ ID NO: 26: FIG. 13).

[Target Nucleic Acid Detection Kit]
The oligonucleotide or primer set can be used as one element of a kit for detecting a target nucleic acid. The kit for detecting a target nucleic acid may contain at least one of the oligonucleotides or primer sets, or may contain a plurality of the oligonucleotides or primer sets. The kit for detecting a target nucleic acid may further contain reagents and materials used for carrying out PCR in addition to the primer set. Such reagents and materials include, for example, an enzyme for PCR (e.g., DNA polymerase), a pH buffer solution, a fluorescent dye, dNTP, _MgCl2 , a positive control DNA, and sterilized water. Furthermore, if necessary, a measurement container may be included.

[Method for detecting target nucleic acid]
The above-mentioned oligonucleotide or primer set can be used for detecting a target nucleic acid that may be contained in a sample.

(sample)
The sample is not particularly limited as long as it may contain the target nucleic acid. When the target nucleic acid is a group of bacteria belonging to a single genus of heat-resistant fungi, examples of the sample include food and related equipment and biological samples. Among them, long-term storage foods (e.g., canned foods, bottled foods, retort foods) that undergo at least one of heat treatment, high pressure treatment, and vacuum packaging treatment during the manufacturing process, manufacturing equipment, containers, and cleaning solutions used in the manufacturing process are preferred.

(Step of amplifying target nucleic acid)
The detection method is not particularly limited as long as it is a method using an oligonucleotide and a primer set to detect the target nucleic acid, but it is preferable that the method includes a step of amplifying the target nucleic acid in the sample by PCR using the oligonucleotide and the primer set. PCR may be performed by appropriately selecting the implementation conditions according to a conventional method, but it is preferable to adopt a touchdown protocol. The touchdown protocol is a protocol in which the annealing temperature is set to a high temperature in the first cycle of the thermal cycle of PCR, the temperature is gradually lowered for each subsequent cycle, and the annealing temperature is maintained until the last cycle after reaching the optimal temperature. As a result, by annealing at a high temperature in the first cycle, a specific amplification product can be preferentially amplified, and by gradually lowering the temperature thereafter, the drop in amplification efficiency can be covered, and a highly specific amplification reaction is likely to occur, including in the subsequent cycles. In touchdown PCR, the annealing temperature in the thermal cycle may be adjusted to a temperature higher than the melting temperature of the primer used and the recommended annealing temperature in the standard protocol of the PCR kit used (for example, a temperature higher than the annealing temperature within a range of 12°C, 11°C, 10°C, or 9°C).

[Method of manufacturing long-term storage foods]
The above oligonucleotide or primer set can be used to detect heat-resistant fungi during the production of long-term storage foods. That is, after the production process of long-term storage foods, which involves at least one of heat treatment, high pressure treatment, and vacuum packaging, a detection process is included in which heat-resistant fungi are detected by the above-mentioned detection method after the production process is completed and before shipping, thereby preventing spoilage and deterioration caused by the generation of heat-resistant fungi during long-term storage and producing safe foods. The timing of the detection process can be, for example, immediately after the production of the long-term storage food or an appropriate time during the storage period, and is preferably immediately after production, but is not particularly limited.

Example 1 (Detection of Bysochlamys sp.)
(1) Primer design In selecting a primer pair for detecting Byssochlamys, PCR was performed on Byssochlamys using a primer set of known universal primers Bt2a (GGTAACCAAATCGGTGCTGCTTTC: SEQ ID NO: 20, corresponding to the portion of nucleotides 352 to 375 of SEQ ID NO: 1) and Bt2b (ACCCTCAGTGTAGTGACCCTTGGC, SEQ ID NO: 39, corresponding to the portion of nucleotides 825 to 848 of SEQ ID NO: 1) (Glass and Donaldson (1995) Applied and Environmental Microbiology, Apr. 1995, p. 1323-1330), and sequence information of the amplified product of the β-tubulin gene of Byssochlamys was obtained. Based on the obtained sequence information, a reverse primer was designed that would produce an amplification product of about 350 bp when the β-tubulin gene of Bysochlamys was amplified by PCR using Bt2a as a forward primer. As a result, Bys-R1 (GGAACATACTTCTTGCCGGCAGC: SEQ ID NO: 2) having a nucleotide sequence complementary to the sequence portion of the β-tubulin gene of Bysochlamys corresponding to the portion of nucleotides 681 to 703 of SEQ ID NO: 1 (NC_026506.1), Bys-R1L, Bys-R1L-2, and Bys-R1L3 having the same nucleotide sequence as Bys-R1 but with one or two nucleotides substituted with LNA were designed (Table 1).

(Table 1 footnote)
Underlined bases: LNA

(2) Detection test for Byssochlamys and other heat-resistant fungi Using the reverse primer designed in (1), a detection test for the heat-resistant fungi shown in Table 2 was carried out. The following primer set was used:
Primer set 1: existing primer set B1F-B1R (ttgggaccaaacaagagaca (SEQ ID NO: 21) and tgtgcacttacacaccagca (SEQ ID NO: 22); Nakayama, Soft Drink Technical Information 2012, No. 1)
Primer set 2: Bt2a-Bys-R1
Primer set 3: Bt2a-Bys-R1L
Primer set 4: Bt2a-Bys-R1L-2
Primer set 5: Bt2a-Bys-R1L-3
Primer set 6: Bt2a-Bys-R1L-4.

KAPA2G (registered trademark) Robust HotStart ReadyMix with dye (Roche), which shows high detection ability even in crude samples, was used as the PCR reaction reagent. A total of 20 μL of reaction solution was prepared, consisting of 1 μL of template DNA sample solution, 10 μM primer (forward, reverse), 1 μL each, distilled water, 7 μL, and PCR Mix, 10 μL. The reaction cycle was changed from the standard reaction cycle in the reagent kit to the following in order to improve the specificity of the amplification reaction.

[Touchdown type cycle]
95°C, 2 min;
(95°C: 15 sec → 68-61°C ^* : 15 sec → 72°C: 25 sec) × 8 cycles;
(95°C: 15 sec → 60°C: 15 sec → 72°C: 25 sec) × 27 cycles;
72°C: 10 min
10℃:
^* The annealing temperature was decreased by 1°C from 68°C to 61°C for each cycle.

5 μL of the PCR reaction mixture was applied to a 2% agarose/TAE (Tris-acetate EDTA Buffer) gel containing Midori Green Advance (Nihon Genetics) for DNA staining, and electrophoresis was performed. After electrophoresis, the gel was irradiated with a blue LED to detect the band pattern of the amplified product (Table 2, Figure 5).

(Footnotes to Table 2)
+: single amplification product of expected length, + ^* : amplification product of expected length and non-specific amplification product, ^* : non-specific amplification product

Comparing the ability of each primer set to distinguish between heat-resistant fungi, when primer set 1 was used, detection of B. lagunculariae (strains 2 and 5) was not confirmed (top row of Figure 5). When primer set 2 was used, non-specific amplification products were detected in Byssochlamys genus, and non-specific amplification occurred in many other heat-resistant fungi (middle row of Figure 5). On the other hand, when primer sets 3 to 6 were used, a single amplification product of the expected chain length (350 bp) at the time of primer design was confirmed for Byssochlamys genus (B. fulva, 1; B. nivea, 3.6; B. lagunculariae, 2.5; B. zollerniae, 4) (Table 2, lanes 1-6 in the bottom row of Figure 5). In particular, when primer set 3 was used, a single amplification product was not formed in Paecilomyces variotii, some of which have been reported to have a perfect stage of the Byssochlamys genus. In particular, it was found that B. lagunculariae (2, 5) can be detected together with other Byssochlamys genus bacteria by using primer sets 3 to 6, whereas existing technology requires the use of individual specific primers for detection.

Example 2 (Detection of Neosartoria sp.)
(1) Primer design In selecting a primer pair for detecting Neosartoria bacteria, PCR was performed on Neosartoria bacteria using the same primer set of Bt2a and Bt2b as in Example 1, and sequence information of the amplified product of the β-tubulin gene of Neosartoria bacteria was obtained. Based on the obtained sequence information, a reverse primer was designed that is expected to produce an amplified product of about 220 bp when the β-tubulin gene of Byssochlamys bacteria is amplified by PCR using Bt2a as a forward primer. PCR was performed on Neosartoria bacteria, and Ne-R1 (CGGAGGAGCCATTGTAGCTGTTATA: SEQ ID NO: 9) and Ne-R2 (CGGAGGAGCCATTGTAGCTATTAA C: SEQ ID NO: 14) have a nucleotide sequence complementary to the nucleotide positions 545 to 569 of the β-tubulin gene of Neosartoria bacteria, and Ne-R1L and Ne-R2L have nucleotide sequences in which one nucleotide of each of Ne-R1 and Ne-R2 has been replaced with LNA (Table 3).

(Footnotes to Table 3)
Underlined bases: LNA

(2) Detection test for Byssochlamys and A. fumigatus Using the reverse primer designed in (1), a detection test for the heat-resistant fungi shown in Table 4 was carried out. The same procedure as in Example 1(1) was carried out except that the following primer set was used.
Primer set 1: N2F-N2R, an existing primer set (ggctctggccagtaagttcg (SEQ ID NO: 23) and ttgtcaccgttggcctagta (SEQ ID NO: 24); Japanese Patent No. 5548385)
Primer set 2: Bt2a-Ne-R1
Primer set 3: Bt2a-Ne-R2
Primer set 4: Bt2a-Ne-R1L
Primer set 5: Bt2a-Ne-R2L

(Footnotes to Table 4)
+: Single amplification product of expected length

Comparing the discrimination ability of each primer set between Neosartoria and A. fumigatus, when primer set 1 was used, a false positive detection result originating from A. fumigatus was obtained (upper left row of Figure 2). In PCR reactions using primer sets 2 to 5, no false positive results originating from A. fumigatus were detected (second and third rows on the left side of Figure 5). Furthermore, in PCR reactions using primer sets 4 and 5, Neosartoria could be discriminated for each primer (first and second rows on the right side of Figure 6). Among them, N. fennelliae (A) is a domestically derived bacterium and its detection is important, but it cannot be detected well using existing technology. However, this example shows that it is possible to discriminate by using primer set 5. In addition, in the test performed in this example without adopting the touchdown protocol, false positives were confirmed in the results of bacteria other than Neosartorius when primer sets 2 and 3 were used, whereas when primer sets 4 and 5 were used, the same results as those in Table 4 were obtained.

(3) Detection test for Byssochlamys genus and other heat-resistant fungi Using the reverse primer designed in (1), a detection test for the heat-resistant fungi shown in Table 5 was carried out. The same procedure as in Example 1(2) was carried out except that the primer sets used were primer sets 1, 4, and 5 shown in (2).

As a result of comparing the detection specificity of each primer set for heat-resistant fungi, primer sets 4 and 5 did not show false positives for A. fumigatus (5, 6) or for Eurotium species (7, 8) that, like Neosartorya species, have Aspergillus as their imperfect stage, and there were no false positives for other heat-resistant fungi, demonstrating that highly specific detection is possible (Figure 7).

Example 3 (Detection of Eurotium sp.)
(1) Primer Design With reference to a partial sequence of the ribosomal RNA coding region of Eurotium sp. (SEQ ID NO: 26, FIG. 13), a forward primer was designed that would produce an amplification product of about 500 bp when amplifying at least a part of the partial sequence by PCR using universal primer ITS4 (TCCTCCGCTTATTGATATGC: SEQ ID NO: 28: corresponding to the 526th to 545th bases of SEQ ID NO: 26 (FIG. 13)) as a reverse primer. As a result, Eur-F1 (CATCCGTGTCTATCTGTACCC: SEQ ID NO: 27) having a nucleotide sequence complementary to the 52nd to 72nd bases of SEQ ID NO: 26 (FIG. 13) and Eur-F1L having the same nucleotide sequence as Eur-F1 but with one nucleotide substituted with LNA were designed (Table 6).

(Footnotes to Table 6)
Underlined bases: LNA

(2) Detection test for the genus Eurotium Using the forward primer Eur-F1L designed in (1) together with the reverse primer ITS4, a detection test for the heat-resistant fungi shown in Table 7 was carried out. The same procedure was carried out except that the primer set Eur-F1L/ITS4 was used.

In the PCR reaction using Eur-F1L/ITS4, all Eurotium species tested were detected (Figure 10).

(3) Detection test for the genus Eurotium and other heat-resistant fungi Using the reverse primer designed in (1), a detection test for the heat-resistant fungi shown in Table 5 was carried out. The same procedure as in (2) was carried out except that the following primer set 1 or 2 was used as the primer set.
Primer set 1: Eur-F1/ITS4
Primer set 2: Eur-F1L/ITS4

A comparison of the detection specificity of each primer set for heat-resistant fungi showed that primer set 1 falsely detected Neosartoria primulina (5), Neosartoria pseudofischeri (6), and Aspergillus fumigatus (17, 18), whereas primer set 2 showed no false detection of Aspergillus species (17-20) or heat-resistant fungi other than Eurotium species (5-16), demonstrating the possibility of highly specific detection (Figure 11).

SEQ ID NO: 1: Nucleotide sequence encoding the β-tubulin gene of Neurospora crassa SEQ ID NO: 2: Nucleotide sequence of oligonucleotide (Bys-R1) for detecting target nucleic acid SEQ ID NO: 3: Partial nucleotide sequence corresponding to Bys-R1 in the β-tubulin gene of B. fulva IFM62195 SEQ ID NO: 4: Partial nucleotide sequence corresponding to Bys-R1 in the β-tubulin gene of B. lagunculariae IFM58746 SEQ ID NO: 5: Partial nucleotide sequence corresponding to Bys-R1 in the β-tubulin gene of B. nivea IFM59486 SEQ ID NO: 6: Partial nucleotide sequence corresponding to Bys-R1 in the β-tubulin gene of B. zollerniae IFM61583 SEQ ID NO: 7: Partial nucleotide sequence corresponding to Bys-R1 in the β-tubulin gene of P. Partial base sequence corresponding to Bys-R1 in the β-tubulin gene of P. variotii NBRC31685 SEQ ID NO: 8: Partial base sequence corresponding to Bys-R1 in the β-tubulin gene of P. variotii NBRC109023 SEQ ID NO: 9: Base sequence of oligonucleotide (Ne-R1L) for detecting target nucleic acid SEQ ID NO: 10: Partial base sequence corresponding to Ne-R1L in the β-tubulin gene of N. primulina NBRC33243 SEQ ID NO: 11: Partial base sequence corresponding to Ne-R1L in the β-tubulin gene of N. quadricincta NBRC9842 SEQ ID NO: 12: Partial base sequence corresponding to Ne-R1L in the β-tubulin gene of N. stramenia NBRC31358 SEQ ID NO: 13: A. Partial base sequence corresponding to Ne-R1L in the β-tubulin gene of N. fumigatus IFM62629 SEQ ID NO: 14: Base sequence of oligonucleotide (Ne-R2L) for detecting target nucleic acid SEQ ID NO: 15: Partial base sequence corresponding to Ne-R2L in the β-tubulin gene of N. fennelliae NBRC32080 SEQ ID NO: 16: Partial base sequence corresponding to Ne-R2L in the β-tubulin gene of N. fischeri NBRC31354 SEQ ID NO: 17: Partial base sequence corresponding to Ne-R2L in the β-tubulin gene of N. hiratsukae NBRC33242 SEQ ID NO: 18: Partial base sequence corresponding to Ne-R2L in the β-tubulin gene of N. pseudofischeri NBRC33244 SEQ ID NO: 19: N. Partial base sequence corresponding to Ne-R2L in the β-tubulin gene of A. udagawae NBRC101678 SEQ ID NO: 20: Base sequence of primer Bt2a SEQ ID NO: 21: Base sequence of primer B1F SEQ ID NO: 22: Base sequence of primer B1R SEQ ID NO: 23: Base sequence of primer N2F SEQ ID NO: 24: Base sequence of primer N2R SEQ ID NO: 25: Base sequence of the ITS region of A. oryzae RIB40 SEQ ID NO: 26: Base sequence of the ITS region of E. echinulatum NBRC5862 SEQ ID NO: 27: Base sequence of oligonucleotide for detecting target nucleic acid (Eur-F1) SEQ ID NO: 28: Base sequence of primer ITS4 SEQ ID NO: 29: Partial base sequence corresponding to Eur-F1 in the ITS region of E. echinulatum NBRC5862 SEQ ID NO: 30: E. Partial base sequence corresponding to Eur-F1 in the ITS region of E. rubrum NBRC4089 SEQ ID NO: 31: Partial base sequence corresponding to Eur-F1 in the ITS region of E. rubrum NBRC7712 SEQ ID NO: 32: Partial base sequence corresponding to Eur-F1 in the ITS region of E. chevalieri NBRC4086 SEQ ID NO: 33: Partial base sequence corresponding to Eur-F1 in the ITS region of E. chevalieri NBRC100022 SEQ ID NO: 34: Partial base sequence corresponding to the primer described in Patent Document 6 in the ITS region of E. echinulatum NBRC5862 SEQ ID NO: 35: Partial base sequence corresponding to the primer described in Patent Document 6 in the ITS region of E. rubrum NBRC4089 SEQ ID NO: 36: E. SEQ ID NO: 37: A partial base sequence of the ITS region of E. rubrum NBRC7712 corresponding to the primer described in Patent Document 6 SEQ ID NO: 38: A partial base sequence of the ITS region of E. chevalieri NBRC100022 corresponding to the primer described in Patent Document 6 SEQ ID NO: 39: Base sequence of primer Bt2b

Claims (13)

At least one of the following oligonucleotides ( 1-1 ) to (4):
a primer for amplifying a target nucleic acid,
the target nucleic acid of the oligonucleotide ( 1-1 ) is at least a partial base sequence of a gene of Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp., or Eupenicillium brefeldianum;
the target nucleic acid of the oligonucleotide (1-2) is at least a partial base sequence of a gene of Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp., Paecilomyces variotii, or Eupenicillium brefeldianum;
The target nucleic acid of the oligonucleotide (2) is at least a partial base sequence of a gene of Neosartorya primulina, Neosartorya quadricincta, or Neosartorya stramenia;
The target nucleic acid of the oligonucleotide (3) is at least a partial base sequence of a gene of Neosartorya fennelliae, Neosartorya fischeri var. fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri, or Neosartorya udagawae;
The target nucleic acid of the oligonucleotide (4) is at least a partial base sequence of a gene of Eurotium chevalieri, Eurotium costiforme, Eurotium herbariorum, Eurotium repens, Eurotium rubrum, or Eurotium amstelodami;
Oligonucleotides for detecting target nucleic acids.
( 1-1 ) An oligonucleotide consisting of the base sequence of SEQ ID NO:2, in which the base at base number 18 is BNA
(1-2) An oligonucleotide having the base sequence of SEQ ID NO: 2, in which the base at base number 21 is BNA
(2) An oligonucleotide having the base sequence of SEQ ID NO:9, in which the 25th base is BNA. (3) An oligonucleotide having the base sequence of SEQ ID NO:14, in which the 24th base is BNA. (4) An oligonucleotide having the base sequence of SEQ ID NO:27, in which the 16th base is BNA. The oligonucleotide of claim 1, wherein the BNA is an LNA. The method comprises the steps of: ( 1-1 ) an oligonucleotide; and (2) a target nucleic acid is at least a partial base sequence of a gene of Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp. , or Eupenicillium brefeldianum;
The oligonucleotide (1-2) is included, and the target nucleic acid is at least a partial base sequence of a gene of Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp., Paecilomyces variotii, or Eupenicillium brefeldianum;
The nucleic acid comprises an oligonucleotide (2), and the target nucleic acid is at least a partial base sequence of a gene of Neosartorya primulina, Neosartorya quadricincta, or Neosartorya stramenia;
A primer set comprising an oligonucleotide (3), wherein the target nucleic acid is at least a partial base sequence of a gene of Neosartorya fennelliae, Neosartorya fischeri var. fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri, or Neosartorya udagawae, or comprising an oligonucleotide (4), wherein the target nucleic acid is at least a partial base sequence of a gene of Eurotium chevalieri, Eurotium costiforme, Eurotium herbariorum, Eurotium repens, Eurotium rubrum, or Eurotium amstelodami.
( 1-1 ) An oligonucleotide consisting of the base sequence of SEQ ID NO:2, in which the base at base number 18 is BNA
(1-2) An oligonucleotide having the base sequence of SEQ ID NO: 2, in which the base at base number 21 is BNA
(2) An oligonucleotide having the base sequence of SEQ ID NO:9, in which the 25th base is BNA. (3) An oligonucleotide having the base sequence of SEQ ID NO:14, in which the 24th base is BNA. (4) An oligonucleotide having the base sequence of SEQ ID NO:27, in which the 16th base is BNA. The primer set according to claim 3, comprising at least one of the oligonucleotides ( 1-1 ) to (3) and a forward primer, wherein the forward primer is an oligonucleotide comprising at least a part of bases 1 to 544 of the base sequence of SEQ ID NO: 1 . The primer set according to claim 4 , wherein the forward primer is an oligonucleotide comprising at least the base sequence of SEQ ID NO: 20. The primer set according to claim 3 , comprising the oligonucleotide (4) and a reverse primer, wherein the reverse primer is an oligonucleotide comprising at least a part of base numbers 73 to 1108 of the base sequence of SEQ ID NO:26. The primer set according to claim 6 , wherein the reverse primer is an oligonucleotide comprising at least the base sequence of SEQ ID NO:28. A method for detecting a target nucleic acid that may be contained in a sample, comprising a step of amplifying a target nucleic acid in the sample by PCR using the primer set according to any one of claims 3 to 7 ,
the primer set comprises an oligonucleotide ( 1-1 ), and the target nucleic acid is at least a partial nucleotide sequence of a gene of Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp., or Eupenicillium brefeldianum;
the primer set comprises oligonucleotide (1-2), and the target nucleic acid is at least a partial nucleotide sequence of a gene of Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp., Paecilomyces variotii, or Eupenicillium brefeldianum;
The primer set includes an oligonucleotide (2), and the target nucleic acid is at least a partial nucleotide sequence of a gene of Neosartorya primulina, Neosartorya quadricincta, or Neosartorya stramenia;
A detection method, wherein the primer set comprises an oligonucleotide (3), and the target nucleic acid is at least a partial base sequence of a gene of Neosartorya fennelliae, Neosartorya fischeri var. fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri, or Neosartorya udagawae, or wherein the primer set comprises an oligonucleotide (4), and the target nucleic acid is at least a partial base sequence of a gene of Eurotium chevalieri, Eurotium costiforme, Eurotium herbariorum, Eurotium repens, Eurotium rubrum, or Eurotium amstelodami. A method for detecting a thermotolerant fungus that may be contained in a sample, comprising a step of amplifying a target nucleic acid in the sample by PCR using the primer set according to any one of claims 3 to 7 ,
the primer set comprises an oligonucleotide ( 1-1 ), and the target nucleic acid is at least a partial nucleotide sequence of a gene of Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp., or Eupenicillium brefeldianum;
the primer set comprises oligonucleotide (1-2), and the target nucleic acid is at least a partial nucleotide sequence of a gene of Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp., Paecilomyces variotii, or Eupenicillium brefeldianum;
The primer set includes an oligonucleotide (2), and the target nucleic acid is at least a partial nucleotide sequence of a gene of Neosartorya primulina, Neosartorya quadricincta, or Neosartorya stramenia;
A method in which the primer set comprises an oligonucleotide (3), and the target nucleic acid is at least a partial base sequence of a gene of Neosartorya fennelliae, Neosartorya fischeri var. fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri, or Neosartorya udagawae, or the primer set comprises an oligonucleotide (4), and the target nucleic acid is at least a partial base sequence of a gene of Eurotium chevalieri, Eurotium costiforme, Eurotium herbariorum, Eurotium repens, Eurotium rubrum, or Eurotium amstelodami. The detection method according to claim 9 , wherein the sample is a long-term storage food that has undergone at least one of heat treatment, high pressure treatment, and vacuum packaging treatment during the manufacturing process. The detection method according to claim 9 or 10 , wherein the sample is a canned, bottled or retort food. The method according to any one of claims 9 to 11 , wherein the detection is carried out immediately after production. A manufacturing process for long-term storage foods that involves at least one of heat treatment, high pressure treatment, and vacuum packaging treatment; and
A detection step of detecting a heat-resistant fungus by the detection method according to any one of claims 9 to 12 after the completion of the production step and before shipping,
the primer set includes oligonucleotide ( 1-1 ), and the thermotolerant bacterium to be detected is one or more bacteria selected from Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp., or Eupenicillium brefeldianum;
the primer set comprises the oligonucleotide (1-2), and the thermotolerant bacterium to be detected is one or more bacteria selected from Byssochlamys fulva, Byssochlamys lagunculariae, Byssochlamys nivea, Byssochlamys sp., Paecilomyces variotii, and Eupenicillium brefeldianum;
the primer set includes an oligonucleotide (2), and the thermotolerant bacterium to be detected is one or more bacteria selected from Neosartorya primulina, Neosartorya quadricincta, and Neosartorya stramenia;
The primer set includes the oligonucleotide (3), and the thermotolerant bacteria to be detected are one or more bacteria selected from Neosartorya fennelliae, Neosartorya fischeri var. fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri, and Neosartorya udagawae; or the primer set includes the oligonucleotide (4), and the thermotolerant bacteria to be detected are one or more bacteria selected from Eurotium chevalieri, Eurotium costiforme, Eurotium herbariorum, Eurotium repens, Eurotium rubrum, and Eurotium amstelodami.
Methods for producing long-term storage foods.

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