JP5934038B2 - Method for detecting Thermoscus fungus - Google Patents

Description

The present invention relates to a method for detecting a fungus of the genus Thermoascus .

  Heat-resistant fungi (fungi) are widely distributed in nature and propagate on agricultural products such as vegetables and fruits, and contaminate foods and drinks made from these agricultural products. Moreover, heat-resistant fungi form ascospores having higher heat resistance in the life cycle than other normal fungi. For example, even when an acid beverage is heat sterilized, heat-resistant fungi survive and multiply, which may cause mold.

  A heat-resistant fungus belonging to the genus Thermoscus is known as one of the main heat-resistant fungi that cause contamination accidents that may be detected from food and drink after heat sterilization. To prevent accidents caused by heat-resistant fungi in foods and drinks and their raw materials, it is important to detect heat-resistant fungi belonging to the genus Thermoscus. Have been wary.

Conventional detection methods for Thermoscus spp. And identification methods from other genera are mainly morphological classification using culture. This method requires a long period of at least 14 days since culture must be continued until morphological characteristics occur. In addition, since morphological identification requires extremely high expertise, it is impossible to deny the risk that identification results differ depending on the judge. In addition, there are cases where damaged cells due to heating or drugs lose their morphogenic ability, and these cells do not form a characteristic shape even after long-term culture, so the accuracy of detection and identification results There was a problem.
As described above, morphological methods that require a long period of time to detect fungi and have problems with accuracy of identification results with other genera include hygiene management of food and drink, ensuring freshness of raw materials, and distribution. From the viewpoint of constraints, it is not always satisfactory. Therefore, it has been demanded to establish a detection method that solves the problems of the conventional quickness and accuracy.

  As a rapid and accurate detection method for fungi, an amplification method (for example, PCR method or LAMP method) targeting a specific base sequence of a gene is known. In addition, the existence of a gene that distinguishes the genus Thermoscus from other genera is known (see, for example, Non-Patent Document 1), and the base sequence of the gene of Thermoscus spp. In a public gene database such as DDBJ Information is registered. However, the DNA base sequence database of Thermoscus fungus is currently fragile, and no highly sensitive primer has been designed to detect Thermoscus fungus quickly and accurately.

J. Houbraken, et al., Studies in Mycology, 2011, vol. 70, p. 1-51

  It is known that fungi having heat resistance exist in addition to Thermoscus spp., And their heat resistance varies depending on the genus and species. Therefore, in the food and beverage industry, etc., determine the control method according to the microbiological characteristics of each bacterial species for the control (prevention, killing, and growth inhibition) of heat-resistant bacteria such as Thermoscus fungi. Is required. Therefore, in order to perform a quick and accurate risk assessment of harmful bacteria in the food and beverage industry and the like, it is important to quickly and accurately detect the thermoscus fungus.

It is an object of the present invention to provide a method capable of quickly and accurately detecting a Thermoscus fungus that is regarded as a harmful fungus in the food and beverage industry and distinguishing it from other genera. The present invention also aims to provide the applicable oligonucleotide pair and detection kit for this method.

  In view of such problems, the present inventors have conducted intensive studies in order to search for a new DNA region that can detect Thermosacus fungus rapidly and accurately. Specifically, the present inventors are molecules based on the base sequences of the RNA polymerase II large subunit gene (hereinafter also referred to as “RPB1 gene” in the present specification) and the β-tubulin gene of the genus Thermoscus. We conducted a intensive study using a phylogenetic approach. As a result, the RPB1 gene and β-tubulin gene of the Thermospus fungus have a specific base sequence that can be clearly distinguished from those of other fungi (hereinafter referred to as “variable site”). I found out). It was also found that Thermoscus fungus can be detected quickly and accurately by targeting these variable sites. The present invention has been completed based on these findings.

The present invention provides at least one oligonucleotide pair selected from the group consisting of an oligonucleotide pair comprising the following oligonucleotides (a) and (b) and an oligonucleotide pair comprising the following oligonucleotides (c) and (d): The present invention relates to a method for detecting a Thermoscus fungus, wherein the Thermoscus fungus is detected by using.
(A) An oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, or one or several bases deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 1, and a thermoscus Oligonucleotide (b) that can be used to detect the genus fungus (b) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 2, or one or several bases deleted, substituted, or inserted in the nucleotide sequence set forth in SEQ ID NO: 2 Or an oligonucleotide that is added and can be used to detect a Thermoscus genus fungus (c) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3, or one or several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 3 Oligonucleotide (d) in which the base of is deleted, substituted, inserted or added and can be used for the detection of Thermoscus fungus Oligonucleotide represented by the nucleotide sequence described in column No. 4, or one or several bases deleted, substituted, inserted or added in the nucleotide sequence described in SEQ ID NO: 4 and Oligonucleotides that can be used for detection

Further, with the present invention, the front Symbol (a) and oligonucleotide or the (c) and oligonucleotide pair consisting of an oligonucleotide of (d) of (b).
Further, the present invention relates to thermo Asker scan genus fungi detection kit comprising a pre Kio Rigo nucleotide pairs.

  The detection method of the present invention can quickly and accurately detect a Thermoscus fungus that is one of the causative bacteria of food and beverage contamination accidents, and distinguish it from other genera. Moreover, the oligonucleotide, oligonucleotide pair, and detection kit of the present invention can be suitably applied to the above method.

It is a figure which shows the molecular phylogenetic tree of the Thermoscus genus fungi created based on the base sequence of the RPB1 gene of a Thermoscus genus fungus, and a beta-tubulin gene. The base sequence of the RPB1 gene of Thermoascus crustaceus , Thermoascus thermophilus and Thermascus aurantiacus , as well as Thermoscus clusterseus and Thermoscus -It is a figure which shows the positional relationship of the base sequence which the oligonucleotide of said (a) and (b) recognizes in the base sequence of the RPB1 gene of a thermophilus and a thermoscus aurantiacus. In the base sequence of the β-tubulin gene of Thermoscus cluster seus and Thermoscus aurantiacus, and in the base sequence of β-tubulin gene of Thermoscus clusterseus and Thermoscus aurantiacus It is a figure which shows the positional relationship of the base sequence which the oligonucleotide of (c) and (d) recognizes. It is an electrophoretic diagram of the PCR product amplified using the oligonucleotide of said (a) and (b) in an Example. It is an electrophoretic diagram of the PCR product amplified using the oligonucleotide of said (a) and (b) in an Example. FIG. 2 is an electrophoretogram of PCR products amplified using the oligonucleotides (c) and (d) in the examples. FIG. 2 is an electrophoretogram of PCR products amplified using the oligonucleotides (c) and (d) in the examples.

  The present invention is stringent to a specific partial base sequence of the RPB1 gene or β-tubulin gene of a Thermoscus fungus, that is, a base sequence site (variable site) specific to the Thermoscus fungus present in this region. This is a method for detecting Thermoscus fungus using a pair of oligonucleotides that can hybridize under various conditions, and detecting Thermoscus fungus quickly and accurately. Here, the “variable site” is a site where base mutations tend to accumulate in the base sequence of the RPB1 gene or β-tubulin gene, and the base sequence of this site is greatly different from other bacterial species.

Thermoscus fungus belongs to the order of Eurotium, and forms a spore of a sexual genus (complete generation) with extremely high heat resistance.
Mature spore spores have a D-value at 90 ° C (the time required to sterilize the initial number of surviving bacteria to 1/10), which is about 30 minutes. 6D sterilization (sterilization that reduces the initial bacterial count to 1/1000000) cannot be performed. Moreover, since Thermoscus spp. Are widely distributed in the soil, there is a high risk of contamination from foods derived from agricultural products into food. Furthermore, since Thermoscus spp. Can be easily grown even with acidic beverage pH, Thermoscus spp. Surviving after heat sterilization may grow in food and cause a spoilage accident.

    In this specification, “RPB1” is an enzyme that synthesizes RNA, and DNA binding ability and RNA synthesis among a plurality of subunits constituting RNA polymerase II that transcribes many of mRNA and snRNA genes using DNA as a template. It is a kind of subunit having a function, and the “RPB1 gene” is a gene encoding RPB1. Further, “β-tubulin” is a protein constituting a microtubule, and “β-tubulin gene” is a gene encoding β-tubulin.

The term “genus” as used herein refers to the base sequence of DNA of Thermoscus spp. And other heat-resistant fungi (preferably RPB1 gene, β-tubulin gene, or RPB1 gene and β-tubulin gene). It means a group of species of Thermoscus fungi that are classified based on molecular phylogenetic trees created using molecular phylogenetic methods.
There is no restriction | limiting in particular as the preparation method of the said molecular phylogenetic tree, It can create by a normal method. For example, using neighbor-joining method, maximum parsimony, maximum likelihood estimation, Bayesian method, unweighted pair group method with Arithmetic mean can do. In the present invention, it is preferable to create a molecular phylogenetic tree using the neighborhood joining method.
The “Thermuscus fungus” in the present invention specifically includes Thermoscus cluster seus, Thermoscus thermophilus, Thermoscus Aurantiacus and the like. Furthermore, even if it is classified into a different taxonomic species such as Byssochlamys verrucosa (see J. Houbraken et al., Studies in Mycology, 2011, vol. 70, p. 1-51). As described later, any one having a specific base sequence in at least one of the RPB1 gene and the β-tubulin gene is included in the “Thermuscus fungus” in the present invention. In addition, J.J. According to Houbraken et al., Bisoclamis varkosa is more closely related to the genus Thermoscus rather than to the genus Bisoclamis, and should be classified as the genus Thermoscus (J. Houbraken et al., Studies in Mycology, 2011, vol. 70, p.

  In this specification, “stringent conditions” include, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W., et al. Russell., Cold Spring Harbor Laboratory Press], for example, 6 × SSC (composition of 1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% Examples include conditions in which a solution containing SDS, 5 × Denhart and 100 mg / mL herring sperm DNA is incubated with a probe at 65 ° C. for 8 to 16 hours and hybridized.

The detection method of the present invention is characterized by using an oligonucleotide pair represented by a nucleic acid corresponding to a variable site in the base sequence of the RPB1 gene or β-tubulin gene of a Thermoscus fungus.
The inventors determined the base sequences of the RPB1 gene and β-tubulin gene of the Thermoscus fungus and analyzed the genetic distance between the species and the homology of the base sequence. That is, the base sequences of the RPB1 gene and β-tubulin gene of Thermoscus spp. Were determined by sequencing, and the matching base region was examined by alignment analysis. As a result, in the base sequences of the RPB1 gene and β-tubulin gene, a conserved region within the same genus is highly conserved, but the conserved property of the base sequence is low between different genera, and a variable site having a unique base sequence depending on the genus was found. . At this variable site, Thermoscus fungus has a genus-specific base sequence. For this reason, this region is useful as a genetic index for rapidly and accurately detecting a Thermoscus fungus.

The detection method of the present invention comprises at least one selected from the group consisting of an oligonucleotide pair comprising the following oligonucleotides (a) and (b) and an oligonucleotide pair comprising the following oligonucleotides (c) and (d): Of oligonucleotide pairs.

(A) An oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, or one or several bases deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 1, and a thermoscus Oligonucleotide (b) that can be used to detect the genus fungus (b) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 2, or one or several bases deleted, substituted, or inserted in the nucleotide sequence set forth in SEQ ID NO: 2 Or an oligonucleotide that is added and can be used to detect a Thermoscus genus fungus (c) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3, or one or several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 3 Oligonucleotide (d) in which the base of is deleted, substituted, inserted or added and can be used for the detection of Thermoscus fungus Oligonucleotide represented by the nucleotide sequence described in column No. 4, or one or several bases deleted, substituted, inserted or added in the nucleotide sequence described in SEQ ID NO: 4 and Oligonucleotides that can be used for detection

By detecting and identifying a Thermoscus fungus using the oligonucleotide pair, it is possible to detect a Thermoscus fungus.

The variable sites in the base sequence of the RPB1 gene of the Thermospus fungus will be described based on the base sequences described in FIG. 2 and SEQ ID NOs: 5 to 7. 2 shows the base sequence of the RPB1 gene of Thermoscus cluster seus CBS181.67, Thermoscus thermophilus CBS528.71 and Thermoscus aurantiacus CBS396.78, and Thermoscus The positional relationship of the base sequences recognized by the oligonucleotides (a) and (b) in the base sequences of the RPB1 gene of cluster seus, thermoscus thermophilus and thermoscus aurantiacus is shown. The base sequence described in SEQ ID NO: 5 represents the base sequence of the RPB1 gene of Thermoscus cluster seus CBS181.67 strain, and the base sequence described in SEQ ID NO: 6 is Thermoscus aurantiacus CBS396. The base sequence of 78 strains of the RPB1 gene is shown, and the base sequence described in SEQ ID NO: 7 is the base sequence of the RPB1 gene of Thermoscus thermophilus CBS528.71.
The oligonucleotide (a) (also referred to herein as The_RPB_F2) and the oligonucleotide (b) (also referred to herein as The_RPB_R2) are each the base described in any of SEQ ID NOs: 5 to 7 The oligonucleotide is capable of hybridizing under stringent conditions to the region from position 67 to position 90 and the region from position 438 to position 462 of the sequence. The present inventors have found that these regions are variable regions with low conserved base sequences between genera, and that the base sequences of these regions are base sequences inherent in Thermoscus fungi.
Therefore, Thermoscus fungi can be detected quickly and accurately at the genus level using the oligonucleotides (a) and (b). Specifically, it can be used as a primer specific to Thermoscus spp. If it is designed so that 5 or more bases of the 3 ′ end of the primer, which is the direction of DNA extension by polymerase, hybridize to this region. , Detection by PCR using these primers becomes possible .

In the present invention, using the oligonucleotides (a) and (b), the presence of a nucleic acid (preferably the variable site) represented by any of the following base sequences (e) to (j) is confirmed. It is preferable to do.
(E) 1 or several (preferably 1 to 25, more preferably 1 to 20) nucleotide sequences of the RPB1 gene represented by SEQ ID NO: 5 or its complementary sequence (f) SEQ ID NO: 5 More preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases are deleted, substituted, inserted or added and can be used for the detection of Thermoscus fungi 70% or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more) with respect to the base sequence described in SEQ ID NO: 5 Or a complementary sequence (g) of the RPB1 gene described in SEQ ID NO: 6 or a complementary sequence (h) thereof. 1 or several (preferably 1 to 25, more preferably 1 to 20, more preferably 1 to 15, particularly preferably 1 to 10, most preferably 1 to 5) in the base sequence described in No. 6. 70% or more (preferably 80% or more) of the base sequence described in SEQ ID NO: 6, wherein the base sequence is deleted, substituted, inserted or added and can be used for the detection of Thermosacus fungus More preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more), and a nucleotide sequence that can be used for detection of Thermoscus fungi, or One or several (preferably 1 to 25) of the complementary sequence (i) the base sequence of the RPB1 gene described in SEQ ID NO: 7 or the complementary sequence (j) of the base sequence described in SEQ ID NO: 7 More preferably 1 to 20, more preferably 1 to 15, particularly preferably 1 to 10, most preferably 1 to 5) bases are deleted, substituted, inserted or added and 70% or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95%) with respect to the base sequence described in SEQ ID NO: 7, which can be used for detection of genus fungi Or more, particularly preferably 97% or more) and a base sequence that can be used for detection of a Thermoscus fungus, or a complementary sequence thereof.

The oligonucleotides (a) and (b) used in the detection method of the present invention are preferably oligonucleotides represented by the nucleotide sequence set forth in SEQ ID NO: 1 or 2. The oligonucleotide used in the detection method of the present invention is a base having 70% or more homology to the base sequence described in SEQ ID NO: 1 or 2 as long as it can be used for detection at the genus level of Thermoscus fungus. It may be an oligonucleotide represented by a sequence or a complementary sequence thereof, the homology is more preferably 80% or more, the homology is more preferably 85% or more, and the homology is 90% or more. More preferably, the homology is particularly preferably 95% or more. In addition, the oligonucleotide that can be used in the present invention has one or several, preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 1 nucleotide sequences in SEQ ID NO: 1 or 2. Also encompassed are oligonucleotides which are deleted, substituted or inserted with 3, more preferably 1 or 2, particularly preferably 1 base, and which can be used for detection at the genus level of Thermoscus fungi. The An appropriate base sequence may be added to the base sequence described in SEQ ID NO: 1 or 2.
The base sequence homology is calculated by the Lipman-Pearson method (Science, 1985, 227, p. 1435) or the like. Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the parameter Unit size to compare (ktup) is set to 2 and the calculation is performed. Can do.

The variable sites in the base sequence of the β-tubulin gene of the genus Thermoscus will be described based on the base sequences shown in FIG. 3 and SEQ ID NOs: 8 and 9. FIG. 3 shows the base sequences of the β-tubulin genes of Thermoscus cluster seus CBS181.67 and Thermoscus aurantiacus CBS396.78, Thermoscus clusterseus and Thermoscus The positional relationship of the base sequence which the oligonucleotide of said (c) and (d) recognizes in the base sequence of the β-tubulin gene of Aurantiacus is shown. Further, the base sequence described in SEQ ID NO: 8 represents the base sequence of the β-tubulin gene of Thermoscus cluster seus CBS181.67 strain, and the base sequence described in SEQ ID NO: 9 is Thermoscus aurantia. The base sequence of the β-tubulin gene of Kas NRRL5861 strain is shown.
The oligonucleotide (c) (also referred to herein as The_F2) and the oligonucleotide (d) (also referred to herein as The_R2) are each in position 33 of the nucleotide sequence set forth in SEQ ID NO: 8. In a stringent condition to a region from position to 53 and a region from position 306 to 328, a region from position 52 to position 72 and a region from position 323 to position 345 in the base sequence set forth in SEQ ID NO: 9. It is a hybridizable oligonucleotide. The present inventors have found that these regions are variable regions with low conserved base sequences between genera, and that the base sequences of these regions are base sequences inherent in Thermoscus fungi.
Therefore, Thermoscus fungi (in particular, Thermoscus cluster seus and Thermoscus aurantiacus) can be detected quickly and accurately using the oligonucleotides (c) and (d). . Specifically, it can be used as a primer specific to Thermoscus spp. If it is designed so that 5 or more bases of the 3 ′ end of the primer, which is the direction of DNA extension by polymerase, hybridize to this region. Identification by the PCR method using these primers becomes possible .

In the present invention, using the oligonucleotides (c) and (d), the presence of a nucleic acid (preferably the variable site) represented by any of the following base sequences (k) to (n) is confirmed. It is preferable to do.
(K) One or several (preferably 1 to 25, more preferably 1 to 25) nucleotide sequences of the β-tubulin gene represented by SEQ ID NO: 8 or its complementary sequence (l) SEQ ID NO: 8 20 bases, more preferably 1-15 bases, particularly preferably 1-10 bases, most preferably 1-5 bases have been deleted, substituted, inserted or added and A base sequence that can be used for detection of at least one selected from the group consisting of Thermoascus cluster seus and Thermoscus Aurantiacus), preferably 70% or more with respect to the base sequence set forth in SEQ ID NO: 8 (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more). A base sequence that can be used for detection of a fungus of the genus Moascus (preferably at least one selected from the group consisting of Thermoascus clusteraeus and Thermoscus aurantiacus), or a complementary sequence thereof (m) SEQ ID NO: 9 1 or several (preferably 1 to 25, more preferably 1 to 20, more preferably 1) in the base sequence of the β-tubulin gene or the complementary sequence (n) SEQ ID NO: 9 1-15, particularly preferably 1-10, most preferably 1-5, bases have been deleted, substituted, inserted or added and a Thermoscus fungus (preferably Thermoscus cluster A base sequence that can be used for detection of at least one selected from the group consisting of Seus and Thermoscus aurantiacus), SEQ ID NO: 9 70% or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more) to the described base sequence. And a base sequence that can be used for the detection of a Thermoscus fungus (preferably, at least one selected from the group consisting of Thermoscus clusteraeus and Thermoscus aurantiacus), or a complementary sequence thereof

The oligonucleotides (c) and (d) used in the detection method of the present invention are preferably oligonucleotides represented by the nucleotide sequence set forth in SEQ ID NO: 3 or 4. Moreover, if the oligonucleotide used in the detection method of the present invention can be used for detection of the genus Thermoscus (in particular, Thermoscus cluster seus and Thermoscus aurantiacus), it is represented by SEQ ID NO: 3 or 4. It may be an oligonucleotide represented by a base sequence having 70% or more homology to the base sequence or a complementary sequence thereof, more preferably homology of 80% or more, and homology of 85% or more. Is more preferable, the homology is more preferably 90% or more, and the homology is particularly preferably 95% or more. The oligonucleotide that can be used in the present invention is 1 or several, preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 1 in the base sequence described in SEQ ID NO: 3 or 4. 3, more preferably 1 or 2, particularly preferably 1 base deletion, substitution or insertion, and Thermoscus fungi (especially Thermoscus cluster seus and Thermoscus Also included are oligonucleotides that can be used to detect Aurantiacus). An appropriate base sequence may be added to the base sequence described in SEQ ID NO: 1 or 2.
The base sequence homology is calculated by the Lipman-Pearson method (Science, 1985, 227, p. 1435) or the like. Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the parameter Unit size to compare (ktup) is set to 2 and the calculation is performed. Can do.

  The binding mode of the oligonucleotide may be not only a phosphodiester bond existing in a natural nucleic acid but also a phosphoramidate bond, a phosphorothioate bond, or the like.

  The oligonucleotide can be prepared by a usual synthesis method such as chemical synthesis using, for example, an automatic DNA synthesizer. It is also possible to excise directly from the gene of Thermoscus sp. Using restriction enzymes or the like, or after cloning and isolating and purifying the gene and then excising using restriction enzymes or the like. It is preferable to prepare by chemical synthesis because it is easy to operate and can obtain a certain quality of oligonucleotide in a large amount and at low cost.

  The presence of the nucleic acid represented by at least one partial base sequence selected from the group consisting of the RPB1 gene and β-tubulin gene of Thermoscus fungus using the oligonucleotide pair, and Thermoscus fungus There is no particular limitation as to the method for detecting, and it can be performed by commonly used genetic engineering techniques such as sequencing, hybridization, PCR, real-time PCR, and LAMP.

Detector oligonucleotides of the invention can be used as nucleic acid primers.

In the detection method of the present invention, at least one base selected from the group consisting of the RPB1 gene and the β-tubulin gene of Thermoscus sp. It is preferable to amplify a part of the nucleic acid represented by the sequence and confirm the presence or absence of the amplification product. The method for amplifying a DNA fragment is not particularly limited. The polymerase chain reaction (PCR) method, real-time PCR method, LCR (Ligase Chain Reaction) method, SDA (Strand Displacement Amplification) method, NASBA (Nucleic Acid Sequence-based Amplification) method Ordinary methods such as RCA (Rolling-circle amplification) method and LAMP (Loop mediated isothermal amplification) method can be used. In the present invention, the PCR method is preferably used.
In the present invention, a case where an amplification reaction is carried out by the PCR method to detect a Thermoscus spp. However, the present invention is not limited to these.

In the present invention, from the oligonucleotides (a) and (b) (preferably, the oligonucleotide represented by the base sequence described in SEQ ID NO: 1 and the oligonucleotide represented by the base sequence described in SEQ ID NO: 2) PCR is performed using the oligonucleotide pair as a nucleic acid primer, and the nucleic acid represented by the base sequence of the RPB1 gene of Thermoscus fungus (preferably any of the base sequences of (e) to (j) above) It is preferable to amplify a part, confirm the presence or absence of the amplification product, and perform detection at the genus level of Thermoscus fungus. Here, the “Thermuscus fungus” in the present invention has the base sequence of the RPB1 gene described in any one of SEQ ID NOs: 5 to 7 in addition to those having the base sequences of SEQ ID NOs: 5 to 7, regardless of taxonomic classification. Homology with any of the nucleotide sequences is 70% or more (preferably 80% or more, more preferably 85% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 97% or more) 1 or several (preferably 1 to 25, more preferably 1 to 20, more preferably 1 to 15, more preferably 1) in the base sequence or the base sequence described in any one of SEQ ID NOs: 5 to 7 (Including 10 to 10 and most preferably 1 to 5) nucleotide sequences deleted, substituted, inserted or added in the RPB1 gene. The base sequence homology is calculated by the Lipman-Pearson method (Science, 1985, 227, p. 1435). Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the parameter Unit size to compare (ktup) is set to 2 and the calculation is performed. Can do.
The oligonucleotides (a) and (b) can amplify a nucleic acid represented by any one of the base sequences (e) to (j) or a part thereof by PCR, and the Thermoscus fungus It can function as a nucleic acid primer for rapid and accurate detection. Therefore, the oligonucleotide pair consisting of the oligonucleotides (a) and (b) can be used as a thermoscus fungus detection oligonucleotide pair.

In the present invention, the oligonucleotides (preferably the sequences) of (c) and (d) above are used to detect Thermoscus fungi (in particular, Thermoscus clusterseus and Thermoscus aurantiacus). PCR was performed using an oligonucleotide pair consisting of the oligonucleotide represented by the nucleotide sequence of No. 3 and the oligonucleotide represented by the nucleotide sequence of SEQ ID No. 4 as nucleic acid primers, and A portion of the nucleic acid represented by the base sequence of the β-tubulin gene (preferably any one of the base sequences (k) to (n) above) is amplified, the presence or absence of the amplified product is confirmed, and Thermoscus It is preferable to detect the genus fungus. Here, the “Thermuscus fungus” in the present invention has the β-tubulin gene sequence shown in SEQ ID NO: 8 or 9 as well as SEQ ID NO: 8 or 9 regardless of taxonomic classification. A base having a homology of 70% or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more) 1 or several (preferably 1 to 25, more preferably 1 to 20, more preferably 1 to 15, and particularly preferably 1 to 10) in the sequence or the nucleotide sequence of SEQ ID NO: 8 or 9 , Most preferably 1 to 5) including a base sequence in which a base is deleted, substituted, inserted or added, is included in the β-tubulin gene. The base sequence homology is calculated by the Lipman-Pearson method (Science, 1985, 227, p. 1435). Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the parameter Unit size to compare (ktup) is set to 2 and the calculation is performed. Can do.
The oligonucleotides (c) and (d) can amplify a nucleic acid represented by any one of the base sequences (k) to (n) or a part thereof by PCR, and rapidly It can function as a nucleic acid primer for accurate detection. Therefore, the oligonucleotide pair consisting of the oligonucleotides of (c) and (d) can be used as the oligonucleotide pair for the detection of Thermoscus fungus.

The PCR conditions are not particularly limited as long as the target DNA fragment can be amplified to a detectable level.
For example, when PCR is performed using an oligonucleotide pair consisting of the oligonucleotides of (a) and (b) as a nucleic acid primer, a preferable PCR condition is a heat denaturation reaction in which a double-stranded DNA is made into a single strand. An annealing reaction for hybridizing the primer pair to the single-stranded DNA at 5 ° C to 98 ° C for 5 seconds or more and 60 seconds or less is 55 ° C or more (preferably 59 ° C or more) and 65 ° C or less (preferably 62 ° C or less). For 60 seconds or less, preferably at 62 ° C. for 60 seconds, and by performing an elongation reaction for allowing DNA polymerase to act at about 72 ° C. for 10 seconds or more and 60 seconds or less. From the viewpoint of preventing false positives from occurring, preferably 30 cycles or less).
When PCR is performed using an oligonucleotide pair consisting of the oligonucleotides (c) and (d) as a nucleic acid primer, a preferable PCR condition is a heat denaturation reaction in which a double-stranded DNA is single-stranded at 95 ° C. or higher. An annealing reaction for hybridizing the primer pair to the single-stranded DNA at 98 ° C. or lower for 5 seconds or longer and 60 seconds or shorter at 55 ° C. or higher (preferably 59 ° C. or higher) or 65 ° C. or lower (preferably 62 ° C. or lower) for 5 seconds or longer 60 seconds or less, preferably 61 ° C. or more and 62 ° C. or less for 60 seconds, and an extension reaction that causes DNA polymerase to act is performed at about 72 ° C. for 10 seconds or more and 60 seconds or less, and these are defined as one cycle for 35 cycles or less ( From the viewpoint of preventing false positives from occurring, preferably 30 cycles or less).

In the present invention, confirmation of gene fragments can be performed by a usual method. Examples include a method for confirming the presence or absence of a band corresponding to the size of the amplified gene by electrophoresis on the amplified product, a method for measuring the amount of amplified product over time, and a method for decoding the base sequence of the amplified product. The present invention is not limited to these methods. In the present invention, a method of performing electrophoresis after gene amplification treatment and confirming the presence or absence of a band corresponding to the size of the amplified gene is preferable. In the present invention, the amplification product can be detected by a usual method. For example, a method of incorporating nucleotides labeled with a radioactive substance during an amplification reaction, a method of using a primer labeled with a fluorescent substance, or the like, and fluorescence intensity is increased by binding to DNA such as ethidium bromide between two amplified DNA strands. Although the method etc. which put in the fluorescent substance which becomes strong are mentioned, this invention is not limited to these methods. In the present invention, a method in which a fluorescent substance whose fluorescence intensity is increased by binding to DNA between the amplified DNA double strands is preferable.
When a specimen contains a fungus to be detected, PCR is performed using the oligonucleotide pair of the present invention as a primer set, and when the obtained PCR product is electrophoresed, a DNA fragment of a specific size is confirmed. Specifically, when the specimen contains Thermoscus fungus, electrophoresis of the PCR product obtained using the oligonucleotide pair consisting of the oligonucleotides (a) and (b) as a nucleic acid primer is performed. A DNA fragment is confirmed with a size of about 400 bp. In addition, when the specimen contains a Thermoscus fungus (particularly, Thermoscus cluster seus and / or Thermoscus aurantiacus), it comprises the oligonucleotides (c) and (d). When a PCR product obtained by using an oligonucleotide pair as a nucleic acid primer is electrophoresed, a DNA fragment is confirmed with a size of about 250 bp. By performing such an operation, it can be confirmed whether or not the detection target fungus is contained in the specimen.

In the present invention, at least one partial base sequence selected from the group consisting of the RPB1 gene and β-tubulin gene of the subject is determined using the oligonucleotide pair, and the partial base sequence is the above (e) to (e) n) by confirming whether it is included in any of the base sequences, the presence of the nucleic acid represented by any of the base sequences (e) to (n) above is confirmed, and It can also be detected. That is, the detection method of the present invention analyzes and determines at least one partial base sequence selected from the group consisting of the RPB1 gene and β-tubulin gene possessed by the subject, and the determined base sequence and the above (e) to (e) (N) is compared with any of the base sequences, and the presence or absence of the nucleic acid represented by any one of the base sequences (e) to (n) is confirmed based on the coincidence or difference. The detection is performed.
For example, PCR is performed using an oligonucleotide pair consisting of the oligonucleotides (a) and (b) as a primer, and a part of the nucleic acid represented by any one of the base sequences (e) to (j) is amplified. Then, the presence or absence of the amplification product is confirmed, the base sequence of the amplification product obtained is compared with any of the base sequences (e) to (j), and any one of the base sequences (e) to (j) The presence of the nucleic acid represented by If the base sequence of the amplification product is included as part of the nucleic acid represented by any of the base sequences (e) to (j) above, the microorganism contained in the specimen is identified as a Thermoscus fungus Can do. In this way, Thermoscus fungi can be detected quickly and accurately at the genus level.
Alternatively, PCR is performed using an oligonucleotide pair consisting of the oligonucleotides (c) and (d) as a primer to amplify a part of the nucleic acid represented by any one of the base sequences (k) to (n) Then, the presence or absence of the amplification product is confirmed, the base sequence of the amplification product obtained is compared with any of the base sequences (k) to (n), and any one of the base sequences (k) to (n) The presence of the nucleic acid represented by If the base sequence of the amplification product is included as a part of the nucleic acid represented by any one of the base sequences (c) to (f), the microorganism contained in the specimen is a Thermoscus fungus (in particular, a thermos Ascus Clusterseus and Thermoscus Aurantiacus). In this way, it is possible to quickly and accurately detect the thermospus fungus.
The method for analyzing and determining the base sequence is not particularly limited, and a commonly used technique of RNA or DNA sequencing can be used.
Specific examples include electrophoresis such as Maxam-Gilbert method and Sanger method, mass spectrometry, hybridization method and the like. Examples of the Sanger method include a method of labeling a primer or a terminator by a radiation labeling method, a fluorescence labeling method, or the like.

  In the present invention, the primer used in the amplification reaction can be chemically synthesized based on the designed sequence or purchased from a reagent manufacturer. Specifically, it can be synthesized using an oligonucleotide synthesizer or the like. Moreover, what was refine | purified using the adsorption column, the high performance liquid chromatography, and the electrophoresis method after a synthesis | combination can also be used. In addition, an oligonucleotide having a base sequence in which one to several bases are substituted, deleted, inserted or added can be synthesized using a conventional method.

There is no restriction | limiting in particular as a test substance used in this invention, Food / beverage products itself, the raw material of food / beverage products, an isolated microbial cell, a cultured cell, food / beverage products, or the packaging container of the raw material etc. can be used.
The method for preparing genomic DNA from a specimen is not particularly limited as long as DNA having a sufficient degree of purification and quantity can be obtained even in an unpurified state for detection of Thermoscus spp., Further separation and extraction Further, it can be used after pretreatment such as concentration and purification. For example, it can be used after treatment with phenol and chloroform, or purification using a commercially available extraction kit to increase the purity of the nucleic acid. In addition, DNA obtained by reverse transcription of RNA in a subject can also be used.

Thermo Asker scan genus fungi detection kit of the present invention, the oligonucleotide pair of the present invention are those which contain as nucleic acid primers. Kits of the present invention, in addition to the prior Kikaku acid primers, depending on the purpose, labeled detection agent, a buffer, a nucleic acid synthetase (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), enzyme substrates (dNTPs, rNTP, etc.) A substance usually used for detecting fungi. The kit of the present invention may include a positive control (positive control) to confirm that it is possible to detect the oligonucleotide pair of the present invention. Examples of the positive control include genomic DNA containing a region amplified by the method of the present invention.

Relates embodiment described above, the present invention further following thermo Asker scan genus detection method of a fungal, oligonucleotide pairs, thermo Asker scan genus fungi detection kit, use, and discloses methods.

<1> At least one oligonucleotide selected from the group consisting of an oligonucleotide pair consisting of the following oligonucleotides (a) and (b) and an oligonucleotide pair consisting of the following oligonucleotides (c) and (d): A method for detecting a Thermoscus fungus, comprising detecting a Thermoscus fungus using a pair.
(A) One or several oligonucleotides (preferably 1 to 5, more preferably 1 to 4, more preferably 1) in the oligonucleotide represented by the base sequence described in SEQ ID NO: 1 or the base sequence described in SEQ ID NO: 1. Are oligonucleotides that have 1 to 3, more preferably 1 or 2, particularly preferably 1) bases deleted, substituted, inserted or added, and can be used for the detection of Thermoscus fungi, or The homology with the nucleotide sequence shown in SEQ ID NO: 1 is 70% or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more), and Thermoscus Oligonucleotide that can be used for detection of genus fungi (b) The oligonucleotide represented by the base sequence described in SEQ ID NO: 2 or the base sequence described in SEQ ID NO: 2 And one or several (preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, still more preferably 1 or 2, particularly preferably 1) bases are deleted, Oligonucleotide which is substituted, inserted or added and can be used for detection of Thermoscus fungus, or has a homology of 70% or more (preferably 80% or more, more preferably 85%) with the nucleotide sequence of SEQ ID NO: 2. % Or more, more preferably 90% or more, particularly preferably 95% or more) and can be used for the detection of Thermosacus fungus (c) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 3, Or 1 or several (preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3) in the base sequence shown in SEQ ID NO: 3. More preferably, 1 or 2 (particularly preferably 1) bases are deleted, substituted, inserted or added, and can be used for the detection of Thermoscus fungi, or the base described in SEQ ID NO: 3 It has a homology with the sequence of 70% or more (preferably 80% or more, more preferably 85% or more, more preferably 90% or more, particularly preferably 95% or more) and can be used for the detection of Thermoscus fungi Oligonucleotide (d) 1 or several (preferably 1 to 5, more preferably 1 to 4) of the oligonucleotide represented by the base sequence set forth in SEQ ID NO: 4 or the base sequence set forth in SEQ ID NO: 4, More preferably, 1 to 3, more preferably 1 or 2, particularly preferably 1) base is deleted, substituted, inserted or added. And the homology with the oligonucleotide which can be used for the detection of a Thermoscus genus fungus, or the base sequence of SEQ ID NO: 4 is 70% or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% Or more, particularly preferably 95% or more) and can be used for detection of Thermoscus fungus

<2> For detection, a part of the nucleic acid represented by the base sequence of the RPB1 gene or β-tubulin gene of Thermoscus fungus is amplified using the at least one oligonucleotide pair, and amplified The detection method according to <1>, wherein the presence or absence of a product is confirmed, and a Thermoscus fungus is detected.
<3> A nucleic acid represented by the base sequence of the RPB1 gene of a Thermoscus fungus by performing a polymerase chain reaction using an oligonucleotide pair comprising the oligonucleotides of (a) and (b) as a nucleic acid primer. The detection method according to <2>, wherein the portion is amplified, the presence or absence of an amplification product is confirmed, and a Thermoscus fungus is detected.
<4> The detection method according to any one of <1> to <3>, wherein the base sequence of the RPB1 gene of the Thermoscus fungus is any of the following base sequences (e) to (j):
(E) 1 or several (preferably 1 to 25, more preferably 1 to 20) nucleotide sequences of the RPB1 gene represented by SEQ ID NO: 5 or its complementary sequence (f) SEQ ID NO: 5 More preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases are deleted, substituted, inserted or added and can be used for the detection of Thermoscus fungi 70% or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more) with respect to the base sequence described in SEQ ID NO: 5 Or a complementary sequence (g) of the RPB1 gene described in SEQ ID NO: 6 or a complementary sequence (h) thereof. 1 or several (preferably 1 to 25, more preferably 1 to 20, more preferably 1 to 15, particularly preferably 1 to 10, most preferably 1 to 5) in the base sequence described in No. 6. 70% or more (preferably 80% or more) of the base sequence described in SEQ ID NO: 6, wherein the base sequence is deleted, substituted, inserted or added and can be used for the detection of Thermosacus fungus More preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more), and a nucleotide sequence that can be used for detection of Thermoscus fungi, or One or several (preferably 1 to 25) of the complementary sequence (i) the base sequence of the RPB1 gene described in SEQ ID NO: 7 or the complementary sequence (j) of the base sequence described in SEQ ID NO: 7 More preferably 1 to 20, more preferably 1 to 15, particularly preferably 1 to 10, most preferably 1 to 5) bases are deleted, substituted, inserted or added and 70% or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95%) with respect to the base sequence described in SEQ ID NO: 7, which can be used for detection of genus fungi Or more, particularly preferably 97% or more) and a base sequence that can be used for the detection of Thermoscus fungi, or its complementary sequence <5> an oligo comprising the oligonucleotides (c) and (d) above Polymerase chain reaction is performed using nucleotide pairs as nucleic acid primers, and a portion of the nucleic acid represented by the base sequence of the β-tubulin gene of the Thermoscus spp. Width, to confirm the presence or absence of an amplification product, performs thermo Asuka scan genus detection of fungi, the detection method of the <2> above, wherein.
<6> The <1>, <2>, and <5> items, wherein the base sequence of the β-tubulin gene of the Thermoscus fungus is any of the following base sequences (k) to (n): One of the detection methods.
(K) One or several (preferably 1 to 25, more preferably 1 to 25) nucleotide sequences of the β-tubulin gene represented by SEQ ID NO: 8 or its complementary sequence (l) SEQ ID NO: 8 (20, more preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases have been deleted, substituted, inserted or added and are used for the detection of Thermoscus fungi 70% or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably the nucleotide sequence that can be used, or the nucleotide sequence set forth in SEQ ID NO: 8 97% or higher) and a base sequence that can be used for detection of Thermoscus fungi, or a complementary sequence thereof (m) the base sequence of the β-tubulin gene described in SEQ ID NO: 9 Or its complementary sequence (n) 1 or several (preferably 1 to 25, more preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10) in the nucleotide sequence of SEQ ID NO: 9. , Most preferably 1 to 5) of the base sequence which is deleted, substituted, inserted or added and can be used for the detection of Thermoscus fungus, 70 against the base sequence set forth in SEQ ID NO: 9 % Or more (preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more) and Base sequences that can be used for detection, or their complementary sequences <7> Selected from the group consisting of Thermoascus cluster seus and Thermoscus aurantiacus by the above detection method Detecting at least one that, the <1>, <2>, <5> and <6> detection method according to any one of Items.

<8> before Symbol (a) and oligonucleotide or the (c) and oligonucleotide pair consisting of an oligonucleotide of (d) of (b).

<9> the oligonucleotide is a nucleic acid primer, oligonucleotide pairs of the <8> above, wherein.
<10> Each of the oligonucleotides (a) and (b) can hybridize to the nucleic acid represented by any one of the base sequences (e) to (j), can function as a nucleic acid primer for detecting, oligonucleotide pairs of <9> above, wherein.
<11> The oligonucleotides (a) and (b) can amplify a part of the nucleic acid represented by any one of the base sequences (e) to (j) by polymerase chain reaction, genus are those which can function as a nucleic acid primer for detecting the fungi, the <10> oligonucleotide pair according to claim.
<12> Each of the oligonucleotides (c) and (d) can hybridize to the nucleic acid represented by any one of the base sequences (k) to (n), can function as a nucleic acid primer for detecting, oligonucleotide pairs of <9> above, wherein.
<13> The oligonucleotides (c) and (d) can amplify a part of the nucleic acid represented by any one of the base sequences (k) to (n) by polymerase chain reaction, genus are those which can function as a nucleic acid primer for detecting the fungi, the <12> oligonucleotide pair according to claim.
<14> the thermo Asker scan genus fungi is at least one selected from the group consisting of thermo Asuka scan-Kurasutaseusu and thermo Asker scan-a uranium tier Kas, the <12> or <13> O according to claim oligo Nucleotide pair.

<15> The <8> ~ <14> thermophilus Asker scan genus fungi detection kit comprising oligonucleotide pair according to any one of claim.

<16> Use of the oligonucleotides (a) and (b) as a nucleic acid primer for detecting a Thermoscus fungus.
<17> Use of the oligonucleotides (a) and (b) above for the production of a nucleic acid primer for detecting a Thermoscus fungus.
<18> A method of using the oligonucleotides (a) and (b) as a nucleic acid primer for detecting a Thermoscus fungus.
<19> Use of the oligonucleotides of (c) and (d) above as a nucleic acid primer for detecting a Thermoscus fungus.
<20> Use of the oligonucleotides of (c) and (d) above for the production of a nucleic acid primer for detecting a Thermoscus fungus.
<21> A method of using the oligonucleotides (c) and (d) as a nucleic acid primer for detecting a Thermoscus fungus.
<22> The above <19> to <21>, wherein the Thermoscus genus fungus is at least one selected from the group consisting of Thermoscus clusteraeus and Thermoscus aurantiacus Use or method.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Test Example 1 Preparation of molecular phylogenetic tree of thermostable fungi including Thermoscus fungus Thermoscus fungus (Thermuscus Aurantiacus, Thermoscus thermophilus, and Thermoscus cluster) by the following method Seusu), and various heat-resistant fungi (Talaromyces flavus (Talaromyces flavus), Talaromyces MACROSS Porras (Talaromyces macrosporus), Talaromyces helix (Talaromyces helicus var. helices), Talaromyces Torakisuperumusu (Talaromyces Trachyspermus), Talaromyces purpureus (Talaromyces purpureus), Talaromyces Walt Mann knee (Talaromyces wortmannii), Talaromyces Bashirisuporasu (Talaromyces bacillisporus), Talaromyces Samofiresu (Talaromyces thermophiles), Talaromyces Rute Scan (Talaromyces luteus), Talaromyces Emerusonii (Talaromyces emersonii), Talaromyces Viso lightheadedness Doi Death (Talaromyces byssochlamydoides), Byssochlamys-Supekutabirisu (Byssochlamys spectabilis), Byssochlamys-Nivea (Byssochlamys nivea), Talaromyces Reisetanasu (Talaromyces leycettanus), Hamigera The base sequences of the RPB1 gene and β-tubulin gene of Hamigera avellanea , Hamigera striata , and Asperguillus fumigatus ) Houbraken et al., Studies in Mycology, 2011, vol. 70, p. The determination was made with reference to the method described in 1-51.

Using a potato dextrose agar medium (PDA medium, manufactured by Difco), each bacterial cell was cultured in the dark at an appropriate temperature (25 ° C., 30 ° C., 37 ° C.) for 5 to 7 days. Gene Toru-kun (for yeast) High
DNA was extracted from the cells using Recovery (manufactured by TAKARA), and prepared with TE buffer so that the DNA concentration was 5 ng / μL.
The base sequence of RPB1 gene of each bacterial species is searched for DNA data bank of Japan (DDBJ: http://www.ddbj.nig.ac.jp/Welcome-j.html) by ARSA and F1843 primer (5'-atttygayggtgaygaratgaac -3 ′: SEQ ID NO: 10) and PCR using a R3096 primer (5′-gracrgtdccrtcatayttracc-3 ′: SEQ ID NO: 11) (denaturation temperature 94 ° C. 30 seconds, annealing temperature 47 ° C. 30 seconds, extension temperature 72 ° C. 1 minute) It was obtained by analysis of the PCR product obtained in 40 cycles). ARSA search of DNA data bank of Japan for the base sequence of β-tubulin gene of each bacterial species, and Bt2a primer (5'-aataggtgccgctttctgg-3 ': SEQ ID NO: 12) and Bt2b primer (5'-agttgtcgggacggaagag-3': SEQ ID NO: 13) (Glass and Donaldson, Appl Environ Microbiol 61: 1323-1330, 1995) using PCR (35 cycles of denaturation temperature 98 ° C. 5 seconds, annealing temperature 55 ° C. 5 seconds, extension temperature 72 ° C. 10 seconds) Obtained by analysis of the resulting PCR product. The PCR product was electrophoresed on a 2% agarose gel to confirm the size, and then purified using a High Pure PCR Product purification kit (trade name, manufactured by Roche). The purified PCR product was terminated using a Terminator Cycle Sequencing Ready Reaction kit (trade name, manufactured by Applied Biosystems), and for the RPB1 gene, a new R2623 primer (5'-gcrttgttsaratccttmarrctc-3 ': SEQ ID NO: 14) was used. -The tubulin gene was labeled using the above-mentioned primers and electrophoresed with an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). For determination of the base sequence from the fluorescence signal during electrophoresis, the software 'ATGC Ver. 4 ′ (Genetyx) was used.
The resulting nucleotide sequence is analyzed using Clustal X (http://clustalw.ddbj.nig.ac.jp/top-j.html), and analyzed by the neighbor binding method using the software NJPLOT. A molecular phylogenetic tree was created. The created molecular phylogenetic tree is shown in FIG.

As shown in FIG. 1, Thermoscus Aurantiacus, Thermoscus thermophilus and Thermoscus clusterseus constitute a single group of bacteria closely related to each other, It is clear that can be distinguished.
In addition, although not shown in FIG. 1, Bisoclamis varcosas classified into the genus Bisoclamis is present in the branch of the Thermoscus fungus, and is supposed to be originally classified as the Thermoscus genus. (See J. Houbraken et al., Studies in Mycology, 2011, vol. 70, p. 1-51).

Test Example 2 Analysis of partial base sequences of RPB1 gene and β-tubulin gene specific to Thermospus fungus Various types of Thermospus fungi determined by the above method, and known RPB1 gene and β- of various fungi Based on the base sequence information of the tubulin gene, alignment analysis was performed using Clustal W (http://clustalw.ddbj.nig.ac.jp/top-j.html), and the RPB1 of the Thermoscus spp. A gene-specific base sequence (The_RPB_F2: atctgccggcgtgatgtgttcctg (SEQ ID NO: 1), The_RPB_R2: gttgtgcagaagccagtagttgacc (SEQ ID NO: 2)), and a base sequence specific to the β-tubulin gene of the Thermospus fungus (The_F2: gatggttgtggt SEQ ID NO: 3) and The_R2: acgaggaacgtacttgttgccat (SEQ ID NO: 4)) were determined.

Example (1) Primer design Primer consisting of an oligonucleotide represented by the base sequence described in SEQ ID NO: 1 based on the base sequence site specific to Thermoscus fungus determined above (The_RPB_F2 primer) A primer composed of an oligonucleotide represented by the base sequence described in SEQ ID NO: 2 (The_RPB_R2 primer), a primer composed of an oligonucleotide represented by the base sequence described in SEQ ID NO: 3 (The_F2 primer), and the sequence described in SEQ ID NO: 4 A primer (The_R2 primer) consisting of an oligonucleotide represented by the nucleotide sequence of the above was designed, and synthesis was requested from Sigma-Aldrich Japan (desalted product, 0.02 μmol scale) and purchased.

(2) Preparation of specimens The fungi used for the evaluation of the effectiveness of the designed primer, that is, Thermoscus fungus and other heat-resistant fungi, and fungi reported in accident cases in food and drink, etc. are shown in Table 1. The strain described in ~ 3 was used. These strains are stocks stored by The Centraalbureau voor Schimmelcultures and managed by the CBS number, strains stored by the Center for Fungal Medicine, Chiba University and controlled by the SUN number, and stored by the Center for Fungal Medicine, Chiba University. The stock managed by the number and the stock stored by the National Institute of Technology and Evaluation managed by the NBRC number were obtained and used.
Each strain was cultured under optimal conditions. As for the culture conditions, potato dextrose medium (trade name: Pearl Core potato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.) was used for 7 days at the optimum temperature of each bacterium.

  Each cultured cell was collected using a platinum loop, and a genomic DNA solution was prepared using a genomic DNA preparation kit (PrepMan ultra (trade name) manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 5 ng / μL.

(3) Detection of Thermoscus spp.
As a DNA template, 1 μL of the fungal genomic DNA solution prepared in Table 1 and 2 prepared above, 25 μL of SapphireAmp Fast PCR Master Mix (trade name, manufactured by Takara Bio Inc.) and 22 μL of sterile distilled water were mixed, and The_RPB_F2 primer (20 pmol / 1 μL) and 1 μL of The_RPB_R2 primer (20 pmol / μL) were added to prepare a PCR solution.
The PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR conditions are as follows: treatment at 97 ° C. for 10 minutes, (i) heat denaturation reaction at 97 ° C. for 1 minute, (ii) annealing reaction at 62 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute. 30 cycles were performed, and the reaction solution was held at 72 ° C. for 10 minutes.

  After PCR, 3.5 μL or 2.5 μL is taken from the PCR solution and mixed well with 0.5 μL of loading buffer (Nucleic Acid sample loading buffer 5 × (manufactured by BIO RAD)), and 2% agarose gel is used. Electrophoresis (135V, 20 minutes; or 100V, 45 minutes). After completion of electrophoresis, the agarose gel was stained with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen), and the presence or absence of the amplified DNA fragment was confirmed. The results are shown in FIGS. The numbers in the figure indicate that the samples were reacted using DNA extracted from the samples with corresponding sample numbers in the table. 4 shows an electrophoretic diagram for the fungal sample shown in Table 1, and FIG. 5 shows an electrophoretic diagram for the fungal sample shown in Table 2. As a molecular weight marker, EZ Load 100bp molecular Ruler (trade name, manufactured by BIO RAD) was used.

  As a result, the gene fragment was confirmed at a size of about 400 bp in the sample containing the genomic DNA of Thermoscus spp. On the other hand, gene fragments were not confirmed in samples containing heat-resistant fungi other than Thermoscus spp. And other fungal genomic DNAs widely detected in food production environments. Therefore, according to the present invention, Thermoscus fungi can be specifically detected at the genus level.

(4) Detection of Thermoscus fungus 2
As a DNA template, 1 μL of the fungal genomic DNA solution prepared in Tables 1 and 3 above, 25 μL of SapphireAmp Fast PCR Master Mix (trade name, manufactured by Takara Bio Inc.) and 22 μL of sterile distilled water were mixed, and The_F2 primer (20 pmol / 1 μL) and 1 μL of The_R2 primer (20 pmol / μL) were added to prepare a PCR solution.
The PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR conditions are as follows: 97 ° C., 10 minutes treatment, (i) heat denaturation reaction at 97 ° C. for 1 minute, (ii) 61 ° C. or 62 ° C., 1 minute annealing reaction, and (iii) 72 ° C., 1 minute 30 cycles of the extension reaction were performed, and the reaction solution was held at 72 ° C. for 10 minutes.

  After PCR, 3.5 μL or 2.5 μL is taken from the PCR solution and mixed well with 0.5 μL of loading buffer (Nucleic Acid sample loading buffer 5 × (manufactured by BIO RAD)), and 2% agarose gel is used. Electrophoresis (135V, 20 minutes; or 100V, 45 minutes). After completion of electrophoresis, the agarose gel was stained with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen), and the presence or absence of the amplified DNA fragment was confirmed. The results are shown in FIGS. The numbers in the figure indicate that the samples were reacted using DNA extracted from the samples with corresponding sample numbers in the table. 6 shows an electrophoretic diagram for the fungal sample shown in Table 1, and FIG. 7 shows an electrophoretic diagram for the fungal sample shown in Table 3. As a molecular weight marker, EZ Load 100bp molecular Ruler (trade name, manufactured by BIO RAD) was used.

  As a result, among the Thermoscus fungi, a sample containing the genomic DNA of Thermoscus cluster seus, Thermoscus aurantiacus or Bisoclamis varkosa was confirmed to have a gene fragment with a size of about 250 bp. On the other hand, gene fragments were not confirmed in samples containing heat-resistant fungi other than the above-mentioned fungi and fungal genomic DNA widely detected from the food production environment.

  From the above results, by using the oligonucleotide of the present invention, it is possible to confirm the presence of the partial base sequence of the RPB1 gene or β-tubulin gene of the Thermospus fungus, and detect the RPB1 gene quickly and accurately. I understand that I can do it. Therefore, according to the method of the present invention, it is possible to collectively detect Thermoscus fungi more rapidly and more accurately than in the conventional method.

Claims (13)

An oligonucleotide pair consisting of the following oligonucleotides (a) and (b), and an oligonucleotide pair consisting of the following oligonucleotides (c) and (d):
A method for detecting a Thermoscus fungus, wherein the Thermoascus fungus is detected using at least one oligonucleotide pair selected from the group consisting of:
(A) An oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, or one base in the nucleotide sequence set forth in SEQ ID NO: 1 has been deleted, substituted, inserted or added, and a Thermoscus fungus (B) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 2, or one base is deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 2 Oligonucleotide that can be used for detection of fungus and Thermoscus fungus (c) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3, or one base is deleted in the nucleotide sequence set forth in SEQ ID NO: 3, Oligonucleotide that is substituted, inserted or added and can be used for detection of Thermoscus fungus (d) represented by the nucleotide sequence set forth in SEQ ID NO: 4 The oligonucleotide, or one base deletion in the nucleotide sequence set forth in SEQ ID NO: 4, substitution, insertion or addition has been provided and an oligonucleotide that can be used to detect thermo Asker scan genus fungi   In order to perform detection, a part of the nucleic acid represented by the nucleotide sequence of the RNA polymerase II large subunit gene or β-tubulin gene of Thermoscus fungus is amplified using the at least one oligonucleotide pair. The detection method according to claim 1, wherein the presence or absence of an amplification product is confirmed, and a Thermoscus fungus is detected.   A nucleic acid represented by the base sequence of the RNA polymerase II large subunit gene of Thermoscus fungus using the oligonucleotide pair consisting of the oligonucleotides of (a) and (b) as a nucleic acid primer The detection method of Claim 2 which amplifies a part of this, confirms the presence or absence of an amplification product, and performs a Thermoscus genus fungus detection. The detection method according to any one of claims 1 to 3, wherein a base sequence of the RNA polymerase II large subunit gene of the Thermoscus fungus is any one of the following base sequences (e) to (j):
(E) the base sequence of the RNA polymerase II large subunit gene shown in SEQ ID NO: 5 or its complementary sequence (f) one or several bases deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 5 A nucleotide sequence that can be used for detection of a Thermoscus fungus, or its complementary sequence (g), the nucleotide sequence of the RNA polymerase II large subunit gene described in SEQ ID NO: 6 or its complementary sequence (h) SEQ ID NO: 6 Or a complementary sequence thereof (i) described in SEQ ID NO: 7 in which one or several bases are deleted, substituted, inserted or added in the base sequence described in the above, and can be used for detection of Thermoscus fungi In the base sequence of RNA polymerase II large subunit gene or its complementary sequence (j) SEQ ID NO: 7 Number of nucleotides are deleted, substituted, inserted or added in and and nucleotide sequences can be used to detect thermo Asker scan genus fungi, or its complementary sequence   One of the nucleic acids represented by the base sequence of the β-tubulin gene of Thermoscus fungus by performing polymerase chain reaction using the oligonucleotide pair consisting of the oligonucleotides of (c) and (d) as a nucleic acid primer. The detection method of Claim 2 which amplifies a part, confirms the presence or absence of an amplification product, and performs a Thermoscus genus fungus detection. The detection method according to any one of claims 1, 2, and 5, wherein a base sequence of the β-tubulin gene of the genus Thermoscus is any one of the following base sequences (k) to (n).
(K) the base sequence of the β-tubulin gene described in SEQ ID NO: 8 or its complementary sequence (1) one or several bases are deleted, substituted, inserted or added in the base sequence described in SEQ ID NO: 8 And a base sequence that can be used for the detection of fungi of the genus Thermoscus, or its complementary sequence (m), the base sequence of the β-tubulin gene described in SEQ ID NO: 9 or its complementary sequence (n), the base described in SEQ ID NO: 9 A base sequence in which one or several bases have been deleted, substituted, inserted or added in the sequence and can be used for detection of a Thermoscus fungus, or a complementary sequence thereof Under Symbol (a) and oligonucleotide or oligonucleotide pair consisting of an oligonucleotide of the following (c) and (d) of (b).
(A) An oligonucleotide represented by the base sequence described in SEQ ID NO: 1 or a base sequence described in SEQ ID NO: 1 with one base deleted, substituted, inserted or added, and Thermoascus ) Oligonucleotide that can be used for detection of genus fungi (b) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 2, or one base in the nucleotide sequence set forth in SEQ ID NO: 2 is deleted, substituted, inserted or An oligonucleotide which is added and can be used for detection of Thermoscus fungus (c) an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 or one base in the nucleotide sequence of SEQ ID NO: 3 Oligonucleotide (d) which is deleted, substituted, inserted or added and can be used for detection of Thermoscus fungus Oligonucleotide represented by a nucleotide sequence, or an oligonucleotide in which one base is deleted, substituted, inserted or added in the nucleotide sequence of SEQ ID NO: 4 and can be used for detection of Thermoscus fungi The oligonucleotide is a nucleic acid primer, oligonucleotide pair of claim 7, wherein. The oligonucleotides (a) and (b) can each hybridize to a nucleic acid represented by any one of the following base sequences (e) to (j), and to detect a Thermoscus fungus of it can function as nuclei acid primers, oligonucleotide pair of claim 8.
(E) the base sequence of the RNA polymerase II large subunit gene shown in SEQ ID NO: 5 or its complementary sequence (f) one or several bases deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 5 A nucleotide sequence that can be used for detection of a Thermoscus fungus, or its complementary sequence (g), the nucleotide sequence of the RNA polymerase II large subunit gene described in SEQ ID NO: 6 or its complementary sequence (h) SEQ ID NO: 6 Or a complementary sequence thereof (i) described in SEQ ID NO: 7 in which one or several bases are deleted, substituted, inserted or added in the base sequence described in the above, and can be used for detection of Thermoscus fungi In the base sequence of RNA polymerase II large subunit gene or its complementary sequence (j) SEQ ID NO: 7 Number of nucleotides are deleted, substituted, inserted or added in and and nucleotide sequences can be used to detect thermo Asker scan genus fungi, or its complementary sequence The oligonucleotides (a) and (b) can amplify a part of the nucleic acid represented by any of the base sequences (e) to (j) by polymerase chain reaction, it is capable of functioning as a nucleic acid primer for detecting, oligonucleotide pair of claim 9, wherein. The oligonucleotides (c) and (d) can each hybridize to a nucleic acid represented by any one of the following base sequences (k) to (n), and to detect a Thermoscus fungus of it can function as nuclei acid primers, oligonucleotide pair of claim 8.
(K) the base sequence of the β-tubulin gene described in SEQ ID NO: 8 or its complementary sequence (1) one or several bases are deleted, substituted, inserted or added in the base sequence described in SEQ ID NO: 8 And a base sequence that can be used for the detection of fungi of the genus Thermoscus, or its complementary sequence (m), the base sequence of the β-tubulin gene described in SEQ ID NO: 9 or its complementary sequence (n), the base described in SEQ ID NO: 9 A base sequence in which one or several bases have been deleted, substituted, inserted or added in the sequence and can be used for detection of a Thermoscus fungus, or a complementary sequence thereof The oligonucleotides (c) and (d) can amplify a part of the nucleic acid represented by any one of the base sequences (k) to (n) by polymerase chain reaction, it is capable of functioning as a nucleic acid primer for detecting, oligonucleotide pair of claim 11, wherein. Thermo Asuka scan (Thermoascus) genus fungi detection kit comprising oligonucleotide pair of any one of claims 7-12.