JP5548385B2 - Detection method of heat-resistant fungi - Google Patents

Description

  The present invention relates to a method for detecting heat-resistant fungi.

  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. In addition, heat-resistant fungi have higher heat resistance than other normal fungi. For example, even when an acid beverage is heat sterilized, the heat-resistant fungi can survive and multiply, causing mold generation. For this reason, heat-resistant fungi are wary of important harmful bacteria that cause serious accidents.

The main heat-resistant fungi bacteria causing contamination accidents, which may also be detected from the heat sterilization treatment after the food or beverage, Byssochlamys (Byssochlamys) genus Talaromyces (Talaromyces) genus Neosartorya (Neosartorya) genus and Hamigera (Hamigera ) Thermostable fungi belonging to the genus are known. Compared to other heat-resistant fungi that form ascospores, fungi belonging to the above four genera have a high heat resistance and are highly likely to survive after heat sterilization. On the other hand, since heat-resistant fungi other than the above four genera can be killed under normal sterilization conditions, there is little possibility of causing a contamination accident unless there is a sterilization defect or the like. Therefore, detection and identification of heat-resistant fungi belonging to these four genera are particularly important for preventing accidents caused by heat-resistant fungi in foods and drinks and their raw materials.
Furthermore, identification of the causative agent of the accident is necessary for investigation and countermeasure of the accident at the time of the occurrence of the harm accident. Therefore, if the four genera of heat-resistant fungi can be identified, it is possible to more quickly detect and identify the causative bacteria of the accident.

  On the other hand, as a conventional method for detecting and identifying heat-resistant fungi, there is a method for detecting and identifying a specimen by culturing a specimen in a PDA medium or the like. However, this method requires about 7 days until colonies are confirmed. In addition, since the identification of the bacterial species is performed based on the characteristic organ morphology of the fungus, it requires about 7 days of culture until morphological characteristics are recognized. Therefore, according to this method, it takes about 14 days to detect and identify heat-resistant fungi. The long time required for detection and identification of heat-resistant fungi is not always satisfactory from the viewpoints of hygiene management of food and drink, ensuring freshness of raw materials, restrictions on distribution, and the like. For this reason, there is a demand for more rapid detection and identification of heat-resistant fungi.

  As a rapid detection and identification method of fungi, a detection method using a PCR (polymerase chain reaction) method is known (for example, see Patent Documents 1 to 4). However, these methods have a problem that it is difficult to detect specific heat-resistant fungi specifically and quickly.

Japanese National Patent Publication No. 11-505728 JP 2006-61152 A JP 2006-304763 A JP 2007-174903 A

  An object of the present invention is to provide a method capable of specifically and rapidly detecting and identifying heat-resistant fungi that are the main causative bacteria of food and beverage contamination.

As described above, one of the factors that make it difficult to detect thermostable fungi is that the PCR method using a conventional known primer gives false positive or false negative results.
The present inventors have intensively studied to search for a new DNA region capable of overcoming this problem and specifically detecting and identifying a specific thermostable fungus. As a result, the β-tubulin gene and 28S rDNA of thermostable fungi have a region with a specific base sequence that can be clearly distinguished from those of other fungi (hereinafter also referred to as “variable region”). The inventors have found that the heat-resistant fungi can be specifically and rapidly detected and identified by targeting this variable region. The present invention has been completed based on this finding.

The present invention, Byssochlamys (Byssochlamys) genus Talaromyces (Talaromyces) genus Neosartorya (Neosartorya) genus, in a method of detecting at least two fungi selected from the group consisting of Aspergillus fumigatus (Aspergillus fumigatus) and Hamigera (Hamigera) genus The present invention relates to a method for detecting heat-resistant fungi comprising at least two steps selected from the group consisting of 1) to 4) below.
1) A step of identifying a fungus belonging to the genus Bysochramys using a nucleic acid represented by the base sequence of the following (A):
2) A step of identifying a fungus belonging to the genus Talaromyces using a nucleic acid represented by any one of the following base sequences (B) to (D):
3) conducting the identification of the following (E) ~ (J) Neosartorya using a nucleic acid represented by any one of the nucleotide sequences of (Neosartorya) fungi belonging to the genus and / or Aspergillus fumigatus (Aspergillus fumigatus),
4) A step of identifying a fungus belonging to the genus Hamigera using a nucleic acid represented by the base sequence (K) below

(A) For detection of fungi belonging to the genus Bisoclamis, wherein one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 24 or a complementary sequence thereof, or in the base sequence or the complementary sequence thereof Usable base sequence (B) The base sequence shown in SEQ ID NO: 25 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof is deleted, substituted or added, and belongs to the genus Talalomyces The base sequence (C) that can be used for detecting fungi (C) The base sequence set forth in SEQ ID NO: 26 or its complementary sequence, or one or several bases in the base sequence or its complementary sequence has been deleted, substituted, or added; Base sequence (D) that can be used for detection of fungi belonging to the genus Talalomyces Or a complementary sequence thereof, or a base sequence (E) SEQ ID NO: 28, which can be used for detection of fungi belonging to the genus Talaromyces, wherein one or several bases are deleted, substituted or added in the base sequence or the complementary sequence Used for the detection of fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria, in which one or several bases are deleted, substituted or added in the described base sequence or its complementary sequence, or the base sequence or its complementary sequence Possible base sequence (F) The base sequence shown in SEQ ID NO: 29 or its complementary sequence, or one or several bases in the base sequence or its complementary sequence is deleted, substituted or added, and belongs to the genus Neosartoria Salts that can be used to detect fungi and / or Aspergillus fumigatus Fungi belonging to the genus Neosartoria, wherein one or several bases are deleted, substituted or added in the base sequence described in the base sequence (G) SEQ ID NO: 30 or a complementary sequence thereof, or in the base sequence or the complementary sequence thereof And / or a base sequence (H) that can be used for detection of Aspergillus fumigatus or the base sequence set forth in SEQ ID NO: 31 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof deleted, substituted, or added Base sequence that can be used for the detection of fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria (I) the base sequence set forth in SEQ ID NO: 32 or its complementary sequence, or one or a number in the base sequence or its complementary sequence Neobases deleted and substituted or added The base sequence described in SEQ ID NO: 33 or its complementary sequence, or the base sequence or its complementary sequence lacking one or several bases can be used to detect fungi belonging to the genus Ria and / or Aspergillus fumigatus. A nucleotide sequence which is deleted, substituted or added and which can be used for detection of fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria (K), the nucleotide sequence of SEQ ID NO: 34 or a complementary sequence thereof, or the nucleotide sequence or its complement A nucleotide sequence in which one or several bases have been deleted, substituted or added in the sequence and can be used for detection of fungi belonging to the genus Hamigera

The present invention also provides a kit for detecting a thermostable fungus comprising at least two oligonucleotide pairs selected from the following groups (1) to (4) and selected from different groups as nucleic acid primers: About.
(1) Oligonucleotide pair of (a) and (b) below (a) The base sequence described in SEQ ID NO: 1 or its complementary sequence, or 70% or more homology with the base sequence or its complementary sequence And an oligonucleotide represented by a base sequence that can be used as a detection oligonucleotide (b) 70% or more homology to the base sequence of SEQ ID NO: 2 or its complementary sequence, or the base sequence or its complementary sequence (2) the following oligonucleotide pairs (c) and (d), the following (g) and (h) oligonucleotide pairs, and the following ( (i) and (h) oligonucleotide pairs, (e) and (f) oligonucleotide pairs, and (j) and (k) below. (C) the nucleotide sequence of SEQ ID NO: 3 or its complementary sequence, or a homology of 70% or more to the nucleotide sequence or its complementary sequence, and can be used as a detection oligonucleotide Oligonucleotide represented by a base sequence (d) As a detection oligonucleotide having 70% or more homology with the base sequence shown in SEQ ID NO: 4 or its complementary sequence, or the base sequence or its complementary sequence Oligonucleotide represented by a usable base sequence (e) Oligonucleotide for detection having 70% or more homology with the base sequence of SEQ ID NO: 5 or its complementary sequence, or the base sequence or its complementary sequence Oligonucleotide represented by a nucleotide sequence that can be used as a nucleotide (f) The nucleotide sequence described in SEQ ID NO: 6 Or a complementary sequence thereof, or an oligonucleotide represented by a base sequence having 70% or more homology with the base sequence or the complementary sequence and usable as a detection oligonucleotide (g) described in SEQ ID NO: 7 An oligonucleotide represented by a nucleotide sequence or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or a complementary sequence thereof and usable as a detection oligonucleotide (h) An oligonucleotide represented by the nucleotide sequence described above or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or a complementary sequence thereof and usable as a detection oligonucleotide (i) SEQ ID NO: The base sequence according to 9, or its complementary sequence, or the base sequence or its complementary sequence (J) a nucleotide sequence represented by SEQ ID NO: 10 or a complementary sequence thereof, or the nucleotide sequence or a nucleotide sequence thereof; Oligonucleotide represented by a nucleotide sequence having 70% or more homology to a complementary sequence and usable as a detection oligonucleotide (k) the nucleotide sequence described in SEQ ID NO: 11 or its complementary sequence, or the nucleotide sequence Or an oligonucleotide represented by a base sequence that has a homology of 70% or more to its complementary sequence and can be used as a detection oligonucleotide (3) an oligonucleotide pair of the following (l) and (m): n) and (o) oligonucleotide pairs, and (v) and (w) oligonucleotide pairs (L) a nucleotide sequence represented by SEQ ID NO: 12 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide. (M) the nucleotide sequence of SEQ ID NO: 13 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide (N) The base sequence described in SEQ ID NO: 14 or a complementary sequence thereof, or has a homology of 70% or more to the base sequence or the complementary sequence and can be used as a detection oligonucleotide. Oligonucleotide represented by the nucleotide sequence (o) The nucleotide sequence of SEQ ID NO: 15 or A complementary sequence, or an oligonucleotide represented by a base sequence that has 70% or more homology to the base sequence or its complementary sequence and can be used as a detection oligonucleotide (v) the base sequence described in SEQ ID NO: 22 Or a complementary sequence thereof, or an oligonucleotide represented by a base sequence having 70% or more homology with the base sequence or the complementary sequence and usable as a detection oligonucleotide (w) described in SEQ ID NO: 23 Oligonucleotide represented by a base sequence or its complementary sequence, or a base sequence having 70% or more homology with the base sequence or its complementary sequence and usable as a detection oligonucleotide (4) And (q) oligonucleotide pairs, the following (r) and (s) oligonucleotide pairs, A group consisting of the following oligonucleotide pairs (t) and (u) (p) having 70% or more homology with the base sequence set forth in SEQ ID NO: 16 or a complementary sequence thereof, or the base sequence or the complementary sequence thereof And an oligonucleotide represented by a base sequence that can be used as a detection oligonucleotide (q) 70% or more homology to the base sequence set forth in SEQ ID NO: 17 or a complementary sequence thereof, or the base sequence or the complementary sequence thereof (R) a nucleotide sequence represented by SEQ ID NO: 18 or a complementary sequence thereof, or 70% or more of the nucleotide sequence or a complementary sequence thereof Oligonucleotides represented by nucleotide sequences that have homology and can be used as oligonucleotides for detection (S) represented by the nucleotide sequence of SEQ ID NO: 19 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide. Oligonucleotide (t) represented by the nucleotide sequence shown in SEQ ID NO: 20 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide (U) the nucleotide sequence set forth in SEQ ID NO: 21 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide Oligonucleotide represented by

  ADVANTAGE OF THE INVENTION According to this invention, the method which can detect and identify the heat-resistant fungi which are the main causative bacteria of the contamination accident of food-drinks specifically and rapidly can be provided.

It is the figure which compared a part of base sequence of the β-tubulin gene of Aspergillus fumigatus, Neosartria fischeri fischeri, and Neosartria fischeri spinosa. It is a figure which shows the base sequence of (beta) -tubulin gene of Hamigera abellanea and cladosporium cladosporides. It is an electrophoretic diagram which shows the identification result of the fungi which belong to the genus Bisoclamis in Example 1 (A-1). It is an electrophoretic diagram using each strain of bisoclamis falba in Example 1 (A-2). It is an electrophoretic diagram using each strain of Bisochramis nivea in Example 1 (A-2). It is an electrophoretic diagram which shows the identification result of the fungi which belong to the Tallaromyces genus in Example 1 (B-1). It is an electrophoretic diagram which shows the identification result of the fungi which belong to the Tallaromyces genus in Example 1 (B-2). It is an electrophoretic diagram which shows the result of identification of the fungi which belong to the Tallaromyces genus in Example 1 (B-2). It is an electropherogram in Example 1 (B-3). It is an electrophoretic diagram in Example 1 (B-4). It is an electrophoretic diagram using each strain of Talaromyces flavus in Example 1 (B-5). It is an electrophoretic diagram using each strain of Talaromyces macrosporus in Example 1 (B-5). It is an electrophoretic diagram which shows the identification result of the fungi which belong to Neosartoria genus in Example 1 (C-1), and Aspergillus fumigatus. It is an electrophoretic diagram which shows the identification result of the fungi which belong to Neosartoria genus and Aspergillus fumigatus in Example 1 (C-2). It is an electrophoretic diagram which shows the discrimination | determination result of the fungi which belong to Neosartoria genus and Aspergillus fumigatus in Example 1 (C-3). It is an electrophoretic diagram which shows the discrimination | determination result of the fungi which belong to Neosartoria genus and Aspergillus fumigatus in Example 1 (C-3). It is an electrophoretic diagram using each strain of Neosartria fischeri fischeri in Example 1 (C-4). It is an electrophoretic diagram using each strain of Neosartria Fischeri grabra in Example 1 (C-4). It is an electrophoretic diagram using each strain of Neosartria hiratsukae in Example 1 (C-4). It is an electrophoretic diagram using each strain of Neosartoria Paulis tensis in Example 1 (C-4). It is an electrophoretic diagram using each strain of Neosartoria Fischeri spinosa in Example 1 (C-4). It is an electrophoretic diagram which shows the identification result of the fungi which belong to the Hamigella genus in Example 1 (D-1). It is an electrophoretic diagram which shows the identification result of the fungi which belong to the Hamigella genus in Example 1 (D-2). It is an electrophoretic diagram which shows the identification result of the fungi which belong to the Hamigella genus in Example 1 (D-3). It is an electrophoretic diagram using each strain of Hamigella striata in Example 1 (D-4). It is an electrophoretic diagram using each strain of Hamigella abernea in Example 1 (D-5). It is an electrophoretic diagram using fungi belonging to the genus Hamigera and the genus Bisoclamis in Example 1 (D-6). It is an electrophoretic diagram using fungi belonging to the genus Hamigera and the genus Bisoclamis in Example 1 (D-6).

Hereinafter, the present invention will be described in detail.
The present invention relates to a partial base sequence of β-tubulin gene of thermostable fungi and / or a base sequence of D1 / D2 region of 28S rDNA, that is, D1 / D2 of β-tubulin gene region and / or 28S rDNA of thermostable fungi. The heat-resistant fungi are identified by using the nucleic acid represented by the nucleotide sequence of the region specific to each genus of the heat-resistant fungi in the D2 region or the species-specific region (variable region). It is a method of identifying and detecting.
More specifically, the present invention is a method for detecting a heat-resistant fungus comprising at least two of the following identification and detection steps 1) to 4).
Performs identification of fungi belonging to Byssochlamys genus using a nucleic acid represented by the nucleotide sequence of 1) Byssochlamys (Byssochlamys) belonging to the genus fungi β- tubulin gene region-specific areas Byssochlamys genus in the (variable region) The process of specifically identifying and detecting fungi belonging to the genus Bisoclamis.
2) It is represented by a base sequence of a region specific to the genus Taralomyces or a species-specific region (variable region) in the β-tubulin gene region of a fungus belonging to the genus Talaromyces and / or the D1 / D2 region of 28S rDNA. Identifying a fungus belonging to the genus Talalomyces using a nucleic acid to specifically identify and detect the fungus belonging to the genus Talalomyces.
3) Neosartorya (Neosartorya) belonging to the genus fungi or Aspergillus fumigatus (Aspergillus fumigatus) of β- tubulin gene region regions specific to Neosartorya genus and / or Aspergillus fumigatus in the or species-specific region of the (variable region) A step of identifying fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria using a nucleic acid represented by the base sequence, and specifically identifying and detecting fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria.
4) It belongs to the genus Hamigera using a nucleic acid represented by the nucleotide sequence of a region specific to the genus Hamigera or a species-specific region (variable region) in the β-tubulin gene region of fungi belonging to the genus Hamigera The process of identifying and detecting fungi belonging to the genus Hamigera by identifying fungi.
The detection method of the present invention comprises at least two of the identification / detection steps of 1) to 4) above, and preferably includes at least three of the identification / detection steps of 1) to 4) above. More preferably, it includes all the identification / detection steps 1) to 4) above.

The “heat-resistant fungi” in the present invention means fungi belonging to the genus Bisoclamis, fungi belonging to the genus Talalomyces, fungi belonging to the genus Neosartoria, fungi belonging to the genus Aspergillus fumigatus and Hamigera. These fungi are irregular ascomycetous fungi belonging to the family Trichocomaceae , and are thermostable fungi that form viable spore spores even after heat treatment at 75 ° C. for 30 minutes. Examples of the fungi belonging to the genus Bisoclamis include Bisochlamys fulva and Bysochramys nivea . Examples of fungi belonging to the Talaromyces genus Talaromyces flavus (Talaromyces flavus), Talaromyces luteus (Talaromyces luteus), Talaromyces Torakisuperumusu (Talaromyces trachyspermus), Talaromyces Walt Mann knee (Talaromyces wortmannii), Talaromyces Bashirisuporasu (Talaromyces bacillisporus), Talaromyces Macross Porras ( Talaromyces macrosporus ). Examples of fungi belonging to the genus Neosartria include Neosartria fischeri spinosa ( Neosartoria fischeri var. Spinosa; hereinafter referred to as Neosartoria fischeri var. Spinosa); Neosartria fischeri var. fischeri glabra (Neosartorya fischeri var glabra;. or less and Neosartorya glabra), Neosartorya Hiratsukae (Neosartorya hiratsukae), Neosartorya Pauli Sten cis (Neosartorya paulistensis), Neosartorya pseudoephedrine fischeri (Neosarto rya peudofischeri ). Examples of the fungi belonging to the genus Hamigera include Hamigera avelnea and Hamigera striata .
The term “Aspergillus fumigatus” in the present invention is a kind of incomplete fungus that is morphologically very similar to anamorph (asexual generation) of Neosartria ficelli but has no teleomorph (sexual generation).

  “Β-tubulin” is a protein constituting a microtubule, and “β-tubulin gene” is a gene encoding β-tubulin. In the present invention, the “variable region” is a region in which base mutations tend to accumulate in the β-tubulin gene or in the D1 / D2 region of 28S rDNA, and the base sequence of this region is a genus or species of fungus. Varies greatly. The variable region of the β-tubulin gene of these fungi or the D1 / D2 region of 28S rDNA is a base sequence unique to these fungi, so that it can be clearly distinguished from those of other fungi.

  The detection method of the present invention is a nucleic acid represented by the following base sequences (A) to (K), that is, a specific region (variable) in the β-tubulin gene of heat-resistant fungi or the D1 / D2 region of 28S rDNA. The nucleic acid corresponding to the base sequence of (region) is used.

In the detection method of the present invention, in order to detect fungi belonging to the genus Bisoclamis, a nucleic acid represented by the base sequence of the following (A), that is, a specific region (variable region) in the β-tubulin gene of the fungus belonging to the genus Bisoclamis The nucleic acid corresponding to the base sequence of) is used.
(A) For detection of fungi belonging to the genus Bisoclamis, wherein one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 24 or a complementary sequence thereof, or in the base sequence or the complementary sequence thereof Usable Base Sequences The inventors have identified the base sequences of various β-tubulin genes from fungi belonging to the genus Bisoclamis and fungi related to the genus Bisoclamis, And genetic distance analysis within the genus Bisoclamis. Furthermore, homology analysis of various determined β-tubulin gene sequences was performed. As a result, a variable region having a base sequence unique to a fungus belonging to the genus Bisoclamis was found in the sequence. In this variable region, fungi belonging to the genus Bisoclamis have a unique base sequence, and therefore it is possible to identify and identify fungi belonging to the genus Bisoclamis.

In the detection method of the present invention, a nucleic acid represented by any one of the following base sequences (B) to (D) for detecting a fungus belonging to the genus Talalomyces, that is, a β-tubulin gene of a fungus belonging to the genus Tallalomyces A nucleic acid corresponding to the base sequence of the specific region (variable region) in the D1 / D2 region of the middle and / or 28S rDNA is used.
(B) For detection of a fungus belonging to the genus Talalomyces wherein one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 25 or a complementary sequence thereof, or the base sequence or the complementary sequence thereof Usable nucleotide sequence (C) The nucleotide sequence described in SEQ ID NO: 26 or its complementary sequence, or one or several bases in the nucleotide sequence or its complementary sequence is deleted, substituted or added and belongs to the genus Talalomyces The base sequence (D) that can be used for detecting fungi (D) The base sequence set forth in SEQ ID NO: 27 or its complementary sequence, or one or several bases in the base sequence or its complementary sequence has been deleted, substituted, or added; Nucleotide sequences that can be used to detect fungi belonging to the genus Talalomyces The base sequences of the D1 / D2 region of various β-tubulin genes and 28S rDNA are identified from fungi that are closely related to the fungi belonging to the genus and Talamomyces, And the genetic distance within Talamomyces was analyzed. Furthermore, homology analysis of the determined various β-tubulin gene sequences and the nucleotide sequence of the D1 / D2 region of 28S rDNA was performed. As a result, a variable region having a base sequence unique to a fungus belonging to the genus Talalomyces was found in the sequence. In this variable region, fungi belonging to the genus Talalomyces have a unique base sequence, so that it is possible to identify and identify fungi belonging to the genus Talalomyces.

In the detection method of the present invention, in order to detect fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus, a nucleic acid represented by any one of the following base sequences (E) to (J), that is, a fungus belonging to the Neosartoria genus Alternatively, a nucleic acid corresponding to the base sequence of a specific region (variable region) in the β-tubulin gene of Aspergillus fumigatus is used.
(E) a fungus belonging to the genus Neosartria, wherein one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 28 or a complementary sequence thereof, or in the base sequence or the complementary sequence thereof, and / or Alternatively, the base sequence (F) that can be used for detection of Aspergillus fumigatus or the complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof is deleted, substituted, or added. And the base sequence (G) that can be used for detecting fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus (G) SEQ ID NO: 30 or its complementary sequence, or one or several of the base sequence or its complementary sequence Neosartria with base deleted, substituted or added A base sequence described in SEQ ID NO: 31 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof, which can be used for detecting fungi and / or Aspergillus fumigatus belonging to In the base sequence described in SEQ ID NO: 32, or a complementary sequence thereof, or the base sequence or a complementary sequence thereof, which is substituted or added and can be used for detection of fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria One or several bases deleted, substituted or added and used for detection of fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus (J) Base sequence described in SEQ ID NO: 33 or its complementary sequence Or the base sequence or its complementary sequence One or several bases deleted, substituted or added, and a base sequence that can be used for detection of fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus Nucleotide sequences of various β-tubulin genes were identified from fungi related to fungi belonging to the genus Neosartria, and between Neosartria and related genus and Neosartria, within Neosartria and within Neosartria We analyzed the genetic distance between the genera closely related to the genus and Neosartria and Aspergillus fumigatus. Furthermore, homology analysis of various determined β-tubulin gene sequences was performed. As a result, a variable region having a base sequence unique to fungi belonging to the genus Neosartoria and Aspergillus fumigatus was found in the sequence. In this variable region, fungi and Aspergillus fumigatus belonging to the genus Neosartoria have a unique base sequence, so that it is possible to identify and identify fungi and Aspergillus fumigatus belonging to the genus Neosartoria.

In the detection method of the present invention, in order to detect fungi belonging to the genus Hamigera, a nucleic acid represented by the following base sequence (K), that is, a specific region (variable region) in the β-tubulin gene of the fungus belonging to the genus Hamigera A nucleic acid corresponding to the base sequence is used.
(K) For detection of fungi belonging to the genus Hamigera, wherein one or several bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 34 or a complementary sequence thereof, or the base sequence or the complementary sequence thereof Usable base sequences The inventors have identified the base sequences of various β-tubulin genes from fungi belonging to the genus Hamigera and fungi close to the fungus belonging to the genus Hamigera. And the genetic distance within the genus Hemigera. Furthermore, homology analysis of various determined β-tubulin gene sequences was performed. As a result, a variable region having a base sequence unique to a fungus belonging to the genus Hamigera was found in the sequence. In this variable region, fungi belonging to the genus Hamigera have a unique base sequence, so that it becomes possible to identify and identify fungi belonging to the genus Hamigera.
The present invention targets these variable regions and nucleic acids and oligonucleotides derived from the variable regions.

The base sequence (A) used in the detection method of the present invention corresponds to a partial base sequence of the β-tubulin gene of a fungus belonging to the genus Bisoclamis.
The nucleotide sequence set forth in SEQ ID NO: 24 and its complementary sequence are the nucleotide sequences of the variable region of the β-tubulin gene isolated and identified from B. clamis nivea. This sequence is specific to fungi belonging to the genus Bisoclamis, and it is possible to specifically identify and identify fungi belonging to the genus Bisoclamis by confirming whether the subject has this base sequence. is there. In addition, a nucleic acid represented by a base sequence that can be used to detect fungi belonging to the genus Bisoclamis, wherein one or several bases are deleted, substituted, or added in the base sequence set forth in SEQ ID NO: 24 or its complementary sequence Similarly, it is possible to specifically identify and identify fungi belonging to the genus Bisoclamis. Nucleic acids represented by these base sequences are particularly preferably used for detecting B. clamis nivea and B. clamis farva.

The base sequence (B) or (C) used in the detection method of the present invention corresponds to a partial base sequence of the β-tubulin gene of a fungus belonging to the genus Talalomyces. The base sequence (D) corresponds to the base sequence of the variable region of the D1 / D2 region of 28S rDNA of fungi belonging to the genus Talalomyces.
The base sequence set forth in SEQ ID NO: 25 and its complementary sequence are the base sequence of the variable region of the β-tubulin gene isolated and identified from Talaromyces flavus. This sequence is specific to fungi belonging to the genus Tallalomyces, and it is possible to specifically identify and identify fungi belonging to the genus Tallalomyces by confirming whether the subject has this base sequence. is there. In addition, a nucleic acid represented by a base sequence that can be used to detect a fungus belonging to the genus Talalomyces, wherein one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 25 or its complementary sequence Similarly, it is possible to specifically identify and identify fungi belonging to the genus Taralomyces. Nucleic acids represented by these base sequences are preferably used for detecting Talaromyces flavus and Talaromyces traxpermus.
The base sequence set forth in SEQ ID NO: 26 and its complementary sequence are the base sequence of the variable region of the β-tubulin gene isolated and identified from Talaromyces luteus. This sequence is specific to fungi belonging to the genus Tallalomyces, and it is possible to specifically identify and identify fungi belonging to the genus Tallalomyces by confirming whether the subject has this base sequence. is there. In addition, a nucleic acid represented by a base sequence described in SEQ ID NO: 26 or a complementary sequence thereof, wherein one or several bases are deleted, substituted or added and can be used for detection of a fungus belonging to the genus Talalomyces Similarly, it is possible to specifically identify and identify fungi belonging to the genus Taralomyces. Nucleic acids represented by these nucleotide sequences are preferably used for detecting Talaromyces luteus, Talaromyces bacilis porus, Talaromyces wortmannie.
The nucleotide sequence set forth in SEQ ID NO: 27 and its complementary sequence are the nucleotide sequences of the variable regions in the D1 / D2 region of 28S rDNA isolated and identified from Talalomyces wortmanny. This sequence is specific to fungi belonging to the genus Tallalomyces, and it is possible to specifically identify and identify fungi belonging to the genus Tallalomyces by confirming whether the subject has this base sequence. is there. In addition, a nucleic acid represented by a base sequence represented by SEQ ID NO: 27 or a complementary sequence thereof, wherein one or several bases are deleted, substituted or added and can be used for detection of fungi belonging to the genus Talalomyces Similarly, it is possible to specifically identify and identify fungi belonging to the genus Taralomyces. The nucleic acids represented by these base sequences are preferably used for detecting Talaromyces wortmanny, Talaromyces flavus, Talaromyces traxpermus, Talaromyces macrosporus.

The base sequences (E), (G) to (J) used in the detection method of the present invention correspond to partial base sequences of β-tubulin genes of fungi belonging to the genus Neosartoria. The base sequence (F) corresponds to a partial base sequence of the β-tubulin gene of Aspergillus fumigatus.
The nucleotide sequence set forth in SEQ ID NO: 28 and its complementary sequence are the nucleotide sequences of the variable region of the β-tubulin gene isolated and identified from Neosartria grabra. The base sequences described in SEQ ID NOs: 30 and 31 and their complementary sequences are partial base sequences of the variable region of the β-tubulin gene isolated and identified from Neosartria fischeri. The base sequences described in SEQ ID NOs: 32 and 33 and their complementary sequences are partial base sequences of the variable region of the β-tubulin gene isolated and identified from Neosartria spinosa. The base sequence shown in SEQ ID NO: 29 and its complementary sequence are the base sequences of the variable region of the β-tubulin gene isolated and identified from Aspergillus fumigatus. The sequence is specific to fungi and / or Aspergillus fumigatus belonging to the genus Neosartria, and by confirming whether the subject has this base sequence, fungi and / or Aspergillus fumigatus belonging to the genus Neosartria Can be specifically identified and identified. Further, in the base sequence described in any of SEQ ID NOs: 28 to 33 or a complementary sequence thereof, fungi and / or Aspergillus fumigatus belonging to Neosartoria genus in which one or several bases are deleted, substituted or added Similarly, fungi and / or Aspergillus fumigatus belonging to the genus Neosartria can be specifically identified and identified using a nucleic acid represented by a base sequence that can be used for detection. Nucleic acids represented by these nucleotide sequences are particularly suitable for detecting Neosartria grabra, Neosartria fischeri, Neosartria spinosa, Neosartria hiratsukae, Neosartria Paulistensis, Neosartria pseudofischeri and Aspergillus fumigatus. It is done.

The base sequence of (K) used in the detection method of the present invention corresponds to a partial base sequence of the β-tubulin gene of a fungus belonging to the genus Hamigera.
The nucleotide sequence set forth in SEQ ID NO: 34 and its complementary sequence are the nucleotide sequences of the variable region of the β-tubulin gene isolated and identified from Hamigella abernea. This sequence is specific to fungi belonging to the genus Hamigera, and it is possible to specifically identify and identify fungi belonging to the genus Hamigera by confirming whether or not the subject has this base sequence. is there. Further, a nucleic acid represented by a base sequence described in SEQ ID NO: 34 or a complementary sequence thereof, wherein one or several bases have been deleted, substituted or added and can be used for detection of fungi belonging to the genus Hamigera Similarly, it is possible to specifically identify and identify fungi belonging to the genus Hamigera. Nucleic acids represented by these base sequences are particularly preferably used for detecting Hamigella abernea and Hamigella striata.
Hereinafter, any one of the base sequences (A) to (K) is collectively referred to as a “base sequence of the variable region of the present invention”.

  The method for identifying thermostable fungi using the nucleic acid represented by the base sequence of the variable region of the present invention is not particularly limited, and may be a commonly used genetic engineering technique such as a sequencing method, a hybridization method, or a PCR method. It can be carried out.

In the detection method of the present invention, in order to identify a thermostable fungus using the nucleic acid represented by the base sequence of the variable region of the present invention, the β1 / tubulin gene region of the subject or D1 / D of 28S rDNA is used. It is preferable to determine the base sequence of the D2 region and confirm whether or not any of the base sequences (A) to (K) is included in the base sequence of the region.
More specifically, in order to identify a fungus belonging to the genus Bisocramis using the nucleic acid represented by the base sequence of the variable region of the present invention, determine the base sequence of the β-tubulin gene region of the subject, It is preferable to confirm whether or not the base sequence of (A) is included in the base sequence of the region. That is, the detection method of the present invention analyzes and determines the base sequence of the β-tubulin gene possessed by the subject, and the determined base sequence and the variable region of the β-tubulin gene described in (A) of the present invention. Comparing the nucleotide sequence and identifying a fungus belonging to the genus Bisoclamis based on the match or difference is included as a preferred embodiment.
In order to identify a fungus belonging to the genus Talamomyces using the nucleic acid represented by the base sequence of the variable region of the present invention, the base sequence of the β-tubulin gene region of the subject or the D1 / D2 region of 28S rDNA It is preferable to determine and confirm whether or not any of the base sequences (B) to (D) is included in the base sequence of the region. That is, the detection method of the present invention analyzes and determines the base sequence of the D1 / D2 region of the β-tubulin gene or 28S rDNA possessed by the subject, and the determined base sequence and the above (B) to (D It is preferable to compare the base sequence of the variable region of the D1 / D2 region of β-tubulin gene or 28S rDNA described in any of the above and identify the fungi belonging to the genus Talalomyces based on the match or difference It is included as an embodiment.
In order to identify fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus using the nucleic acid represented by the base sequence of the variable region of the present invention, the base sequence of the β-tubulin gene region of the subject is determined. It is preferable to confirm whether or not any of the base sequences (E) to (J) is contained in the base sequence of the region. That is, the detection method of the present invention analyzes and determines the base sequence of the β-tubulin gene possessed by the subject, and the determined base sequence and the β of any one of the above (E) to (J) of the present invention. -Comparing the nucleotide sequence of the variable region of the tubulin gene and identifying fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria based on the match or difference is included as a preferred embodiment.
In order to identify a fungus belonging to the genus Hamigella using the nucleic acid represented by the base sequence of the variable region of the present invention, the base sequence of the β-tubulin gene region of the subject is determined, and the base sequence of the region It is preferable to confirm whether or not the base sequence (K) is contained therein. That is, the detection method of the present invention analyzes and determines the base sequence of the β-tubulin gene possessed by the subject, and the determined base sequence and the variable region of the β-tubulin gene described in (K) of the present invention. And the identification of the fungi belonging to the genus Hamigera based on the match or difference is included as a preferred embodiment.
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, in order to detect and identify thermostable fungi using the nucleic acid represented by the base sequence of the variable region of the present invention, the base sequence of the variable region of the present invention, that is, (A) to (A) to Use a detection oligonucleotide that can hybridize to the nucleic acid represented by any one of the base sequences of (K) and can function as a nucleic acid probe or nucleic acid primer for specifically detecting heat-resistant fungi. Can do.
More specifically, in order to detect and identify fungi belonging to the genus Bisoclamis, it can hybridize to the nucleic acid represented by the base sequence of the variable region of the present invention, that is, the base sequence of (A), In addition, a detection oligonucleotide that can function as a nucleic acid probe or a nucleic acid primer for specifically detecting fungi belonging to the genus Bisoclamis can be used.
In order to detect and identify a fungus belonging to the genus Tallalomyces, it may hybridize to the nucleic acid represented by the base sequence of the variable region of the present invention, that is, any one of the base sequences (B) to (D). A detection oligonucleotide that can function as a nucleic acid probe or nucleic acid primer for specifically detecting fungi belonging to the genus Talalomyces can be used.
In order to detect and identify fungi and / or Aspergillus fumigatus belonging to the genus Neosartria, the nucleotide sequence of the variable region of the present invention, that is, the nucleic acid represented by any one of the nucleotide sequences (E) to (J) And a detection oligonucleotide that can function as a nucleic acid probe or a nucleic acid primer for specifically detecting fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria.
In order to detect and identify fungi belonging to the genus Hamigera, the fungi that can hybridize to the nucleic acid represented by the base sequence of the variable region of the present invention, that is, the (K) base sequence, and belong to the genus Hamiger An oligonucleotide for detection that can function as a nucleic acid probe or a nucleic acid primer for specifically detecting a nucleoside can be used.
The detection oligonucleotide of the present invention may be any as long as it can be used for detection of the above heat-resistant fungi. That is, it can be used as a nucleic acid primer or a nucleic acid probe for the detection of thermostable fungi, or in the base sequence of the variable region of the D1 / D2 region of β-tubulin gene or 28S rDNA of thermostable fungi under stringent conditions Any oligonucleotide that can be hybridized may be used. Here, examples of the “stringent conditions” include the conditions described later.

The detection oligonucleotide of the present invention is a region selected from the base sequence of the variable region of the D1 / D2 region of the β-tubulin gene or 28S rDNA of the present invention, and (1) the thermostable fungi (2) The GC content of the oligonucleotide is approximately 30% to 80%, and (3) the possibility of self-annealing of the oligonucleotide is low. (4) An oligonucleotide capable of hybridizing to a region satisfying the four conditions of Tm value (melting temperature) of about 55 ° C. to 65 ° C. is preferable.
More specifically, when detecting a fungus belonging to the genus Bisocramis, a region in the nucleic acid represented by the base sequence of (A) above, wherein (1) the base sequence of the gene unique to the fungus belonging to the genus Bisocramis is (2) the oligonucleotide has a GC content of approximately 30% to 80%, (3) the possibility of self-annealing of the oligonucleotide is low, and (4) the Tm of the oligonucleotide. Oligonucleotides that can hybridize to a region that satisfies the four conditions of approximately 55 ° C. to 65 ° C. are preferable.
When detecting a fungus belonging to the genus Talalomyces, it is a region in the nucleic acid represented by any one of the base sequences (B) to (D), and (1) a base of a gene unique to the fungus belonging to the genus (2) the oligonucleotide has a GC content of approximately 30% to 80%, (3) the possibility of self-annealing of the oligonucleotide is low. An oligonucleotide capable of hybridizing to a region satisfying the four conditions of Tm value of approximately 55 ° C. to 65 ° C. is preferable.
When detecting fungi and / or Aspergillus fumigatus belonging to the genus Neosartria, it is a region in the nucleic acid represented by any one of the base sequences (E) to (J), and (1) Neosartria and / or Including a region where the base sequence of a gene unique to Aspergillus fumigatus appears continuously around 10 bases, (2) the GC content of the oligonucleotide is approximately 30% to 80%, (3) the possibility of self-annealing of the oligonucleotide (4) Oligonucleotides that can hybridize to regions satisfying the four conditions of (4) Tm value of the oligonucleotide is about 55 ° C. to 65 ° C. are preferable.
When detecting fungi belonging to the genus Hamigera, the region in the nucleic acid represented by the base sequence of (K) above, (1) the base sequence of the gene unique to the fungus belonging to the genus Haemiella continues about 10 bases. (2) the oligonucleotide has a GC content of approximately 30% to 80%, (3) the possibility of oligonucleotide self-annealing is low, and (4) the oligonucleotide has a Tm value of approximately 55 ° C. An oligonucleotide capable of hybridizing to a region satisfying the four conditions of ˜65 ° C. is preferable.
In the above (1), “a region in which the base sequence of a gene unique to a fungus belonging to the genus Bisoclamis appears continuously about 10 bases” refers to a region between different genera among the variable regions of the β-tubulin gene of the present invention. Is particularly low (that is, the specificity of the fungus belonging to the genus Bisocramis is particularly high), and means a region in which the base sequence unique to the fungus belonging to the genus Bisocramis appears continuously for about 10 bases. In addition, the “region where the base sequence of a gene unique to a fungus belonging to the genus Talalomyces appears continuously about 10 bases” refers to the β-tubulin gene of the present invention or the variable region of the D1 / D2 region of 28S rDNA. The region where the conserved base sequence between different genera is particularly low (that is, the specificity of the fungus belonging to the genus Tallalomyces is particularly high), and the region where the base sequence unique to the fungus belonging to the genus Talalomyces appears continuously for around 10 bases Means. In addition, the “region in which the base sequence of a gene unique to a fungus belonging to the genus Neosartoria or Aspergillus fumigatus appears continuously about 10 bases” refers to different intergenus among the variable regions of the β-tubulin gene of the present invention. Is particularly low (that is, the specificity of fungi belonging to the genus Neosartoria or Aspergillus fumigatus is particularly high) It means the area that appears. The “region where the base sequence of a gene unique to fungi belonging to the genus Hamigera appears continuously in about 10 bases” refers to the base sequence between different genera in the variable region of the β-tubulin gene of the present invention. Is particularly low (that is, the specificity of the fungus belonging to the genus Hemigera is particularly high), and means a region where the base sequence unique to the fungus belonging to the genus Hamigera appears continuously over about 10 bases.
In addition, in the above (3), “the possibility of oligonucleotide self-annealing is low” means that the primers are not expected to bind to each other based on the base sequence of the primers.
The number of bases of the detection oligonucleotide of the present invention is not particularly limited, but is preferably 13 to 30 bases, and more preferably 18 to 23 bases. The Tm value of the oligonucleotide at the time of hybridization is preferably in the range of 55 ° C to 65 ° C, and more preferably in the range of 59 ° C to 62 ° C. The GC content of the oligonucleotide is preferably 30% to 80%, more preferably 45% to 65%, and most preferably around 55%.

As the detection oligonucleotide of the present invention, the following oligonucleotides (a) to (w) are more preferable.
By using the oligonucleotide shown in the following (a) or (b), fungi belonging to the genus Bisoclamis can be detected.
(A) represented by the nucleotide sequence of SEQ ID NO: 1 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or its complementary sequence and usable as a detection oligonucleotide Oligonucleotide (b) represented by the nucleotide sequence of SEQ ID NO: 2 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide Oligonucleotide

By using the oligonucleotide shown in any one of the following (c) to (k), fungi belonging to the genus Talalomyces can be detected.
(C) represented by the nucleotide sequence of SEQ ID NO: 3 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide. Oligonucleotide (d) represented by the nucleotide sequence set forth in SEQ ID NO: 4 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide Oligonucleotide (e) the nucleotide sequence set forth in SEQ ID NO: 5 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide (F) the nucleotide sequence set forth in SEQ ID NO: 6 or its complementary sequence, An oligonucleotide having a homology of 70% or more with respect to the base sequence or its complementary sequence and represented by a base sequence that can be used as a detection oligonucleotide (g) the base sequence of SEQ ID NO: 7 or its complementary sequence, Or an oligonucleotide represented by a nucleotide sequence having 70% or more homology with the nucleotide sequence or its complementary sequence and usable as a detection oligonucleotide (h) the nucleotide sequence of SEQ ID NO: 8 or its complement Or an oligonucleotide represented by a nucleotide sequence having 70% or more homology to the nucleotide sequence or its complementary sequence and usable as a detection oligonucleotide (i) the nucleotide sequence of SEQ ID NO: 9 or 70% or more homology with the complementary sequence, or the base sequence or the complementary sequence And an oligonucleotide (j) represented by a base sequence that can be used as a detection oligonucleotide (j) having a base sequence of SEQ ID NO: 10 or a complementary sequence thereof, or 70% or more homology with the base sequence or the complementary sequence An oligonucleotide represented by a base sequence that can be used as an oligonucleotide for detection (k), the base sequence set forth in SEQ ID NO: 11 or a complementary sequence thereof, or a homology of 70% or more with the base sequence or the complementary sequence thereof Oligonucleotides represented by a base sequence that can be used as detection oligonucleotides

By using any of the following oligonucleotides (l) to (o), fungi belonging to the genus Neosartoria and Aspergillus fumigatus can be detected.
(L) represented by the nucleotide sequence set forth in SEQ ID NO: 12 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide Oligonucleotide (m) represented by the nucleotide sequence set forth in SEQ ID NO: 13 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide (N) the nucleotide sequence of SEQ ID NO: 14 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide (O) the nucleotide sequence of SEQ ID NO: 15 or its complementary sequence Or an oligonucleotide represented by a base sequence that has 70% or more homology to the base sequence or its complementary sequence and can be used as a detection oligonucleotide; and any one of the following (v) and (w) Aspergillus fumigatus can be specifically detected by using this oligonucleotide.
(V) represented by the nucleotide sequence set forth in SEQ ID NO: 22 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide. Oligonucleotide (w) represented by the nucleotide sequence set forth in SEQ ID NO: 23 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide Oligonucleotide

By using any of the following oligonucleotides (p) to (u), it is possible to detect fungi belonging to the genus Hamigera.
(P) represented by the nucleotide sequence of SEQ ID NO: 16 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or its complementary sequence and usable as a detection oligonucleotide. Oligonucleotide (q) represented by the nucleotide sequence set forth in SEQ ID NO: 17 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide Oligonucleotide (r) the nucleotide sequence set forth in SEQ ID NO: 18 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide The nucleotide sequence shown in SEQ ID NO: 19 or a complementary sequence thereof Or an oligonucleotide represented by a nucleotide sequence having 70% or more homology to the nucleotide sequence or its complementary sequence and usable as a detection oligonucleotide (t) or the complement thereof. Or an oligonucleotide represented by a nucleotide sequence having 70% or more homology to the nucleotide sequence or its complementary sequence and usable as a detection oligonucleotide (u) An oligonucleotide represented by a complementary sequence thereof, or a nucleotide sequence having 70% or more homology with the base sequence or the complementary sequence and usable as a detection oligonucleotide

That is, as the detection oligonucleotide of the present invention, an oligonucleotide represented by the base sequence described in any one of SEQ ID NOs: 1 to 23 or a complementary sequence thereof, and the base sequence described in any of SEQ ID NOs: 1 to 23 Alternatively, an oligonucleotide represented by a base sequence having 70% or more homology with its complementary sequence, which can be used for detection of heat-resistant fungi is preferable. The base sequence that can be used for detection of thermostable fungi has 70% or more homology to the base sequence described in any of SEQ ID NOs: 1 to 23, and includes a nucleic acid primer for detection of thermostable fungi, Nucleic acid probes that can be used for detection of thermostable fungi, and those that can hybridize to the D1 / D2 region of the β-tubulin gene of each thermostable fungus or the 28S rDNA of fungi belonging to the genus Taralomyces under stringent conditions Good. Here, “stringent conditions” include, for example, the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell., Cold Spring Harbor Laboratory Press], for example, 6 × SSC (composition of 1 × SSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. And the conditions of constant temperature for 8 to 16 hours and hybridization. As long as the oligonucleotide used in the present invention can be used for the detection of heat-resistant fungi as described above, the homology is more preferably 75% or more, and the homology is 80% or more. The homology is more preferably 85% or more, the homology is more preferably 90% or more, and the homology is particularly preferably 95% or more.
In addition, the oligonucleotide for detection of the present invention can be used as a base for the oligonucleotide represented by any one of SEQ ID NOS: 1 to 23 as long as it can be used for detection of heat-resistant fungi. Also included are mutations such as deletions, insertions or substitutions, and modified oligonucleotides. In addition, the oligonucleotide of the present invention is represented by a nucleotide sequence obtained by modifying or modifying a nucleotide sequence described in any of SEQ ID NOS: 1 to 23 such as deletion, insertion or substitution of a base. Oligonucleotides that can be used to detect heat-resistant fungi as primers and probes for the detection of heat-resistant fungi, and the β-tubulin gene of each heat-resistant fungus under the stringent conditions It may be a base sequence capable of hybridizing to the D1 / D2 region of 28S rDNA. Here, “stringent conditions” include, for example, the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell., Cold Spring Harbor Laboratory Press], for example, 6 × SSC (1 × SSC composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. And the conditions of constant temperature for 8 to 16 hours and hybridization. One or several oligonucleotides represented by any one of the nucleotide sequences of SEQ ID NOS: 1 to 23 are preferably used as oligonucleotides that have been subjected to such mutations such as deletion, insertion or substitution of bases, or modifications. 1 to 5, more preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, particularly preferably 1 mutation, modification such as deletion, insertion or substitution of one base Also included are oligonucleotides made. In addition, an appropriate base sequence may be added to the base sequence described in any one of SEQ ID NOs: 1 to 23. The base sequence homology is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985) or the like. Specifically, using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (manufactured by software development), the calculation is performed by performing an analysis with the parameter unit size to compare (ktup) as 2. Can do.

  The oligonucleotides shown in the above (a) and (b) are oligonucleotides that specifically hybridize with the variable region of the β-tubulin gene of fungi belonging to the genus Bisoclamis. Therefore, by using the oligonucleotide, the fungi belonging to the genus Bisoclamis can be detected specifically, rapidly and simply.

  The oligonucleotides represented by the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 are present in the β-tubulin gene region and are complementary to a specific nucleotide sequence of a fungus belonging to the genus Bisoclamis, that is, a part of the variable region. Oligonucleotide. The oligonucleotides represented by the base sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 can specifically hybridize with a part of DNA and RNA of fungi belonging to the genus Bisoclamis.

  The variable region of the β-tubulin gene of fungi belonging to the genus Bisoclamis will be described in detail using Bisoclamis nivea as an example. As described above, the partial base sequence of the β-tubulin gene of B. clamis nivea is represented by SEQ ID NO: 24. Among these, the region from the 20th position to the 175th position of the partial base sequence of the β-tubulin gene of fungi belonging to the genus Bisoclamis is a region having a particularly low conserved base sequence between fungal genera and having a base sequence unique to the genus The present inventors have found that. The oligonucleotides (a) and (b) correspond to regions from position 33 to position 52 and position 159 to position 178, respectively, of the base sequence set forth in SEQ ID NO: 24. Therefore, by hybridizing the oligonucleotide to the β-tubulin gene of a fungus belonging to the genus Bisocramis, the fungus belonging to the genus Bisoclamis can be specifically detected.

  The oligonucleotides (c) to (k) are oligonucleotides that specifically hybridize with the β-tubulin gene of fungi belonging to the genus Talalomyces or the variable region of the D1 / D2 region of 28S rDNA. Therefore, by using the oligonucleotide pair, it is possible to specifically, quickly and easily detect the fungus belonging to the genus Taralomyces.

  The oligonucleotides represented by the nucleotide sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4 are present in the β-tubulin gene region, and are specific nucleotide sequences specific to Talaromyces flavus and Talalomyces traxpermus in fungi belonging to the genus Talalomyces, An oligonucleotide complementary to a portion of the variable region. The oligonucleotides represented by the nucleotide sequences of SEQ ID NOs: 5-6 and SEQ ID NOs: 10-11 are present in the β-tubulin gene region, and among the fungi belonging to the genus Talaromyces, Talaromyces luteus, Talaromyces wortmanny, Talalomyces It is an oligonucleotide complementary to a base sequence specific to Bacillus porus, that is, a part of the variable region. The oligonucleotides represented by the nucleotide sequences set forth in SEQ ID NOs: 7 to 8 and SEQ ID NO: 9 are present in the D1 / D2 region in 28S rDNA, and among the fungi belonging to the genus Talaromyces, Talaromyces flavus, Talalomyces wortmanny, Talalomyces It is an oligonucleotide complementary to a base sequence specific to Trachyspermus or Talaromyces macrosporus, that is, a part of the variable region. These oligonucleotides can specifically hybridize to a part of DNA and RNA of fungi belonging to the genus Talalomyces.

The variable region of the β-tubulin gene of a fungus belonging to the genus Talalomyces will be described in detail using Talalomyces flavus and Talalomyces luteus as examples. As described above, the partial base sequence of the β-tubulin gene of Talalomyces flavus is represented by SEQ ID NO: 25, and the partial base sequence of the β-tubulin gene of Talaromyces luteus is represented by SEQ ID NO: 26. Among these, the variable region of the partial base sequence of the β-tubulin gene of Talalomyces flavus is a region from positions 10 to 40, and the region from positions 70 to 100 is a sequence with particularly high conservation as Talalomyces flavus and Talalomyces traxpermus The present inventors have found that this is a region having no similar sequence in other fungi. Furthermore, the variable region of the partial base sequence of the β-tubulin gene of Talalomyces luteus is the region from 120 to 160 and the region from 295 to 325 are genetically related Talalomyces luteus, Talalomyces wortmanny, Talalomyces The present inventors have found that this is a region that is highly conserved with Basilisporus and has no sequence similar to other fungi.
The oligonucleotides (c) and (d) correspond to the variable regions from the 15th position to the 34th position and from the 76th position to the 98th position, respectively, in the base sequence set forth in SEQ ID NO: 25. The oligonucleotides (e) and (f) and the oligonucleotides (j) and (k) are variable regions from the 133th position to the 153rd position and from the 304th position to the 325th position, respectively, in the base sequence set forth in SEQ ID NO: 26. Corresponding to Therefore, a fungus belonging to the genus Tallalomyces can be detected by hybridizing the oligonucleotide to the β-tubulin gene of a fungus belonging to the genus Talalomyces.

The variable region of the D1 / D2 region of 28S rDNA of fungi belonging to the genus Talalomyces will be described in detail with Talalomyces wortmanny as an example. As described above, the partial base sequence of the D1 / D2 region of 28S rDNA of Talalomyces wortmannny is represented by SEQ ID NO: 27. Among these, the regions from the 300th to 350th positions and the 450th to 510th positions of the partial base sequence of the D1 / D2 region of the 28S rDNA of fungi belonging to the genus Talalomyces are Talalomyces wort Manny, Talalomyces traxpermus, Talalomyces flavus, Talalomyces macross. The present inventors have found that this is a region that is particularly conserved in porous and has no similar sequence in other fungi.
The oligonucleotides represented by (g), (h) and (i) correspond to the variable regions from position 326 to position 345 to position 460 to position 478, respectively, of the base sequence set forth in SEQ ID NO: 27. . Therefore, a fungus belonging to the genus Tallalomyces can be detected by hybridizing the oligonucleotide to the D1 / D2 region of 28S rDNA of a fungus belonging to the genus Talalomyces.

  The oligonucleotides (l) to (o) are oligonucleotides that specifically hybridize with the variable region of the β-tubulin gene of fungi belonging to the genus Neosartoria and Aspergillus fumigatus. Therefore, by using the oligonucleotides (l) to (o), the fungi belonging to the genus Neosartoria and Aspergillus fumigatus can be specifically, rapidly and easily detected.

  The oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOs: 12 to 15 are present in the β-tubulin gene region, and include Neosartria Fischeri, Neosartria spinosa, Neosartria grabra, Neosartria hiratsukae, Neosartria paulistensis, It is an oligonucleotide complementary to a part of the variable region, that is, a base sequence specific to fungi belonging to the genus Neosartria such as Neosartria pseudofiseri and Aspergillus fumigatus. These oligonucleotides can specifically hybridize to DNA and RNA parts of fungi belonging to the genus Neosartoria and Aspergillus fumigatus.

The variable region of the β-tubulin gene of fungi belonging to the genus Neosartria will be described in detail using a neosartria grabula as an example. As described above, the partial base sequence of the Neo-Sartria grabra β-tubulin gene is represented by SEQ ID NO: 28. Among these, the region from the 1st position to the 110th position, the region from the 140th position to the 210th position, and the region from the 350th position to the 380th position of the partial base sequence of the β-tubulin gene of fungi belonging to the genus Neosartoria are interfungal regions. The present inventors have found that the nucleotide sequence is particularly low in conservation and has a unique nucleotide sequence between genera. The partial base sequence of the β-tubulin gene of Aspergillus fumigatus is represented by SEQ ID NO: 29. Of these, the region from position 1 to position 110, the region from position 140 to position 210, and the region from position 350 to position 380 of the partial base sequence of the β-tubulin gene of Aspergillus fumigatus are among the fungal sequences. The present inventors have found that the region has a particularly low conservation and has a unique base sequence between species.
The oligonucleotides represented by (l) and (m) are the nucleotide sequence of SEQ ID NO: 28, the region from positions 84 to 103, and positions 169 to 188, the base sequence of SEQ ID NO: 29, respectively. Each region corresponds to the region from the 83rd position to the 102nd position, and from the 166th position to the 186th position. The oligonucleotides represented by (n) and (o) are the nucleotide sequence of SEQ ID NO: 28, the region from position 144 to position 163, the region from position 358 to position 377, and the nucleotide sequence of SEQ ID NO: 29, respectively. Each of the sequences corresponds to the region from the 141st position to the 160th position, from the 356th position to the 376th position. Therefore, fungi and Aspergillus fumigatus belonging to the genus Neosartoria can be specifically detected by hybridizing the oligonucleotide to β-tubulin genes of fungi and Aspergillus fumigatus belonging to the genus Neosartoria.

The oligonucleotides represented by (v) and (w) are oligonucleotides that specifically hybridize with the variable region of the Aspergillus fumigatus β-tubulin gene.
The oligonucleotides represented by the nucleotide sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23 are present in the β-tubulin gene region and are specific to Aspergillus fumigatus, ie, an oligonucleotide complementary to a part of the variable region It is. These oligonucleotides can specifically hybridize to a part of Aspergillus fumigatus DNA and RNA, but cannot hybridize to fungal DNA and RNA belonging to the genus Neosartoria.

Another variable region of the β-tubulin gene of Aspergillus fumigatus will be described. The region from position 20 to position 50 and the region from position 200 to position 230 of the partial base sequence of the β-tubulin gene of Aspergillus fumigatus described in SEQ ID NO: 29 are between fungi, particularly fungi belonging to the genus Neosartoria. The present inventors have found that the conservation of the base sequence is particularly low.
The oligonucleotides represented by (v) and (w) correspond to the regions from position 23 to position 44 and position 200 to position 222, respectively, in the base sequence set forth in SEQ ID NO: 29. The oligonucleotide can be hybridized with the β-tubulin gene of Aspergillus fumigatus, but cannot be hybridized with DNA and RNA of fungi belonging to the genus Neosartoria. This will be described in detail with reference to FIG. FIG. 1 is a diagram comparing a part of the base sequences of β-tubulin genes of Aspergillus fumigatus, Neosartria fischeri and Neosartria spinosa described in SEQ ID NOs: 29 to 33. As shown in FIG. 1, the region recognized by the oligonucleotides of SEQ ID NOs: 22 and 23 in the β-tubulin gene of Aspergillus fumigatus and the region of the β-tubulin gene of fungi belonging to the genus Neosartoria corresponding to this region Are compared, it can be seen that the homology of the base sequence is very low compared to other regions. Therefore, by using the oligonucleotide represented by (v) and / or (w), it is possible to identify whether the fungus contained in the subject is a fungus belonging to the genus Neosartoria or Aspergillus fumigatus.

  For example, in the sample that showed a positive reaction by the detection method using the oligonucleotides (l) to (o), the oligonucleotides (v) and / or (w) are included in the sample. Species can be identified and detected. As described above, the oligonucleotides represented by the above (v) and (w) can specifically hybridize to a part of Aspergillus fumigatus DNA and RNA, but the DNA and RNA of fungi belonging to the genus Neosartria. Some cannot hybridize. Therefore, when the oligonucleotide shown in (v) and / or (w) is hybridized to DNA or RNA contained in the subject, the fungus contained in the subject is Aspergillus fumigatus, and when not hybridized, The fungus contained in the specimen can be distinguished from the fungus of the genus Neosartoria.

  Further, in the present invention, in a sample that shows a positive reaction by the detection method using the oligonucleotides (l) to (o), the difference in the temperature range in which the fungi belonging to the genus Neosartoria and Aspergillus fumigatus can grow is used. Or by using the oligonucleotide (v) and / or (w), the bacterial species contained in the sample can be identified and detected.

The upper limit of the growth temperature of fungi belonging to the genus Neosartoria is about 45 ° C. In contrast, Aspergillus fumigatus can grow at 50 ° C or higher. Utilizing this difference in the temperature range in which growth is possible, for example, the following species can be used to detect the bacterial species. However, the present invention is not limited to this.
For samples that showed a positive reaction by the detection method using the oligonucleotides (l) to (o) of the present invention, mycelia were planted from a single colony into PDA medium, PDB medium (potato dextrose liquid medium), etc. Incubate at ~ 52 ° C. After culturing for 1-2 days, check for mycelial elongation. Based on the difference in the temperature range where growth is possible, it can be identified as Aspergillus fumigatus when growth is confirmed, and as fungi belonging to the genus Neosartoria when growth is not confirmed. In addition, although there is no restriction | limiting in particular in the confirmation method of proliferation, Extension of the mycelium by a stereomicroscope, extension of the mycelium in a liquid medium, formation of a mycelium, formation of conidia is mentioned.

  The oligonucleotides indicated by the above (p) to (u) are oligonucleotides that specifically hybridize with the variable region of the β-tubulin gene of fungi belonging to the genus Hamigella. Therefore, by using the oligonucleotide, it is possible to specifically, quickly and easily detect the fungi belonging to the genus Hemigera.

  The oligonucleotides represented by the nucleotide sequences shown in SEQ ID NOs: 16 to 21 are present in the β-tubulin gene region, and are oligonucleotide sequences complementary to fungi belonging to the genus Hamigera, ie, complementary to a part of the variable region. It is a nucleotide. These oligonucleotides can specifically hybridize to a part of the DNA and RNA of fungi belonging to the genus Hamigera.

The variable region of the β-tubulin gene of a fungus belonging to the genus Hamigella will be described in detail using Hamigera abernea as an example. As described above, the partial base sequence of the β-tubulin gene of Hamigera abernea is represented by SEQ ID NO: 34. Among these, the region from position 350 to position 480, the region from position 1 to position 25, and the region from position 180 to position 200 of the partial base sequence of the β-tubulin gene of fungi belonging to the genus Hamigera are fungal genera. The present inventors have found that nucleotide sequence conservation is particularly low, and there is a unique nucleotide sequence between genera.
The oligonucleotides represented by (p) and (q) correspond to regions from the 358th position to the 377th position, respectively, from the 440th position to the 459th position in the base sequence set forth in SEQ ID NO: 34. The oligonucleotides represented by (r) and (s) correspond to regions from the 2nd to the 22nd position and from the 181st to the 200th position, respectively, in the base sequence set forth in SEQ ID NO: 34. The oligonucleotides represented by (t) and (u) correspond to the region from position 172 to position 191 to position 397 to position 416, respectively, of the base sequence set forth in SEQ ID NO: 34. Therefore, by hybridizing the oligonucleotide to the β-tubulin gene of a fungus belonging to the genus Hemigera, the fungus belonging to the genus Hemigera can be specifically detected.

In addition, as shown in FIG. 2, regions having high homology with the regions from 358 to 377 and from 440 to 459 of the β-tubulin gene of Hamigera abernea are cladosporium cladosporoides. ) And other β-tubulin genes of fungi belonging to the genus Cladosporium . However, a region having high homology with the region from position 181 to position 200 of the β-tubulin gene of Hamigella abernea does not exist in the β-tubulin gene of fungi belonging to the genus Cladosporium. Therefore, by using the oligonucleotides (p) to (u) in combination, it is possible to detect fungi belonging to the genus Hamigella and fungi belonging to the genus Cladosporium.
That is, the oligonucleotides (s), (t) and (u) can hybridize with the variable region of the β-tubulin gene of fungi belonging to the genus Hamigella, but the fungus belonging to the genus Cladosporium. It cannot hybridize with the variable region of the β-tubulin gene. Therefore, for example, for the sample that showed a positive reaction by the detection method using the oligonucleotides (p) and (q), the oligonucleotides shown in (r) and (s) and / or (t) and By using the oligonucleotide shown in (u), it is possible to identify whether the bacterial species contained in the sample is of the genus Hamigera or Cladosporium.

In addition, “fungi belonging to the genus Cladosporium” are filamentous imperfect fungi, and do not form offspring spores with high heat resistance. In addition, it is widely distributed in the living environment and food factories, and synthesizes melanin pigment, which is the cause of black stains. Examples of fungi belonging to the genus Cladosporium include Cladosporium cladosporodes and Cladosporium sphaerospermum .

In the detection method of the present invention, among the detection oligonucleotides (a) to (w) above, the following oligonucleotides (a1) to (w1) are preferable, and the nucleotide sequences represented by SEQ ID NOs: 1 to 23 are used. The represented oligonucleotide is more preferred.
(A1) an oligonucleotide represented by the base sequence described in SEQ ID NO: 1, or an oligonucleotide represented by a base sequence that has 70% or more homology with the base sequence and can be used as a detection oligonucleotide (B1) the oligonucleotide represented by the base sequence shown in SEQ ID NO: 2 or the oligonucleotide represented by the base sequence that has 70% or more homology with the base sequence and can be used as a detection oligonucleotide (C1) the oligonucleotide represented by the base sequence set forth in SEQ ID NO: 3 or the oligonucleotide represented by the base sequence that has 70% or more homology with the base sequence and can be used as a detection oligonucleotide (D1) the oligonucleotide represented by the base sequence shown in SEQ ID NO: 4 or 7 for the base sequence % Oligonucleotide represented by a nucleotide sequence having a homology of at least% and usable as a detection oligonucleotide (e1), or an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5, or 70 relative to the nucleotide sequence % Oligonucleotide having a homology of at least% and represented by a base sequence that can be used as a detection oligonucleotide (f1) The oligonucleotide represented by the base sequence set forth in SEQ ID NO: 6, or 70 relative to the base sequence Oligonucleotide represented by a nucleotide sequence having a homology of at least% and usable as a detection oligonucleotide (g1) An oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 7, or 70 relative to the nucleotide sequence Oligo represented by a base sequence that has a homology of at least% and can be used as a detection oligonucleotide Oligonucleotide represented by the nucleotide sequence of nucleotide (h1) SEQ ID NO: 8, or an oligonucleotide represented by a nucleotide sequence having 70% or more homology with the nucleotide sequence and usable as a detection oligonucleotide Nucleotide (i1) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 9 or Oligonucleotide represented by the nucleotide sequence having 70% or more homology with the nucleotide sequence and usable as a detection oligonucleotide Nucleotide (j1) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 10 or an oligonucleotide represented by a nucleotide sequence having 70% or more homology to the nucleotide sequence and usable as a detection oligonucleotide Nucleotide (k1) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 11, or the base Oligonucleotide represented by the nucleotide sequence having 70% or more homology to the sequence and usable as a detection oligonucleotide (l1) Oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 12, or the base Oligonucleotide represented by a nucleotide sequence having a homology of 70% or more to the sequence and usable as a detection oligonucleotide (m1) Oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 13, or the base Oligonucleotide (n1) represented by a nucleotide sequence having a homology of 70% or more to the sequence and usable as a detection oligonucleotide, or an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 14 A base that has a homology of 70% or more to a sequence and can be used as a detection oligonucleotide Oligonucleotide represented by the sequence (o1) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 15, or a nucleotide having a homology of 70% or more to the nucleotide sequence and usable as a detection oligonucleotide Oligonucleotide represented by the sequence (p1) Oligonucleotide represented by the base sequence set forth in SEQ ID NO: 16 or a base that has a homology of 70% or more to the base sequence and can be used as a detection oligonucleotide Oligonucleotide represented by sequence (q1) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide having a homology of 70% or more with the nucleotide sequence and usable as a detection oligonucleotide Oligonucleotide (r1) represented by a sequence Oligo represented by the base sequence set forth in SEQ ID NO: 18 Oligonucleotide or oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 19 having a homology of 70% or more to the nucleotide sequence and represented by a nucleotide sequence that can be used as a detection oligonucleotide (S1) Oligonucleotide represented by the nucleotide sequence shown in SEQ ID NO: 20 as a nucleotide or a nucleotide sequence having a homology of 70% or more to the nucleotide sequence and represented by a nucleotide sequence that can be used as an oligonucleotide for detection Oligonucleotide represented by the nucleotide sequence of nucleotide (SEQ ID NO: 21) or nucleotide represented by nucleotide sequence having 70% or more homology with the nucleotide sequence and usable as a detection oligonucleotide Oligonucleotide for detection having a homology of 70% or more with respect to the nucleotide or the base sequence Oligonucleotide represented by a nucleotide sequence that can be used as a peptide (v1) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 22, or a detection oligo having 70% or more homology with the nucleotide sequence Oligonucleotide represented by a nucleotide sequence that can be used as a nucleotide (w1) Oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 23, or a detection oligo having 70% or more homology with the nucleotide sequence Oligonucleotides represented by nucleotide sequences that can be used as nucleotides

As described later, in the detection method of the present invention, the detection oligonucleotide of the present invention can be suitably used as a nucleic acid probe or a nucleic acid primer.
The binding mode of the detection 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 for detection used in the present invention 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 thermostable fungi using a restriction enzyme or the like, or after cloning and isolating and purifying the gene and then excising using a restriction enzyme 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.

  In the present invention, an oligonucleotide pair consisting of the oligonucleotide (a) and the oligonucleotide (b) can be used as an oligonucleotide pair for detecting fungi belonging to the genus Bisoclamis. Among them, it is preferable to use an oligonucleotide pair consisting of the oligonucleotide (a1) and the oligonucleotide (b1), and the oligonucleotide pair represented by the nucleotide sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 is used. More preferably it is used.

In the present invention, an oligonucleotide pair comprising the oligonucleotide (c) and the oligonucleotide (d), an oligonucleotide pair comprising the oligonucleotide (e) and the oligonucleotide (f), g) an oligonucleotide pair comprising the oligonucleotide (h) and the oligonucleotide (h), an oligonucleotide pair comprising the oligonucleotide (i) and the oligonucleotide (h), and the oligonucleotide (j) Can be used as an oligonucleotide pair for detecting fungi belonging to the genus Talalomyces. Among them, an oligonucleotide pair consisting of the oligonucleotide (c1) and the oligonucleotide (d1), an oligonucleotide pair consisting of the oligonucleotide (e1) and the oligonucleotide (f1), (g1) An oligonucleotide pair consisting of the oligonucleotide (i) and the oligonucleotide (h1), an oligonucleotide pair consisting of the oligonucleotide (i1) and the oligonucleotide (h1), or the oligonucleotide (j1) and the above It is preferable to use an oligonucleotide pair consisting of the oligonucleotide of (k1), an oligonucleotide pair represented by the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 The oligonucleotide pair represented by The oligonucleotide pair represented by the base sequence described in No. 7 and SEQ ID NO: 8, the oligonucleotide pair represented by the base sequence described in SEQ ID NO: 9 and SEQ ID NO: 8, or the sequence described in SEQ ID NO: 10 and SEQ ID NO: 11 It is more preferable to use an oligonucleotide pair represented by a base sequence.
By using the oligonucleotide pair shown in the above (c) and (d), Talaromyces flavus and Talaromyces traxpermus can be specifically detected. Further, by using the oligonucleotide pair shown in the above (e) and (f) or the oligonucleotide pair shown in the above (j) and (k), the Talamomyces luteus, Talaromyces wortmannii, and Talalomyces basilisporus are specifically identified. Can be detected automatically. Furthermore, by using the oligonucleotide pairs shown in the above (g) and (h), it is possible to specifically detect Talaromyces flavus, Talalomyces wortmanny, Talalomyces traxpermus, Talalomyces macrosporus. In addition, by using the oligonucleotide pairs shown in (i) and (h) above, it is possible to specifically detect Talaromyces flavus, Talaromyces traxpermus, Talaromyces macrosporus.

In the present invention, an oligonucleotide pair consisting of the oligonucleotide (l) and the oligonucleotide (m), an oligonucleotide pair consisting of the (n) oligonucleotide and the (o) oligonucleotide, and The oligonucleotide pair comprising the oligonucleotide (v) and the oligonucleotide (w) can be used as an oligonucleotide pair for detecting fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus.
Among them, in order to detect fungi belonging to the genus Neosartoria and Aspergillus fumigatus, the oligonucleotide pair consisting of the oligonucleotides (l) and (m) above, or the oligonucleotide pair (n) and (o) above It is preferable to use an oligonucleotide pair, and it is more preferable to use an oligonucleotide pair consisting of the oligonucleotides (l1) and (m1) or an oligonucleotide pair consisting of the oligonucleotides (n1) and (o1). It is more preferable to use the oligonucleotide pair represented by the base sequences described in SEQ ID NO: 12 and SEQ ID NO: 13, or the oligonucleotide pair represented by the base sequences described in SEQ ID NO: 14 and SEQ ID NO: 15. The oligonucleotide pair consisting of the oligonucleotide (l) and the oligonucleotide (m) and the oligonucleotide pair consisting of the oligonucleotide (n) and the oligonucleotide (o) are Neosartoria Fiselli, Neo It is particularly preferably used for detection of fungi belonging to the genus Neosartria such as Sartoria spinosa, Neosartria glabra, Neosartria hiratsukae, Neosartria Paulistensis, Neosartria pseudofiseri, and Aspergillus fumigatus.
In order to detect Aspergillus fumigatus, it is preferable to use the oligonucleotide pair consisting of the oligonucleotides (v) and (w), and the oligonucleotide pair consisting of the oligonucleotides (v1) and (w1) It is more preferable to use, and it is further preferable to use an oligonucleotide pair represented by the nucleotide sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 23. The oligonucleotide pair consisting of the oligonucleotide (v) and the oligonucleotide (w) is preferably used for detection of Aspergillus fumigatus.

In the present invention, an oligonucleotide pair consisting of the oligonucleotide (p) and the oligonucleotide (q), an oligonucleotide pair consisting of the oligonucleotide (r) and the oligonucleotide (s), and An oligonucleotide pair composed of the oligonucleotide (t) and the oligonucleotide (u) can be used as an oligonucleotide pair for detecting a fungus belonging to the genus Hamigera. Among them, an oligonucleotide pair consisting of the oligonucleotide (p1) and the oligonucleotide (q1), an oligonucleotide pair consisting of the oligonucleotide (r1) and the oligonucleotide (s1), or ( It is preferable to use an oligonucleotide pair consisting of the oligonucleotide of t1) and the oligonucleotide of (u1), and the oligonucleotide pair represented by the nucleotide sequence of SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and the sequence It is more preferable to use the oligonucleotide pair represented by the base sequence described in No. 19 or the oligonucleotide pair represented by the base sequences described in SEQ ID No. 20 and SEQ ID No. 21.
The oligonucleotide pair represented by (p) and (q), the oligonucleotide pair represented by (r) and (s), and the oligonucleotide pair represented by (t) and (u) are: It is suitably used for detection of Hamigella abernea and Hamigella strata.

  In the detection method of the present invention, in order to identify a thermostable fungus using the nucleic acid represented by the base sequence of the variable region of the present invention, the base sequence represented by any one of the base sequences (A) to (K) is used. The detection oligonucleotide that hybridizes with the nucleic acid to be detected under stringent conditions is labeled, and the obtained detection oligonucleotide is hybridized with the nucleic acid extracted from the test object under stringent conditions and hybridized. It is preferable to measure the label of the detection oligonucleotide. In this case, as the detection oligonucleotide that hybridizes under stringent conditions with the nucleic acid represented by any one of the base sequences (A) to (K), the detection oligonucleotide and oligo of the present invention described above are used. Nucleotide pairs can be used, and preferred ranges are the same.

The detection oligonucleotide and oligonucleotide pair used in the present invention can be used as a nucleic acid probe. The nucleic acid probe can be prepared by labeling the oligonucleotide with a label. The label is not particularly limited, and usual labels such as radioactive substances, enzymes, fluorescent substances, luminescent substances, antigens, haptens, enzyme substrates, insoluble carriers and the like can be used. The labeling method may be end labeling, labeling in the middle of the sequence, or labeling on a sugar, phosphate group, or base moiety. Since the nucleic acid probe specifically hybridizes with the β-tubulin gene of the heat-resistant fungus or a part of the variable region of 28S rDNA, the heat-resistant fungus in the specimen can be detected quickly and easily. As a means for detecting such a label, for example, when the nucleic acid probe is labeled with a radioisotope, it is labeled with a chemiluminescent substance such as autoradiography, and when it is labeled with a fluorescent substance, such as a fluorescence microscope. In some cases, analysis using a photosensitive film, digital analysis using a CCD camera, and the like can be given.
The detection oligonucleotide of the present invention thus labeled is hybridized with a nucleic acid extracted from the test object by a conventional method under stringent conditions, and then the hybridized detection oligonucleotide is extracted. Heat-resistant fungi can be detected by measuring the label. Here, examples of the stringent conditions include the conditions described above. As a method for measuring the label of the nucleic acid probe hybridized with the nucleic acid, a usual method (FISH method, dot blot method, Southern blot method, Northern blot method, etc.) can be used.

  For example, the oligonucleotide represented by (l) and / or (m), the oligonucleotide represented by (n) and / or (o) or the oligonucleotide represented by (v) and / or (w) It can be labeled to form a nucleic acid probe. The oligonucleotide represented by the above (l) and / or (m) or the nucleic acid probe comprising the oligonucleotide represented by (n) and / or (o) is a fungus belonging to the genus Neosartoria and a β-tube of Aspergillus fumigatus Since it hybridizes specifically with a part of the variable region of the phosphorus gene, fungi belonging to the genus Neosartoria and Aspergillus fumigatus in the subject can be detected quickly and easily. In addition, the nucleic acid probe comprising the oligonucleotide represented by (v) and / or (w) specifically hybridizes with a part of the variable region of the β-tubulin gene of Aspergillus fumigatus, It cannot hybridize to the DNA and RNA of the fungi to which it belongs. Therefore, by confirming whether these oligonucleotides are hybridized, it is possible to quickly and easily identify whether the fungus in the specimen is a fungus belonging to the genus Neosartoria or Aspergillus fumigatus.

  In addition, the detection oligonucleotide used in the present invention can be bound to a solid support and used as a capture probe. In this case, a sandwich assay can be performed with a combination of a capture probe and a labeled nucleic acid probe, or the target nucleic acid can be labeled and captured.

  In the detection method of the present invention, in order to identify a thermostable fungus using the nucleic acid represented by the base sequence of the variable region of the present invention, the base sequence represented by any one of the base sequences (A) to (K) is used. It is also a preferred embodiment that a nucleic acid composed of a part or all of the nucleic acid is subjected to gene amplification and the presence or absence of the amplification product is confirmed. In this case, the oligonucleotide for detection and the oligonucleotide pair of the present invention can be used as a nucleic acid primer and a nucleic acid primer pair, and the preferred range is also the same. Among these, detection is preferably performed by performing gene amplification using the detection oligonucleotide pair of the present invention as a nucleic acid primer pair and confirming gene amplification.

  More specifically, in order to identify a fungus belonging to the genus Bisoclamis, a nucleic acid consisting of a part or all of the nucleic acid represented by the base sequence of (A) above is gene-amplified, and the presence or absence of an amplification product is determined. It is preferable to confirm. In this case, it includes a region in the nucleic acid represented by the base sequence of (A), wherein (1) the base sequence of the gene unique to the fungus belonging to the genus Bisoclamis appears continuously around 10 bases, 2) the GC content of the oligonucleotide is approximately 30% to 80%, (3) the possibility of self-annealing of the oligonucleotide is low, and (4) the Tm value of the oligonucleotide is approximately 55 ° C to 65 ° C. An oligonucleotide that can hybridize to a region that satisfies the four conditions is preferably used as the nucleic acid primer. Especially, it is preferable to use the oligonucleotide and the oligonucleotide pair of (a) and / or (b) as the nucleic acid primer and the nucleic acid primer pair, and the oligonucleotide and the oligonucleotide of (a1) and / or (b1). It is more preferable to use the pair as a nucleic acid primer and a nucleic acid primer pair, and it is more preferable to use an oligonucleotide represented by the base sequences described in SEQ ID NOs: 1 and 2 as the nucleic acid primer and the nucleic acid primer pair.

In order to identify a fungus belonging to the genus Tallalomyces, a nucleic acid consisting of a part or all of the nucleic acid represented by any one of the base sequences (B) to (D) is amplified, and the amplification product It is preferable to check the presence or absence. In this case, it is a region in the nucleic acid represented by any one of the base sequences (B) to (D), and (1) the base sequence of a gene unique to a fungus belonging to the genus Talalomyces is continuous for about 10 bases. (2) the oligonucleotide has a GC content of approximately 30% to 80%, (3) the possibility of oligonucleotide self-annealing is low, and (4) the oligonucleotide has a Tm value of approximately 55 ° C. It is preferable to use, as a nucleic acid primer, an oligonucleotide that can hybridize to a region that satisfies the four conditions of ˜65 ° C. Among them, it is preferable to use any one of the oligonucleotides (c) to (k) as a nucleic acid primer, and it is more preferable to use any one of the oligonucleotides (c1) to (k1) as a nucleic acid primer. It is more preferable to use the oligonucleotide represented by the base sequence described in SEQ ID NOs: 3 to 11 as a nucleic acid primer. In addition, the oligonucleotide pair shown in (c) and (d), the oligonucleotide pair shown in (e) and (f), the oligonucleotide pair shown in (g) and (h), It is preferable to use the oligonucleotide pair represented by (i) and (h) or the oligonucleotide pair represented by (j) and (k) as a nucleic acid primer pair, and (c1) and (d1) The oligonucleotide pair indicated by (e1) and (f1), the oligonucleotide pair indicated by (g1) and (h1), and the above (i1) and (h1) It is more preferable to use the oligonucleotide pair shown above or the oligonucleotide pair shown in the above (j1) and (k1) as a nucleic acid primer pair. The oligonucleotide pair represented by the base sequence described in 4, the oligonucleotide pair represented by the base sequence described in SEQ ID NO: 5 and SEQ ID NO: 6, and the base sequence described in SEQ ID NO: 7 and SEQ ID NO: 8 Oligonucleotide pairs, oligonucleotide pairs represented by the nucleotide sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 8, and oligonucleotide pairs represented by the nucleotide sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 11 are used as nucleic acid primer pairs Is more preferable.
In addition, from the viewpoint of detection specificity and detection sensitivity, the oligonucleotide pair shown in (i) and (h) above or the oligonucleotide pair shown in (j) and (k) above is used as a nucleic acid primer pair. It is preferable to use the oligonucleotide pair represented by the above (i1) and (h1) or the oligonucleotide pair represented by the above (j1) and (k1) as the nucleic acid primer pair. It is more preferable to use the oligonucleotide pair represented by the nucleotide sequences described in SEQ ID NO: 9 and SEQ ID NO: 8 or the oligonucleotide pair represented by the nucleotide sequences described in SEQ ID NO: 10 and SEQ ID NO: 11 as the nucleic acid primer pair.

In order to identify fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria, a nucleic acid consisting of a part or all of the nucleic acid represented by any one of the base sequences (E) to (J) is It is preferable to amplify and confirm the presence or absence of the amplification product. In this case, the region in the nucleic acid represented by any one of the base sequences (E) to (J), (1) the base sequence of a gene unique to fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria (2) the GC content of the oligonucleotide is about 30% to 80%, (3) the possibility of oligonucleotide self-annealing is low, (4) It is preferable to use an oligonucleotide that can hybridize to a region that satisfies the four conditions of Tm value of approximately 55 ° C. to 65 ° C. as a nucleic acid primer. Among these, it is preferable to use any one of the oligonucleotides (l) to (o) and (v) to (w) as a nucleic acid primer, and the above (l1) to (o1) or (v1) to (w1) ) Is more preferably used as a nucleic acid primer, and oligonucleotides represented by the nucleotide sequences of SEQ ID NOS: 12 to 15 and 22 to 23 are more preferably used as a nucleic acid primer. For example, by performing gene amplification using the oligonucleotides (l) and (m) or the oligonucleotides (n) and (o) as nucleic acid primers and confirming gene amplification, Fungi and Aspergillus fumigatus can be detected. In order to detect fungi belonging to the genus Neosartria, which is a particular problem in food accidents, the oligonucleotides of (v) and (w) were further used as nucleic acid primers for the samples in which these fungi were detected by the above method. By performing gene amplification treatment and confirming gene amplification, it is possible to distinguish between fungi belonging to the genus Neosartoria or Aspergillus fumigatus.
In addition, the oligonucleotide pair represented by (l) and (m), the oligonucleotide pair represented by (n) and (o), or the oligonucleotide pair represented by (v) and (w) Is preferably used as a nucleic acid primer pair, the oligonucleotide pair represented by (l1) and (m1), the oligonucleotide pair represented by (n1) and (o1), or the (v1) and (w1) Is preferably used as a nucleic acid primer pair, the oligonucleotide pair represented by the nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 13, the nucleotide sequence of SEQ ID NO: 14 and SEQ ID NO: 15 An oligonucleotide pair represented by the nucleotide sequence of SEQ ID NO: 22 and SEQ ID NO: 23 is used as a nucleic acid primer. Further preferably used in pairs.
In particular, when detecting fungi belonging to the genus Neosartoria and Aspergillus fumigatus, the oligonucleotide pairs shown in (n) and (o) above are used as nucleic acid primer pairs in terms of detection specificity and detection sensitivity. It is more preferable to use the oligonucleotide pair represented by the above (n1) and (o1) as a nucleic acid primer pair, and the oligonucleotide pair represented by the nucleotide sequences described in SEQ ID NO: 14 and SEQ ID NO: 15 More preferably, it is used as a nucleic acid primer pair. When detecting Aspergillus fumigatus, from the viewpoint of detection specificity and detection sensitivity, the oligonucleotide pairs shown in (v) and (w) are preferably used as nucleic acid primer pairs, and (v1) and The oligonucleotide pair represented by (w1) is more preferably used as the nucleic acid primer pair, and the oligonucleotide pair represented by the nucleotide sequences described in SEQ ID NO: 22 and SEQ ID NO: 23 is more preferably used as the nucleic acid primer pair. .

In order to identify fungi belonging to the genus Hamigera, it is preferable to amplify a nucleic acid consisting of a part or all of the nucleic acid represented by the base sequence of (K) and confirm the presence or absence of the amplified product. . In this case, it includes a region in the nucleic acid represented by the base sequence of (K), wherein (1) the base sequence of a gene unique to a fungus belonging to the genus Hamigella appears continuously around 10 bases ( 2) the GC content of the oligonucleotide is approximately 30% to 80%, (3) the possibility of self-annealing of the oligonucleotide is low, and (4) the Tm value of the oligonucleotide is approximately 55 ° C to 65 ° C. An oligonucleotide that can hybridize to a region that satisfies the four conditions is preferably used as the nucleic acid primer. Especially, it is preferable to use any one of the oligonucleotides (p) to (u) as a nucleic acid primer and a nucleic acid primer pair, and any one of the oligonucleotides (p1) to (u1) is used as a nucleic acid primer and It is preferably used as a nucleic acid primer pair, and more preferably an oligonucleotide represented by the base sequence described in SEQ ID NOs: 16 to 21 is used as a nucleic acid primer. For example, by performing gene amplification using the oligonucleotides (p) and (q) as nucleic acid primers and confirming gene amplification, fungi belonging to the genus Hamigera and fungi belonging to the genus Cladosporium Can be detected. In order to detect fungi belonging to the genus Hamigera, which are particularly problematic in food accidents, the oligonucleotides of (r) and (s) or the (t) and (t) By performing gene amplification using the oligonucleotide (u) as a nucleic acid primer and confirming gene amplification, it is possible to identify whether the fungus belongs to the genus Hamigera or the genus Cladosporium.
In addition, the oligonucleotide pair indicated by (p) and (q), the oligonucleotide pair indicated by (r) and (s), or the oligonucleotide pair indicated by (t) and (u) Is preferably used as a nucleic acid primer pair, the oligonucleotide pair represented by (p1) and (q1), the oligonucleotide pair represented by (r1) and (s1), or the above (t1) and (u1 Is preferably used as a nucleic acid primer pair, the oligonucleotide pair represented by the nucleotide sequence of SEQ ID NO: 16 and SEQ ID NO: 17, the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 19 Or an oligonucleotide pair represented by the nucleotide sequence described in SEQ ID NO: 20 and SEQ ID NO: 21 A further preferred.
In particular, from the viewpoint of detection specificity, the oligonucleotide pair represented by (r) and (s) or the oligonucleotide pair represented by (t) and (u) is preferably used as a nucleic acid primer pair. Preferably, the oligonucleotide pair represented by (r1) and (s1) or the oligonucleotide pair represented by (t1) and (u1) is more preferably used as the nucleic acid primer pair. More preferably, the oligonucleotide pair represented by the base sequence described in No. 19 or the oligonucleotide pair represented by the base sequences described in SEQ ID No. 20 and SEQ ID No. 21 is used as the nucleic acid primer pair. From the viewpoint of detection sensitivity, it is preferable to use the oligonucleotide pair represented by (t) and (u) as a nucleic acid primer pair, and the oligonucleotide pair represented by (t1) and (u1) is used as a nucleic acid primer. It is more preferable to use as a pair, and it is more preferable to use an oligonucleotide pair represented by the base sequences described in SEQ ID NO: 20 and SEQ ID NO: 21 as a nucleic acid primer pair.

  In the nucleic acid primer of the present invention, the oligonucleotide can be used as a nucleic acid primer as it is, or the oligonucleotide can be labeled with a label and used as a nucleic acid primer. Examples of the label and the labeling method include the same as in the case of the nucleic acid probe.

  In the present invention, the gene amplification treatment is preferably performed by a polymerase chain reaction (PCR) method. The conditions for the PCR reaction are not particularly limited as long as the target DNA fragment can be amplified to a detectable level. As a preferable example of PCR reaction conditions, when detecting fungi belonging to the genus Bisoclamis, for example, a heat denaturation reaction in which a double-stranded DNA is made into a single strand is performed at 95 to 98 ° C. for 10 to 60 seconds, An annealing reaction for hybridizing the pair to single-stranded DNA is performed at 59-61 ° C. for about 60 seconds, an extension reaction for allowing DNA polymerase to act is performed at about 72 ° C. for about 60 seconds, and these are defined as one cycle for about 30 Perform ~ 35 cycles. When detecting fungi belonging to the genus Talalomyces, for example, a heat denaturation reaction in which double-stranded DNA is made into a single strand is carried out at 95 to 97 ° C. for about 10 to 60 seconds, and the primer pair is hybridized to the single-stranded DNA. An annealing reaction is performed at 55 to 61 ° C. for about 60 seconds, an extension reaction for allowing DNA polymerase to act is performed at about 72 ° C. for about 60 seconds, and these are defined as one cycle for about 30 to 35 cycles. When detecting fungi belonging to the genus Neosartoria and Aspergillus fumigatus using the oligonucleotides (l) and (m) or the oligonucleotides (n) and (o), for example, one double-stranded DNA is used. A heat denaturation reaction for forming a strand is performed at 95 to 98 ° C. for 10 to 60 seconds, an annealing reaction for hybridizing the primer pair to the single-stranded DNA is performed at 59 to 61 ° C. for about 60 seconds, and an extension reaction that causes DNA polymerase to act is performed. This is performed at about 72 ° C. for about 60 seconds, and these are defined as one cycle for about 30 to 35 cycles. When detecting the fungus belonging to the genus Neosartoria or Aspergillus fumigatus using the oligonucleotides (v) and (w), a heat denaturation reaction for converting a double-stranded DNA into a single strand at 95 to 98 ° C. An annealing reaction for hybridizing a primer pair to single-stranded DNA for about 60 seconds at 59-61 ° C. for about 60 seconds, an extension reaction for allowing DNA polymerase to perform at about 72 ° C. for about 60 seconds, and performing one cycle 30 to 35 cycles are performed. When detecting fungi belonging to the genus Hamigella using the oligonucleotides represented by the above (p) and (q), for example, a heat denaturation reaction in which a double-stranded DNA is made into a single strand is carried out at 95 to 98 ° C. at 10 ° C. An annealing reaction for hybridizing the primer pair to the single-stranded DNA for about 60 seconds at about 59-63 ° C. for about 60 seconds, and an extension reaction for allowing DNA polymerase to act on at about 72 ° C. for about 60 seconds. About 30 to 35 cycles are performed. When detecting fungi belonging to the genus Hamigera using the oligonucleotides represented by (r) and (s) or the oligonucleotides represented by (t) and (u), for example, double-stranded DNA A heat denaturation reaction to make a single strand is carried out at 95 to 98 ° C. for 10 to 60 seconds, an annealing reaction for hybridizing the primer pair to the single-stranded DNA is carried out at about 59 to 63 ° C. for about 60 seconds, and extension to allow DNA polymerase to act The reaction is performed at about 72 ° C. for about 60 seconds, and these are defined as one cycle for about 30 to 35 cycles.

  In the present invention, gene fragment amplification can be confirmed by a usual method. For example, a method of incorporating a nucleotide to which a label such as a radioactive substance is bound during a gene amplification reaction, a method of performing electrophoresis on a PCR reaction product to confirm the presence or absence of a band corresponding to the size of the amplified gene, a base sequence of the PCR reaction product And a method in which a fluorescent substance is inserted between the amplified DNA double strands to emit light, but 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 base sequence set forth in SEQ ID NO: 24, the number of bases from position 33 to position 178 is 146 bases. Therefore, when the specimen contains a fungus belonging to the genus Bisoclamis, a PCR reaction is performed using the oligonucleotide pair (a) and (b) as a primer set, and the obtained PCR reaction product is subjected to electrophoresis. Then, amplification of a DNA fragment of about 150 bp specific to fungi belonging to the genus Bisoclamis is observed. By performing this operation, fungi belonging to the genus Bisoclamis can be detected and identified.

  In the base sequence described in SEQ ID NO: 25, the number of bases from the 15th position to the 98th position is 83 bases, and in the base sequence described in SEQ ID NO: 26, the odd number of salts from the 133rd position to the 325th position is 192 bases. In the base sequence shown in SEQ ID NO: 27, the number of bases from positions 326 to 478 is 152 bases. Therefore, when the sample contains Talaromyces flavus and / or Talaromyces traxpermus, PCR reaction is performed using the oligonucleotide pairs (c) and (d) as a primer set, and the obtained PCR reaction product is electrophoresed. The amplification of about 80 bp DNA fragment specific to these fungi is observed. When the subject includes Talaromyces luteus, Talalomyces wortmannii and / or Talaromyces bacilis porus, the oligonucleotide pair (e) and (f) or the oligonucleotide pair (j) and (k) is used as a primer set. As a result, a PCR reaction product was obtained, and the obtained PCR reaction product was subjected to electrophoresis. As a result, amplification of a DNA fragment of about 200 bp specific to these fungi was observed. Further, when the subject includes Talaromyces macrosporus, Talaromyces wortmannii, Talaromyces flavus and / or Talaromyces traxpermus, a PCR reaction is performed using the oligonucleotide pairs of (g) and (h) as a primer set, When the obtained PCR reaction product is subjected to electrophoresis, amplification of a DNA fragment of about 150 bp specific to these fungi is observed. When the subject contains Talalomyces macrosporus, Talalomyces flavus and / or Talalomyces traxpermus, PCR reaction is carried out using the oligonucleotide pairs of (i) and (h) as primer sets, and the resulting PCR reaction product When electrophoresis is performed, amplification of a DNA fragment of about 150 bp specific to these fungi is observed. By performing this operation, fungi belonging to the genus Talalomyces can be detected and identified. In addition, when the subject includes Talalomyces wortmanny, the oligonucleotide pair (e) and (f) or the oligonucleotide pair (j) and (k), and the above (g) and (h) When the oligonucleotide pair is used at the same time, two bands of about 200 bp and about 150 bp are confirmed.

When the specimen contains fungi belonging to the genus Neosartria and / or Aspergillus fumigatus, PCR reaction is performed using the oligonucleotide pairs of (l) and (m) as a primer set, and the obtained PCR reaction product When electrophoresis is performed, amplification of a DNA fragment of about 100 bp specific to these fungi is observed. Further, when PCR reaction was performed using the oligonucleotide pairs (n) and (o) as a primer set, and the obtained PCR reaction product was subjected to electrophoresis, about 200 bp DNA specific to these fungi was obtained. Fragment amplification is observed. By performing this operation, fungi belonging to the genus Neosartoria and Aspergillus fumigatus can be detected and detected.
Further, when Aspergillus fumigatus is included in the specimen, PCR reaction is performed using the oligonucleotide pairs (v) and (w) as a primer set, and the obtained PCR reaction product is subjected to electrophoresis. An amplification of a DNA fragment of about 200 bp specific to the fungus of the present invention is observed. However, when fungi belonging to the genus Neosartria are included, amplification of DNA fragments is not observed even when PCR reaction is performed using the oligonucleotide pairs (v) and (w) as a primer set. Therefore, only Aspergillus fumigatus can be detected by performing a PCR reaction using the oligonucleotide pair (v) and (w) as a primer set.

For confirmation of gene amplification, when the oligonucleotide pair shown in (p) and (q) above was used, the number of bases from position 358 to position 459 in the base sequence set forth in SEQ ID NO: 34 was about 100 bases. is there. Therefore, when the specimen contains fungi belonging to the genus Hamigera, a PCR reaction is performed using the oligonucleotide pairs of (p) and (q) as a primer set, and the obtained PCR reaction product is subjected to electrophoresis. Amplification of a DNA fragment of about 100 bp specific to these fungi is observed.
In the base sequence shown in SEQ ID NO: 34, the number of bases from the 2nd position to the 200th position is about 200 bases. Moreover, when the oligonucleotide pair shown by said (t) and (u) is used, in the base sequence of sequence number 34, the base number from the 172nd position to the 416th position is 245 bases. Therefore, when the subject contains a fungus belonging to the genus Hamigella, the PCR reaction is performed using the oligonucleotide pair (r) and (s) or the oligonucleotide pair (t) and (u) as a primer set. When the obtained PCR reaction product was electrophoresed, amplification of a DNA fragment of about 200 bp or about 240 bp specific to this fungus was observed. By performing this operation, fungi belonging to the genus Hemigera can be detected and detected.

In the detection method of the present invention, detection using the oligonucleotide pair (c) and (d), the oligonucleotide pair (g) and (h), or the oligonucleotide pair (i) and (h) It is also preferable to detect a fungus belonging to the genus Tallaromyces by simultaneously using the method and the detection method using the oligonucleotide pair of (e) and (f) or the oligonucleotide pair of (j) and (k). It is. By using a plurality of oligonucleotide pairs in combination, it is possible to comprehensively detect fungi belonging to the genus Talalomyces. Among them, the detection method using the oligonucleotide pair (c1) and (d1), the oligonucleotide pair (g1) and (h1), or the oligonucleotide pair (i1) and (h1), It is more preferable to use a combination of the detection method using the oligonucleotide pair (e1) and (f1) or the oligonucleotide pair (j1) and (k1), and the nucleotide sequences described in SEQ ID NOs: 3 and 4 And a detection method using the oligonucleotide pair represented by SEQ ID NO: 7 and 8 and the oligonucleotide pair represented by the nucleotide sequence of SEQ ID NOs: 9 and 8; , An oligonucleotide pair represented by the base sequences set forth in SEQ ID NOs: 5 and 6, or an oligonucleotide pair represented by the base sequences set forth in SEQ ID NOs: 10 and 11 It is more preferable to use in combination with a detection method using the fault pair.
Specifically, as described above, by using the oligonucleotide pairs (c) and (d), it is possible to specifically detect Talaromyces flavus and Talalomyces traxpermus. In addition, by using the oligonucleotide pairs of (e) and (f) or (j) and (k), it is possible to specifically detect Talalomyces wortmanny, Talalomyces luteus and Talalomyces bacilis porus. Furthermore, by using the oligonucleotide pairs (g) and (h), it is possible to specifically detect Talaromyces flavus, Talalomyces traxpermus, Talalomyces wortmanny, and Talalomyces macrosporus. In addition, by using the oligonucleotide pairs of (i) and (h), it is possible to specifically detect Talaromyces flavus, Talaromyces traxpermus, and Talaromyces macrosporus. Therefore, the oligonucleotide pair (c) and (d), the oligonucleotide pair (g) and (h), or the oligonucleotide pair (i) and (h), and the (e) and (f) ) Or a combination of the oligonucleotide pairs (j) and (k) above, it is possible to comprehensively detect fungi belonging to the genus Tallalomyces, which are particularly problematic in food accidents.
In particular, in terms of detection sensitivity, the detection method using the oligonucleotide pair (i) and (h) and the detection method using the oligonucleotide pair (j) and (k) are used in combination. Preferably, the detection method using the oligonucleotide pair (i1) and (h1) and the detection method using the oligonucleotide pair (j1) and (k1) are more preferably used in combination. A combination of the detection method using the oligonucleotide pair represented by the nucleotide sequences described in Nos. 9 and 8 and the detection method using the oligonucleotide pair represented by the nucleotide sequences described in SEQ ID Nos. 10 and 11 More preferably, it is used.
In the present invention, “the gene amplification treatment step is used at the same time” means that two oligonucleotide pairs are mixed and the mixture is added as a primer to one reaction system, and the gene amplification treatment as described above is performed. It means that a process is performed. By using two oligonucleotide pairs at the same time, fungi belonging to the genus Taralomyces can be detected more rapidly and comprehensively. The mixing ratio of the two oligonucleotide pairs is not particularly limited, but is preferably 1: 1 to 1: 2.

In the detection method of the present invention, the detection method using the oligonucleotide pair indicated by (l) and (m) and / or the oligonucleotide pair indicated by (n) and (o), It is also a preferred embodiment that the detection method using the oligonucleotide pair shown in v) and (w) is used in combination. As described above, the detection method using the oligonucleotide pair shown in (l) and (m) and the oligonucleotide pair shown in (n) and (o) are fungi and Aspergillus belonging to the genus Neosartoria. Since the detection method using the oligonucleotide pairs shown in (v) and (w) above can detect Aspergillus fumigatus, fungi detected from the specimen by combining these methods. Is a fungus belonging to the genus Neosartoria or Aspergillus fumigatus. Among them, the detection method using the oligonucleotide pair shown by the above (l1) and (m1) and / or the oligonucleotide pair shown by the above (n1) and (o1), and the above (v1) and (w1) It is more preferable to use in combination with the detection method using the oligonucleotide pair shown in (5), and the oligonucleotide pair represented by the nucleotide sequence shown in SEQ ID NO: 12 and 13 and / or SEQ ID NO: 14 and 15 More preferably, the detection method using the oligonucleotide pair represented by the described base sequence and the detection method using the oligonucleotide pair represented by the base sequence described in SEQ ID NOs: 22 and 23 are used in combination. .
In particular, in terms of detection sensitivity, the detection method using the oligonucleotide pairs (n) and (o) and the detection method using the oligonucleotide pairs (v) and (w) are used in combination. It is more preferable that the detection method using the oligonucleotide pair (n1) and (o1) and the detection method using the oligonucleotide pair (v1) and (w1) are more preferably used in combination. A combination of a detection method using the oligonucleotide pair represented by the nucleotide sequences set forth in SEQ ID NOs: 14 and 15 and a detection method using the oligonucleotide pair represented by the nucleotide sequences set forth in SEQ ID NOs: 22 and 23 More preferably, it is used.

In the detection method of the present invention, the detection method using the oligonucleotide pairs (p) and (q) and the oligonucleotide pairs (r) and (s) or the (t) and (u) It is also a preferred embodiment that the detection method using the prepared oligonucleotide pair is used in combination. By combining these methods, only fungi belonging to the genus Hamigera can be specifically detected from the subject. That is, by combining these methods, it is possible to identify whether the fungus detected from the subject is the genus Hamigella or Cladosporium. Since fungi belonging to the genus Hamigera do not produce mycotoxins (mold poison), the risk of mycotoxins can be predicted by detecting both genera. In addition, since both genera have different heat resistance, by detecting the difference between genera, it is possible to make a prediction as to whether the bacterial cells are mixed before heating. It can be used to elucidate the cause, such as confirmation. Among them, the detection method using the oligonucleotide pair (p1) and (q1), the oligonucleotide pair (r1) and (s1) or the oligonucleotide pair shown by (t1) and (u1) It is more preferable to use in combination with a detection method using a detection method using an oligonucleotide pair represented by the nucleotide sequence described in SEQ ID NOs: 16 and 17, and a nucleotide sequence described in SEQ ID NOs: 18 and 19. It is more preferable to use in combination with the detection method using the oligonucleotide pair represented by the above or the oligonucleotide pair represented by the nucleotide sequences of SEQ ID NOS: 20 and 21.
In particular, from the viewpoint of detection sensitivity, the detection method using the oligonucleotide pairs (p) and (q) and the detection method using the oligonucleotide pairs shown in (t) and (u) are provided. The detection method using the oligonucleotide pairs (p1) and (q1) and the detection method using the oligonucleotide pairs shown in (t1) and (u1) are preferably combined. More preferably, the detection method using the oligonucleotide pair represented by the nucleotide sequences set forth in SEQ ID NOs: 16 and 17 and the oligonucleotide pair represented by the nucleotide sequences set forth in SEQ ID NOs: 20 and 21 are used. More preferably, it is used in combination with the detection method used.

  Next, one embodiment of the heat-resistant fungus detection method of the present invention will be described in detail, but the present invention is not limited to this.

1) Analysis of accident-causing bacteria Foods and drinks that may cause contamination accidents due to heat-resistant bacteria include foods and drinks that cannot be sterilized under strong conditions because they contain sugars and proteins. Examples of such foods and drinks include foods and drinks made from agricultural products such as fruits and fruit juices and livestock products such as milk.
DNA is recovered from the mycelia detected from the beverage that caused the accident. Thereafter, gene amplification treatment such as PCR reaction is performed using a primer for detecting heat-resistant bacteria. For example, the oligonucleotide pair represented by (a) and (b), the oligonucleotide pair represented by (t) and (u), the oligonucleotide pair represented by (n) and (o), (i ) And (h) and the oligonucleotide pair indicated by (j) and (k) are used for gene amplification treatment such as PCR reaction. Perform electrophoresis after gene amplification, and check for the presence of reaction products. At the same time, a part of the collected bacterial cells is inoculated into chloramphenicol-added PDA and cultured at 50 ° C.
When the reaction product is confirmed in a system using a fungal detection primer belonging to the genus Neosartoria (the oligonucleotide pair shown in (n) and (o) above), confirm the elongation of the mycelium cultured at 50 ° C., Check if it is Neosartoria or Aspergillus fumigatus. Alternatively, detection is carried out using Neosartria and Aspergillus fumigatus detection oligonucleotides. For example, PCR reaction is carried out using the oligonucleotides shown in (v) and (w) as primers, and Aspergillus fumigatus is found when a reaction product of about 200 bp is found, and Neosartria is found when no reaction product is found. It is determined that the fungus belonging to the genus is positive.
When a reaction product is confirmed in a system using a primer for detecting a fungus belonging to the genus Bisoclamis (the oligonucleotide pair shown in the above (a) and (b)), it is determined that the fungus belonging to the genus Bisoclamis is positive.
When a reaction product is confirmed in a system using a primer for detecting fungi belonging to the genus Hamigera (the oligonucleotide pair indicated by the above (t) and (u)), it is determined that the fungus belonging to the genus Hamigera is positive.
The reaction product is confirmed in a system using a primer for detecting fungi belonging to the genus Talalomyces (the oligonucleotide pair indicated by (j) and (k) and / or the oligonucleotide pair indicated by (i) and (h)). If so, it is determined that the fungus belonging to the genus Taralomyces is positive.
Since heat-resistant fungi survive even under heat sterilization conditions, it is highly possible that heat-resistant fungi are mixed in from the raw materials and processes. Therefore, when the heat-resistant fungi are positive, it is necessary to carefully examine the cleanliness of the raw materials and the manufacturing environment. On the contrary, when the heat-resistant fungi are negative, it is caused by poor sterilization or contamination after sterilization, so it is necessary to review the sterilization process and confirm the confidentiality of the container (cause investigation).

2) Microbial examination of raw materials The examination of fungi of normal raw materials requires two tests: general fungi (samples are not heat shock treated) and heat-resistant fungi (samples are heat shocked). However, according to the present invention, it is not necessary to heat-treat the specimen. That is, only a general fungus is inspected, and when a mycelium is confirmed, it can be determined whether or not it is a heat-resistant fungus by the method 1) using the mycelium. The conventional method usually requires about 7 days for detection of heat-resistant fungi. However, according to the present invention, detection can be performed within 2 days after confirmation of mycelia for about 3 days. Can be accelerated. Further, in the conventional method, it takes about 7 days for the identification of the bacterial species after detection of the thermophilic fungi, but in the method of the present invention, the genus level can be identified simultaneously with the detection.

In the method for detecting heat-resistant fungi of the present invention, the subject is not particularly limited, and food / beverage products themselves, raw materials of food / beverage products, isolated cells, cultured cells, etc. can be used.
The method for preparing DNA from a specimen is not particularly limited as long as DNA having a sufficient degree of purification and quantity sufficient for detection of heat-resistant fungi can be obtained, and ordinary methods and commercially available preparation kits are used. be able to. In addition, DNA obtained by reverse transcription of RNA in a subject can also be used.

  According to the method for detecting a thermostable fungus of the present invention, it is possible to perform the process from the specimen preparation step to the fungus detection step in a short time of about 5 to 12 hours.

In addition, the heat-resistant fungus identification kit of the present invention is selected from oligonucleotide pairs in the following categories (1) to (4), and at least two oligonucleotide pairs selected from different groups are used as nucleic acid primers. It contains.
(1) oligonucleotide pair of (a) and (b) (2) oligonucleotide pair of (c) and (d), oligonucleotide pair of (g) and (h), (i) and ( h) oligonucleotide pair, (e) and (f) oligonucleotide pair, and (j) and (k) oligonucleotide pair (3) (l) and (m) oligonucleotide pairs A group consisting of the pair of oligonucleotides (n) and (o) and the pair of oligonucleotides (v) and (w) (4) the pair of oligonucleotides (p) and (q), ) And (s) oligonucleotide pairs, and the group consisting of the above (t) and (u) oligonucleotide pairs. The heat-resistant fungus identification kit of the present invention is selected from the above categories (1) to (4). Tao Preferably, at least one pair of rigonucleotides is contained for each section. The oligonucleotide pairs (c) and (d), the oligonucleotide pairs (g) and (h) or the oligonucleotide pairs (i) and (h), and the (e) and (f) It is also a preferable aspect that the oligonucleotide pair of (j) and the oligonucleotide pair of (j) and (k) are included as a nucleic acid primer. The oligonucleotide pairs (a) and (b), the oligonucleotide pairs (i) and (h), the oligonucleotide pairs (j) and (k), the (n) and (o) It is also a preferred embodiment that the oligonucleotide pair, the oligonucleotide pair (t) and (u), and the oligonucleotide pair (v) and (w) are included as nucleic acid primers.
This kit can be used for detection and identification methods of heat-resistant fungi. In addition to the nucleic acid primer, the kit of the present invention may be a label detection substance, a buffer, a nucleic acid synthase (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), an enzyme substrate (dNTP, rNTP, etc.), etc. Contains substances commonly used for detection and identification of fungi. This kit may contain a positive control for confirming that the gene amplification reaction proceeds normally with the detection oligonucleotide pair of the present invention. Examples of the positive control include DNA containing a region amplified by the detection oligonucleotide of the present invention.

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

Example 1
(A-1) Detection and identification of fungi belonging to the genus Bisoclamis Determination of base sequence of β-tubulin partial length Base sequences of various β-tubulin genes of the genus Bisocramis were determined by the following method. DNA was extracted using Gen Torkun-kun (manufactured by TAKARA BIO INC.) From B. clamis microbial cells cultured in the dark on a potato dextrose agar slant at 30 ° C. for 7 days. PCR amplification of the target site was performed using PuRe Taq ™ Ready-To-Go PCR Beads (manufactured by GE Health Care UK LTD) as a primer, Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3 ′: SEQ ID NO: 35), Bt2b (5 '-ACCCTCAGTGTAGTGACCCTTGGC-3': SEQ ID NO: 36) (Glass and Donaldson, Appl Environ Microbiol 61: 1323-1330, 1995) was used. The amplification conditions were as follows: β-tubulin partial length was denaturation temperature 95 ° C., annealing temperature 59 ° C., extension temperature 72 ° C., and 35 cycles. The PCR product was purified using Auto Seg ™ G-50 (Amersham Pharmacia Biotech). The PCR product was labeled using BigDye terminator Ver. 1.1 (trade name: Applied Biosystems) and electrophoresed with ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Software “ATGC Ver. 4” (manufactured by Genetyx) was used to determine the base sequence from the fluorescent signal during electrophoresis.
Alignment analysis using DNA analysis software (trade name: DNAsis pro, manufactured by Hitachi Software Co., Ltd.) based on the base sequence information of the known β-tubulin gene of Bisocramis nivea and various fungi determined by sequencing method And a specific region (SEQ ID NO: 24) in the β-tubulin gene containing a base sequence specific to a fungus belonging to the genus Bisoclamis was determined.

2. Detection and identification at the genus level of fungi belonging to the genus Bisoclamis (1) Primer design Of the base sequence region specific to the fungus belonging to the genus Bisoclamis obtained above, of the fungi belonging to the genus Talalomyces on the 3 'end side From the region with particularly high specificity, 1) there are about several bases unique to the genus, 2) the GC content is approximately 30% to 80%, 3) the possibility of self-annealing is low, and 4) A partial region satisfying the four conditions of Tm value of about 55 to 65 ° C. was examined. Based on this base sequence, a pair of primer pairs was designed, and the effectiveness of Bisoclamis detection and genus identification by PCR reaction was examined using DNA extracted from various cells as a template. That is, it was examined that a DNA amplification reaction was observed at about 150 bp in the reaction using Bisochlamis genus DNA as a template, and no amplification product was observed in reactions using other mold genomic DNA as a template. As a result, DNA amplification was specifically observed at about 150 bp in Bisoclamis nivea and Bisoclamis farba, and amplification products were not recognized in reactions using other mold genomic DNA as a template. From this result, it was confirmed that exhaustive detection of the genus Bisocramis, that is, identification at the genus level of the genus Bisocramis was possible. The primer pair whose effectiveness was confirmed is a primer pair composed of oligonucleotides represented by the nucleotide sequences described in SEQ ID NOS: 1 and 2. The primer used was purchased from Sigma Aldrich Japan (salt desalted product, 0.02 μmol scale).

(2) Preparation of specimens The fungi listed in Tables 1 and 2 were used as fungi used for evaluating the effectiveness of the designed primers, that is, the genus Bisocramis and other heat-resistant molds and general molds. These fungi were stored and used by Chiba University School of Medicine, Center for Fungal Medicine, and managed by IFM numbers.
Each cell 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.) is used for the indicated temperature, that is, general mold is 25 ° C, heat-resistant mold and Aspergillus fumigatus are cultured at 30 ° C for 7 days. did.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (trade name: PrepMan ultra, manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 1. The primer (0.02 pmol / μl) 0.5 μl and the primer (0.02 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 2 were added to prepare 25 μl of a PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 59 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 2 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. The electropherograms of the agarose gel are shown in FIGS. 3 (a) and 3 (b). 3A shows an electrophoretic diagram for the fungal sample shown in Table 1, and FIG. 3B shows an electrophoretic diagram for the fungal sample shown in Table 2. The numbers in the figure indicate that the samples were reacted using DNA extracted from the samples with corresponding sample numbers in the table.
As a result, amplification of a gene fragment of about 150 bp was confirmed in the sample containing genomic DNA of fungi belonging to the genus Bisoclamis (lanes 1 to 4 in FIG. 3B). On the other hand, amplification of the gene fragment was not confirmed in the sample not containing the genomic DNA of the fungus belonging to the genus Bisoclamis. From the above results, fungi belonging to the genus Bisoclamis can be specifically detected by using the oligonucleotide of the present invention.

(A-2) Detection and identification of fungi belonging to the genus Bisoclamis (1) Preparation of primers Consisting of oligonucleotides represented by the nucleotide sequences described in SEQ ID NOS: 1 and 2 designed in Example 1 (A-1) Primers were used.

(2) Preparation of Specimens In order to confirm the detection specificity of the oligonucleotides (a) and (b) to fungi belonging to the genus Bisoclamis, each strain of bisoclamis falba shown in FIG. 4 and each of bisoclamis nivea shown in FIG. A strain was used. These fungi were used after being managed by NBRC number by the National Institute of Technology and Product Evaluation Technology and by CBS number by The Centraalbureau voor Schimmelcultures.
Each cell 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 and cultured at 30 ° C. for 7 days.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (trade name: PrepMan ultra, manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 1. The primer (0.02 pmol / μl) 0.5 μl and the primer (0.02 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 2 were added to prepare 25 μl of a PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 59 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 4 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen). The presence or absence of the amplified DNA fragment was confirmed. The electropherograms of the agarose gel are shown in FIGS. FIG. 4 shows an electrophoretic diagram for a sample of each strain of bisoclamis falba, and FIG. 5 shows an electrophoretic diagram for a sample of each strain of bisoclamis nivea.
As a result, specific amplified DNA fragments were confirmed in all the strains of Bisoclamis falba and Bisoclamis nivea used. Therefore, it can be seen that by using the oligonucleotide of the present invention, fungi belonging to the genus Bisoclamis can be specifically detected with high accuracy regardless of the type of strain.

(B-1) Detection and identification of fungi belonging to the genus Talaromyces Analysis of base sequences specific to fungi belonging to the genus Talalomyces The base sequences of various β-tubulin genes of Talalomyces and the D1 / D2 region of 28S rDNA were determined by the following method. DNA was extracted from Genus Talromamyces cells cultured in a dark place on a potato dextrose agar slope medium at 25 ° C. for 7 days using Gen Torukun TM (manufactured by Takara Bio Inc.). PCR amplification of the target site was performed using PuRe Taq ™ Ready-To-Go PCR Beads (manufactured by GE Health Care UK LTD), with Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3 ′) as the primer and β-tubulin partial length : SEQ ID NO: 35), Bt2b (5′-ACCCTCAGTGTAGTGACCCTTGGC-3 ′: SEQ ID NO: 36) (Glass and Donaldson, Appl Environ Microbiol 61: 1323-1330, 1995), and the D1 / D2 region of 28S rDNA is NL1 (5 '-GCATATCAATAAGCGGAGGAAAAG-3': SEQ ID NO: 37) and NL4 (5'-GGTCCGTGTTTCAAGACGG-3 ': SEQ ID NO: 38) (The fungal homorph: Mitotic and plemorphic speciation in fungal systematics., Wallingford: CAB international.) Were used. Amplification conditions were β-tubulin partial length of denaturation temperature 95 ° C., annealing temperature 59 ° C., extension temperature 72 ° C., 35 cycles, 28S rDNA D1 / D2 region at knitting temperature 95 ° C., annealing temperature 55 ° C., extension temperature 72 ° C. , 35 cycles. The PCR product was purified using Auto Seg ™ G-50 (Amersham Pharmacia Biotech). The PCR product was labeled using BigDye terminator Ver. 1.1 (trade name: Applied Biosystems) and electrophoresed with ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Software “ATGC Ver. 4” (manufactured by Genetyx) was used to determine the base sequence from the fluorescent signal during electrophoresis.
Β-tubulin gene of various fungi (Talaromyces flavus, Talaromyces luteus, Talaromyces wortmannii) determined by sequencing method, nucleotide sequence information of D1 / D2 region of 28S rDNA, and known β-tubulin genes of various fungi Based on the nucleotide sequence information of D1 / D2 region of 28S rDNA and DNA analysis software (trade name: DNAsis pro, manufactured by Hitachi Software Co., Ltd.) A specific region (SEQ ID NO: 25 to 27) in the D1 / D2 region of the β-tubulin gene and 28S rDNA containing the correct nucleotide sequence was determined.

2. Detection of fungi belonging to the genus Talalomyces and identification at the genus level (1) Primer design Among the base sequence regions (SEQ ID NOs: 25 to 27) specific to the fungi belonging to the genus Talalomyces obtained above, the 3 ′ end side From the region where the specificity of the fungus belonging to the genus Taralomyces is particularly high, 1) there are about several bases unique to the genus, 2) the GC content is approximately 30% to 80%, and 3) self-annealing 4) A partial region satisfying the four conditions of Tm value of about 55 to 65 ° C. was examined. Based on this nucleotide sequence, 5 primer pairs were designed for the β tubulin gene and 5 primer pairs were designed for the D1 / D2 region of 28S rDNA, and DNA extracted from various cells was used as a template. The effectiveness of the detection and identification of the genera Talamomyces by PCR was investigated. As a result, DNA amplification was observed in the size expected from the designed primer pair specifically in Talaromyces flavus and Talaromyces trachyspermus in 1 out of 5 primers of β-tubulin gene. It was. The primer pairs whose effectiveness was confirmed are SEQ ID NOS: 3 and 4.
Subsequently, by combining multiple primer pairs, exhaustive detection of the genera of Talaromyces, that is, reactions using Talamomyces DNA as a template, showed a DNA amplification reaction in the size expected from the designed primer pair, and other mold genomes It was examined that no amplification product was observed in the reaction using DNA as a template. As a result, by using a mixture of two primer pairs in five β-tubulin primers and two primer pairs in five primer sets in the D1 / D2 region of 28S rDNA, comprehensive detection of Talamomyces sp. It was confirmed that identification at the genus level of Talaromyces was possible. The primer pair whose effectiveness was confirmed is a primer pair composed of an oligonucleotide represented by the base sequences described in SEQ ID NOs: 5 to 11. The primer used was purchased from Sigma Aldrich Japan (salt desalted product, 0.02 μmol scale).

(2) Preparation of specimens The fungi listed in Table 3 were used as the fungi used for the evaluation of the specificity of the designed primer, that is, the genera Tallomyces and other heat-resistant molds and general molds. These fungi were stored and used by Chiba University School of Medicine, Center for Fungal Medicine, and managed by IFM numbers.
Each cell 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 culturing for 7 days at the indicated temperature, that is, general mold at 25 ° C. and heat-resistant mold at 30 ° C.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (PrepMan ultra (trade name) manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence shown in SEQ ID NO: 3. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 4 were added to prepare a 25 μl PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were 30 cycles of (i) heat denaturation reaction at 95 ° C for 10 seconds, (ii) annealing reaction at 59 ° C for 1 minute, and (iii) extension reaction at 72 ° C for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 10 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. The electropherogram of the agarose gel is shown in FIG.
As a result, amplification of a gene fragment of about 80 bp was confirmed in the samples (1 lane and 2 lanes) containing genomic DNA of Talaromyces flavus and Talalomyces traxpermus. On the other hand, amplification of the gene fragment was not confirmed in the sample not containing the genomic DNA of the bacterium belonging to the genus Taralomyces. From the above results, fungi belonging to the genus Talaromyces can be specifically detected by using the oligonucleotides (c) and (d).

(B-2) Detection and identification of fungi belonging to the genus Talalomyces (1) Preparation of primers Comprising oligonucleotides represented by the nucleotide sequences described in SEQ ID NOs: 5 to 8 designed in Example 1 (B-1) Primers were used.

The fungi belonging to the preparation Talaromyces genus (2) specimens, Talaromyces flavus (Talaromyces flavus), Talaromyces Torakisuperumusu (Talaromyces Trachyspermus), was used Talaromyces wortmannii knee (Talaromyces Wortmannii), and Talaromyces luteus (Talaromyces luteus). In order to confirm the specificity of the oligonucleotides (e) to (h) for the β-tubulin gene of fungi belonging to the genus Taralomyces, the fungi shown in Tables 4 and 5 were also used. These fungi were stored and used by Chiba University School of Medicine, Center for Fungal Medicine, and managed by IFM numbers.
Each cell 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 and cultured at 30 ° C. for 7 days.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (trade name: PrepMan ultra, manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 25 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 20 μl of sterile distilled water are mixed and represented by the nucleotide sequence shown in SEQ ID NO: 5. Primer (20 pmol / μl), 1.0 μl of a primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 6, and a primer represented by the base sequence described in SEQ ID NO: 7 (20 pmol / μl) μl) 1.0 μl and 1.0 μl of a primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 8 were added to prepare 50 μl of a PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were 30 cycles of (i) a heat denaturation reaction at 97 ° C. for 10 seconds, (ii) an annealing reaction at 59 ° C. for 1 minute, and (iii) an extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 2 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. The electropherograms of the agarose gel are shown in FIG. 7 (a), FIG. 7 (b) and FIG. 7 (a) shows an electrophoretic diagram for the fungal sample shown in Table 4, FIG. 7 (b) shows an electrophoretic diagram for the fungal sample shown in Table 5, and the numbers in FIG. This indicates that the sample was reacted using DNA extracted from the sample having the corresponding sample number. FIG. 8 shows an electrophoretic diagram of only a sample of a fungus belonging to the genus Tallalomyces.
As a result, among the fungi belonging to the genus Talalomyces, samples containing genomic DNA of Talalomyces flavus, Talalomyces traxpermus or Talalomyces wortmannii (lanes 1 and 2 and 5 to 8 in FIG. 7 (a), and 1 in FIG. 7 (b). (~ 2 and 5-8 lanes), amplification of a gene fragment of about 150 bp was confirmed. This gene fragment was amplified by using primers represented by the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8. In the sample containing genomic DNA of Talalomyces wortmannii or Talalomyces luteus (3-4 and 7-8 lanes in FIG. 7 (a) and 3-4 and 7-8 lanes in FIG. 7 (b)), 200 bp A certain degree of amplification of the gene fragment was confirmed. This gene fragment was amplified by using primers represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6. On the other hand, amplification of the gene fragment was not confirmed in the sample not containing the genomic DNA of the fungus belonging to the genus Taralomyces. From the above results, by simultaneously using the oligonucleotides (e) and (f) and the above (g) and (h), it is possible to specifically and comprehensively detect fungi belonging to the genus Tallalomyces.

(B-3) Detection and identification of fungi belonging to the genus Taralomyces (1) Preparation of primers Comprising oligonucleotides represented by the nucleotide sequences of SEQ ID NOs: 8 and 9 designed in Example 1 (B-1) Primers were used.

(2) Preparation of specimen In order to confirm the detection specificity of the oligonucleotides (h) and (i) above, fungi belonging to the genus Talalomyces shown in Table 6 and other fungi were used. These fungi are obtained from fungi depositors such as those managed by NBRC number by the National Institute for Product Evaluation Technology and those managed by CBS number by The Centraalbureau voor Schimmelcultures. ,used.
Each cell 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 heat-resistant mold containing Talaromyces and Aspergillus fumigatus at 30 ° C. for 7 days, and 25 other general molds. Cultured at 7 ° C. for 7 days.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (PrepMan ultra (trade name) manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 8 The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 9 were added to prepare a 25 μl PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were 30 cycles of (i) 95 ° C for 1 minute heat denaturation reaction, (ii) 59 ° C for 1 minute annealing reaction, and (iii) 72 ° C for 1 minute extension reaction for one cycle. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 10 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. An electropherogram of the agarose gel is shown in FIG. The numbers in the figure indicate samples that have been reacted using DNA extracted from the samples with corresponding sample numbers listed in Table 6.
As a result, amplification of a gene fragment of about 150 bp was confirmed only for the samples (1 to 3 lanes) containing the genomic DNAs of Talaromyces flavus, Talaromyces macrosporus and Talalomyces traxpermus, among fungi belonging to the genus Talalomyces. On the other hand, amplification of the gene fragment was not confirmed in the sample not containing the genomic DNA of the bacterium belonging to the genus Taralomyces. From the above results, it can be seen that by using the oligonucleotide of the present invention, only a specific bacterial species can be specifically detected among the fungi belonging to the genus Talalomyces.

(B-4) Detection and identification of fungi belonging to the genus Talalomyces (1) Primer Primer comprising oligonucleotides represented by the nucleotide sequences described in SEQ ID NOs: 10 and 11 designed in Example 1 (B-1) used.

(2) Preparation of specimen In order to confirm the detection specificity of the oligonucleotides (j) and (k) above, fungi belonging to the genus Tallaromyces and other fungi as in Table 6 of Example 1 (B-3) used.
Each cell 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 heat-resistant mold containing Talaromyces and Aspergillus fumigatus at 30 ° C. for 7 days, and 25 other general molds. Cultured at 7 ° C. for 7 days.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (PrepMan ultra (trade name) manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 10. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 11 were added to prepare a 25 μl PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) heat denaturation reaction at 95 ° C for 1 minute, (ii) annealing reaction at 55 ° C for 1 minute, and (iii) extension reaction at 72 ° C for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 10 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. The electropherogram of the agarose gel is shown in Fig. 9-2. The numbers in the figure indicate samples that have been reacted using DNA extracted from the samples with corresponding sample numbers listed in Table 6.
As a result, amplification of a gene fragment of about 200 bp was confirmed only for a sample (4-6 lanes) containing genomic DNAs of Talaromyces bacilis porus, Talaromyces wortmannii and Talaromyces luteus among fungi belonging to the genus Talalomyces. On the other hand, amplification of the gene fragment was not confirmed in the sample not containing the genomic DNA of the bacterium belonging to the genus Taralomyces. From the above results, it can be seen that by using the oligonucleotide of the present invention, only a specific bacterial species can be specifically detected among the fungi belonging to the genus Talalomyces.

(B-5) Detection and identification of fungi belonging to the genus Taralomyces (1) Preparation of primers Comprising oligonucleotides represented by the nucleotide sequences of SEQ ID NOs: 7 and 8 designed in Example 1 (B-1) Primers were used.

(2) Preparation of specimen In order to confirm the detection specificity of the oligonucleotides (g) and (h) to fungi belonging to the genus Talaromyces, each strain of Talaromyces flavus shown in FIG. Each strain was used. These fungi were obtained by using the NBRC number managed by the National Institute of Technology and Product Evaluation Technology and those managed by the Centraalbureau voor Schimmelcultures using the CBS number.
Each cell was cultured under optimal conditions. Regarding the culture conditions, potato dextrose medium (trade name: Pearl Core potato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.) was used and cultured at 25 ° C. for 7 days.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (PrepMan ultra (trade name) manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.) and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence shown in SEQ ID NO: 7 The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 8 were added to prepare a 25 μl PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 61 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 4 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen). The presence or absence of the amplified DNA fragment was confirmed. The electropherograms of the agarose gel are shown in FIGS.
As a result, specific amplified DNA fragments were confirmed in all strains of Talaromyces flavus and Talaromyces macrosporus used. Therefore, it can be seen that by using the oligonucleotide of the present invention, fungi belonging to the genus Tallalomyces can be specifically detected with high accuracy without depending on the type of strain.

(C-1) Detection and identification of fungi belonging to the genus Neosartoria and Aspergillus fumigatus Determination of the base sequence of the β-tubulin partial length The base sequences of the β-tubulin genes of Neosartria glabra, Neosartria fischeri, Neosartria spinosa and Aspergillus fumigatus were determined by the following method. The cells were cultured in the dark on a potato dextrose agar slope medium at 30 ° C. for 7 days. DNA was extracted from the cells using Gen Torukun TM (manufactured by Takara Bio Inc.). PCR amplification of the target site was performed using PuRe Taq ™ Ready-To-Go PCR Beads (manufactured by GE Health Care UK LTD) as a primer, Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3 ′: SEQ ID NO: 35), Bt2b (5 '-ACCCTCAGTGTAGTGACCCTTGGC-3': SEQ ID NO: 36) (Glass and Donaldson, Appl Environ Microbiol 61: 1323-1330, 1995) was used. The amplification conditions were as follows: β-tubulin partial length was denaturation temperature 95 ° C., annealing temperature 59 ° C., extension temperature 72 ° C., and 35 cycles. The PCR product was purified using Auto Seg ™ G-50 (Amersham Pharmacia Biotech). The PCR product was labeled using BigDye terminator Ver. 1.1 (trade name: Applied Biosystems) and electrophoresed with ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Software “ATGC Ver. 4” (manufactured by Genetyx) was used to determine the base sequence from the fluorescent signal during electrophoresis.
Based on the base sequence information of the β-tubulin genes of Neosartria grabra, Neosartria fischeri, Neosartria spinosa and Aspergillus fumigatus determined by sequencing methods, and the base sequence information of known β-tubulin genes of various fungi , DNA analysis software (trade name: DNAsis pro, manufactured by Hitachi Software Co., Ltd.) is used for alignment analysis, and β-tubulin gene containing a base sequence specific to fungi belonging to the genus Neosartoria and Aspergillus fumigatus A specific region (SEQ ID NO: 28-33) was determined.

2. Detection of fungi and Aspergillus fumigatus belonging to the genus Neosartoria (1) Primer design Conservation of both bacteria at the 3 'end of the base sequence region specific to fungi belonging to the genus Neosartoria and Aspergillus fumigatus obtained above 1) There are about several bases unique to the genus from the region with particularly high properties. 2) The GC content is about 30% to 80%. 3) The possibility of self-annealing is low. 4) Tm. A partial region satisfying the four conditions of approximately 55 to 65 ° C. was examined. Four primer pairs were designed based on this base sequence, and the effectiveness of collective detection of Neosartria and Aspergillus fumigatus by PCR reaction was examined using DNA extracted from various cells as a template. That is, in the reaction using Neosartria and Aspergillus fumigatus DNA as a template, a DNA amplification reaction was observed in the size expected from the designed primer pair, and in other reactions using mold DNA as a template, amplification products were observed. Considered not. As a result, specific amplification of DNA was observed in Neosartria and Aspergillus fumigatus using two pairs of primers, and amplification products were not found in reactions using other mold genomic DNA as a template. From these results, it was confirmed that the two pairs of primers can collectively detect Neosartria and Aspergillus fumigatus. The primer pairs whose effectiveness was confirmed were a primer pair consisting of an oligonucleotide represented by the base sequences described in SEQ ID NOs: 12 and 13 and a primer consisting of an oligonucleotide represented by the base sequences described in SEQ ID NOs: 14 and 15 It is a pair. The primer used was purchased from Sigma Aldrich Japan (salt desalted product, 0.02 μmol scale).

(2) Preparation of specimens The fungi listed in Table 7 were used as fungi belonging to the genus Neosartoria and Aspergillus fumigatus. In order to confirm the specificity of the oligonucleotides (l) and (m) for the β-tubulin gene of these fungi, the fungi shown in Table 7 were also used. These strains are managed by the independent administrative agency RIKEN using the JCM number, the strain managed by the Institute of Molecular Cell Biology, the University of Tokyo using the IAM number, and the National Institute of Technology and Evaluation, the IFO number. A strain derived from a fermentation laboratory fungus was obtained and used for evaluation.
Each cell was cultured under optimal conditions. As for the culture conditions, potato dextrose medium (trade name: PDA medium (Pearl Core potato dextrose agar medium), manufactured by Eiken Chemical Co., Ltd.) was used for heat-resistant mold and Aspergillus fumigatus at 30 ° C, and other general mold at 25 ° C for 7 days. Cultured.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (PrepMan ultra (trade name) manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 12. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 13 were added to prepare a 25 μl PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were 30 cycles of (i) a heat denaturation reaction at 98 ° C. for 10 seconds, (ii) an annealing reaction at 59 ° C. for 1 minute, and (iii) an extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 10 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. An electropherogram of the agarose gel is shown in FIG.
As a result, amplification of a gene fragment of about 100 bp was confirmed in samples containing genomic DNA of fungi belonging to the genus Neosartoria or Aspergillus fumigatus (1, 2 and 7 lanes). On the other hand, amplification of the gene fragment was not confirmed in the sample not containing any genomic DNA of fungi belonging to the genus Neosartoria and Aspergillus fumigatus. From the above results, fungi belonging to the genus Neosartoria and Aspergillus fumigatus can be specifically detected by using the oligonucleotides (l) and (m).

(6) Discrimination using a growth temperature difference between fungi belonging to the genus Neosartoria and Aspergillus fumigatus For samples in which amplification of gene fragments was confirmed by the above method, hyphae were obtained from a single colony in PDA medium (trade name: potato dextrose medium, Eiken Chemical Co., Ltd.) was inoculated, cultured at 50 ° C. for 1 day, and observed by a mycelia confirmation method using a stereomicroscope. As a result, active hyphae growth was observed in the samples inoculated with Aspergillus fumigatus, but no hyphae growth was observed in the samples inoculated with fungi belonging to the genus Neosartoria. From these results, it was possible to distinguish fungi belonging to the genus Neosartoria and Aspergillus fumigatus using the difference in the temperature range where they can grow.

(C-2) Detection and identification of fungi and Aspergillus fumigatus belonging to the genus Neosartria (1) Preparation of primers Primers consisting of oligonucleotides were used.

(2) Preparation of specimens The fungi listed in Tables 8 and 9 were used as fungi belonging to the genus Neosartoria and Aspergillus fumigatus. In order to confirm the specificity of the oligonucleotides (n) and (o) to fungi belonging to the genus Neosartoria and the β-tubulin gene of Aspergillus fumigatus, other fungi shown in Tables 8 and 9 were also used. These fungi were stored and used by the Fungal Medicine Research Center, Chiba University School of Medicine, and managed by numbering.
Each cell 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 30 ° C for heat-resistant mold and Aspergillus fumigatus, and at 25 ° C for other general molds for 7 days.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (trade name: PrepMan ultra, manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence shown in SEQ ID NO: 14. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 15 were added to prepare a 25 μl PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were 30 cycles of (i) a heat denaturation reaction at 97 ° C. for 10 seconds, (ii) an annealing reaction at 59 ° C. for 1 minute, and (iii) an extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 2 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. The electropherograms of the agarose gel are shown in FIGS. 13 (a) and 13 (b). FIG. 13 (a) shows an electrophoretic diagram for the fungal sample shown in Table 8, and FIG. 13 (b) shows an electrophoretic diagram for the fungal sample shown in Table 9. The numbers in the figure indicate that the samples were reacted using DNA extracted from the samples with corresponding sample numbers in the table.
As a result, amplification of a gene fragment of about 200 bp was confirmed in the sample containing genomic DNA of fungi belonging to the genus Neosartoria and Aspergillus fumigatus. On the other hand, amplification of the gene fragment was not confirmed in the sample not containing any genomic DNA of fungi belonging to the genus Neosartoria and Aspergillus fumigatus. From the above results, fungi belonging to the genus Neosartoria and Aspergillus fumigatus can be specifically detected by using the oligonucleotides (n) and (o).

(6) Discrimination using the growth temperature difference between fungi belonging to the genus Neosartria and Aspergillus fumigatus For samples in which amplification of the gene fragment was confirmed by the above method, hyphae were obtained from a single colony in PDA medium (trade name: potato dextrose agar medium) , Manufactured by Eiken Chemical Co., Ltd.), cultured at 50 ° C. for 1 day, and observed for hyphal elongation with a stereomicroscope. As a result, active hyphae growth was observed in the samples inoculated with Aspergillus fumigatus, but no hyphae growth was observed in the samples inoculated with fungi belonging to the genus Neosartoria. From these results, it was possible to distinguish fungi belonging to the genus Neosartoria and Aspergillus fumigatus using the difference in the temperature range where they can grow.

(C-3) Discrimination between fungi belonging to the genus Neosartria and Aspergillus fumigatus using the oligonucleotides (v) and (w) (1) Design of primers for detecting Aspergillus fumigatus Neosartria shown in SEQ ID NOs: 28 to 33 Regions where differences in the base sequences of both species existed were determined using alignment analysis (DNAsis Pro) of the base sequences of β-tubulin genes of Grabra, Aspergillus fumigatus, Neosartoria Fischeri and Neosartoria spinosa. Among the determined region of the base sequence specific to Aspergillus fumigatus, from the region where both bacteria are particularly conserved on the 3 ′ end side, 1) There are several base sequences unique to Aspergillus fumigatus. 2) Examination of partial regions satisfying the following four conditions: the GC content is approximately 30% to 80%, 3) the possibility of self-annealing is low, and 4) the Tm value is approximately 55 to 65 ° C. went. Two primer pairs were designed based on this base sequence, and the effectiveness of distinguishing between Neosartria and Aspergillus fumigatus by PCR reaction was examined using DNA extracted from various cells as a template. In other words, in the reaction using Aspergillus fumigatus DNA as a template, a DNA amplification reaction was observed in the size expected from the designed primer pair, and the amplification product was not observed in the reaction using Neosartoria genomic DNA as a template. Went. As a result, DNA amplification was specifically observed in Aspergillus fumigatus with one primer pair, and no amplification product was observed in reactions using genomic DNA of other fungi as a template. The primer pair whose effectiveness was confirmed was a primer pair consisting of oligonucleotides represented by the nucleotide sequences of SEQ ID NOS: 22 and 23. The primer used was purchased from Sigma Aldrich Japan (salt desalted product, 0.02 μmol scale).

(2) Preparation of specimens The fungi listed in Table 10 and Table 11 were used as fungi belonging to the genus Neosartoria and Aspergillus fumigatus. For reference, fungi other than those belonging to the genus Neosartoria shown in Tables 10 and 11 and fungi other than Aspergillus fumigatus were also used. The strain was preserved by the Research Center for Fungal Medicine, Chiba University, and a strain managed by numbering was obtained and used for testing.
Each cell 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 30 ° C for heat-resistant mold and Aspergillus fumigatus, and at 25 ° C for other general molds for 7 days.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (trade name: PrepMan ultra, manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 22 The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 23 were added to prepare a 25 μl PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 59 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After the PCR reaction, 2.5 μl was taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and the DNA was extracted with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen). After staining, the presence or absence of the amplified DNA fragment was confirmed. Electropherograms of the agarose gel are shown in FIGS. 14 shows an electrophoretic diagram for the fungal sample shown in Table 10, and FIG. 15 shows an electrophoretic diagram for the fungal sample shown in Table 11. The numbers in the figure indicate that the samples were reacted using DNA extracted from the samples with corresponding sample numbers in the table.
As shown in FIGS. 14 and 15, an amplified fragment of about 200 bp was clearly confirmed only in the sample containing Aspergillus fumigatus genomic DNA. On the other hand, an amplified fragment of about 200 bp was not confirmed in samples containing genomic DNA of other fungi including fungi of the genus Neosartoria.
From these results, Aspergillus fumigatus can be specifically detected by performing gene amplification using the oligonucleotides (v) and (w) and confirming the presence or absence of the amplification product.

(C-4) Detection and identification of fungi belonging to the genus Neosartoria and Aspergillus fumigatus (1) Preparation of primers SEQ ID NOs: 14-15 and 22-23 designed in Examples (C-1) and (C-3) The primer which consists of oligonucleotide represented by the base sequence as described in 1 was used.

(2) Preparation of specimen Confirm the detection specificity of the oligonucleotides (n) and (o) for fungi belonging to the genus Neosartoria, and the detection specificity of the oligonucleotides (v) and (w) for Aspergillus fumigatus. Therefore, each strain of Neosartria fischeri shown in FIG. 16, each strain of Neosartria gravura shown in FIG. 17, each strain of Neosartria hiratsukae shown in FIG. 18, each strain of Neosartria paulistensis shown in FIG. Each strain of Neosartria spinosa shown in FIG. 20 was used. Aspergillus fumigatus was used as a positive control for the reaction system using the oligonucleotides (v) and (w). In addition, these fungi are obtained from fungus depository organizations, such as those managed by the National Institute of Technology and Product Evaluation Technology Base under the NBRC number and those managed by the Centraalbureau voor Schimmelcultures as CBS numbers, and Chiba Strains stored at the University Fungal Medicine Research Center and managed with IFM numbers were obtained and used as test bacteria.
Each cell was cultured under optimal conditions. Regarding the culture conditions, potato dextrose medium (trade name: Pearl Core potato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.) was cultured at 30 ° C. for heat-resistant mold and Aspergillus fumigatus at 25 ° C. for 14 days.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (trade name: PrepMan ultra, manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence shown in SEQ ID NO: 14. The primer (0.02 pmol / μl) 0.5 μl and the primer (0.02 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 15 were added to prepare 25 μl of a PCR reaction solution. Similarly, the primer was changed to 0.5 μl of the primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 22 and 0.5 μl of the primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 23. A PCR reaction solution was prepared in the same manner as described above except that.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 59 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 4 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen). The presence or absence of the amplified DNA fragment was confirmed. The electropherograms of the agarose gel are shown in FIGS. 16 shows an electrophoretic diagram for a sample of each strain of Neosartria fischeri, FIG. 17 shows an electrophoretic diagram for a sample of each strain of Neosartria grabra, and FIG. 18 shows each of the strains of Neosartria hiratsukae. FIG. 19 shows an electrophoretic diagram for a sample of each strain of Neosartria Paulistensis, and FIG. 20 shows an electrophoretic diagram for a sample of each strain of Neosartria spinosa.
As a result, in the reaction system using the primers represented by the base sequences described in SEQ ID NOs: 14 and 15, the Neosartria Fischeri, Neosartria grabra, Neosartria hiratsukae, Neosartria Paulistensis and Neosartria spinosa used Specific amplified DNA fragments were confirmed in all strains (FIGS. 16 (a), 17 (a), 18 (a), 19 (a), and 20 (a)). Therefore, it can be seen that by using the oligonucleotide of the present invention, fungi belonging to the genus Neosartria can be specifically detected with high accuracy regardless of the type of strain.
In the reaction system using the primers represented by the nucleotide sequences set forth in SEQ ID NOs: 22 and 23, specific amplified DNA fragments were confirmed only in samples using Aspergillus fumigatus genomic DNA as a template. On the other hand, no amplification of DNA fragments was confirmed in any sample using each strain of the genus Neosartria (FIG. 16 (b), FIG. 17 (b), FIG. 18 (b), FIG. 19 (b), FIG. 20 (b)). Therefore, it can be seen that Aspergillus fumigatus can be specifically detected by performing gene amplification using the oligonucleotides (v) and (w) and confirming the presence or absence of the amplification product.

(D-1) Detection and identification of fungi belonging to the genus Hamigera Determination of the base sequence of the β-tubulin partial length The base sequence of the β-tubulin gene of Hamigella abernea and Cladosporium cladsporoides was determined by the following method. On a potato dextrose agar slant culture medium, Hamigera abernea was cultured in the dark for 7 days at 30 ° C. and Cladosporium cladosporoides at 25 ° C. for 7 days. DNA was extracted from the cells using Gen Torukun TM (manufactured by Takara Bio Inc.). PCR amplification of the target site was performed using PuRe Taq ™ Ready-To-Go PCR Beads (manufactured by GE Health Care UK LTD) as a primer, Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3 ′: SEQ ID NO: 35), Bt2b (5 '-ACCCTCAGTGTAGTGACCCTTGGC-3': SEQ ID NO: 36) (Glass and Donaldson, Appl Environ Microbiol 61: 1323-1330, 1995) was used. The amplification conditions were as follows: β-tubulin partial length was denaturation temperature 95 ° C., annealing temperature 59 ° C., extension temperature 72 ° C., and 35 cycles. The PCR product was purified using Auto Seg ™ G-50 (Amersham Pharmacia Biotech). The PCR product was labeled using BigDye terminator Ver. 1.1 (trade name: Applied Biosystems) and electrophoresed with ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Software “ATGC Ver. 4” (manufactured by Genetyx) was used to determine the base sequence from the fluorescent signal during electrophoresis.
DNA analysis software based on the base sequence information of the β-tubulin gene of Hamigera abernea and Cladosporium cladesporoides determined by sequencing method and the base sequence information of known β-tubulin genes of various fungi Ware (trade name: DNAsis pro, manufactured by Hitachi Software Co., Ltd.), a specific region in the β-tubulin gene containing a base sequence specific to Humigera abernea belonging to the genus Hamigera (SEQ ID NO: SEQ ID NO: 34) was determined.

2. Detection of fungi belonging to fungi belonging to the genus Hamigera (1) Primer design From the region of the base sequence specific to Hamigera avernea obtained above, from the region where the preservability of both bacteria is particularly high on the 3 ′ end side, 1 ) The base sequence unique to the genus exists around several bases 2) The GC content is approximately 30% to 80%, 3) The possibility of self-annealing is low, and 4) The Tm value is approximately 55 to 65 ° C. A partial region satisfying the following four conditions was examined. Five primer pairs were designed based on this base sequence, and the effectiveness of detection of fungi belonging to the genus Hamigera by PCR reaction was examined using DNA extracted from various cells as a template. As a result, DNA amplification was specifically observed in Hamigera abernea and Cladosporium cladosporodes with one primer pair, and no amplification product was observed in reactions using other mold genomic DNA as a template. From this result, it was confirmed that the detection of the genus Humigera was possible. The primer pair whose effectiveness was confirmed was a primer pair consisting of oligonucleotides represented by the nucleotide sequences set forth in SEQ ID NOs: 16 and 17. The primer used was purchased from Sigma Aldrich Japan (salt desalted product, 0.02 μmol scale).

(2) Preparation of specimens The fungi listed in Table 12 were used as fungi used in the evaluation of the effectiveness of the designed primer, that is, the genus Hamigera and other heat-resistant fungi and general fungi. These fungi were stored and used by the Center for Fungal Medicine, Chiba University School of Medicine, and managed by IFM numbers and T numbers.
Each cell 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 culturing for 7 days at the indicated temperature, that is, general mold at 25 ° C. and heat-resistant mold at 30 ° C.

(3) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (PrepMan ultra (trade name) manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.) and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 16. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 17 were added to prepare a 25 μl PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were 30 cycles of (i) a heat denaturation reaction at 98 ° C. for 10 seconds, (ii) an annealing reaction at 63 ° C. for 1 minute, and (iii) an extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified gene fragment After PCR reaction, 10 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. An electropherogram of the agarose gel is shown in FIG. The numbers in the figure indicate samples that have been reacted using DNA extracted from the samples with corresponding sample numbers listed in Table 12.
As a result, amplification of a gene fragment of about 100 bp was confirmed in a sample (1 lane) containing fungal genomic DNA belonging to the genus Hamigera. On the other hand, amplification of the gene fragment was not confirmed in the sample not containing the genomic DNA of the fungus belonging to the genus Hamigera. From the above results, fungi belonging to the genus Hemigera can be specifically detected by using the oligonucleotides (p) and (q).

(D-2) Detection and identification of fungi belonging to the genus Hamigera (a) Preparation of primers Comprising the oligonucleotides represented by the nucleotide sequences described in SEQ ID NOs: 16 and 17 designed in Example 1 (D-1) Primers were used.

(B) Preparation of Specimens As the fungi belonging to the genus Hamigera and the fungus belonging to the genus Cladosporium, Hamigera avelnea and H. cladasporioides ( Cladosporium cladosporoides ) described in Table 13 were used. In order to show the specificity of the oligonucleotides (p) and (q) for the β-tubulin gene of fungi belonging to the genus Hamigera and fungi belonging to the genus Cladosporium, other fungi shown in Table 13 were also used. These fungi were stored and used by the Center for Fungal Medicine, Chiba University School of Medicine, and managed by IFM numbers and T numbers. As a positive control, Hamigera avellanea (strain name: T34) was used as a template DNA.
Each cell 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.) is used at the optimum temperature, ie, 25 ° C for general mold, 30 ° C for heat-resistant mold and Aspergillus fumigatus for 7 days. Cultured.

(C) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (trade name: PrepMan ultra, manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(D) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.) and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 16. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 17 were added to prepare a 25 μl PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were 30 cycles of (i) a heat denaturation reaction at 97 ° C. for 10 seconds, (ii) an annealing reaction at 63 ° C. for 1 minute, and (iii) an extension reaction at 72 ° C. for 1 minute. Cycled.

(E) Confirmation of amplified gene fragment After PCR reaction, 2 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. An electropherogram of the agarose gel is shown in FIG. The numbers in the figure indicate samples that have been reacted using DNA extracted from the samples with corresponding sample numbers listed in Table 13.
As a result, amplification of a gene fragment of about 100 bp was confirmed in samples containing genomic DNA of fungi belonging to the genus Hamigera and fungi belonging to the genus Cladosporium. On the other hand, amplification of the gene fragment was not confirmed in the samples that did not contain any genomic DNA of fungi belonging to the genus Hamigera and fungi belonging to the genus Cladosporium. From the above results, by using the oligonucleotides (p) and (q), it is possible to specifically detect fungi belonging to the genus Hamigella and fungi belonging to the genus Cladosporium.

(D-3) Detection and identification of fungi belonging to the genus Hamigera (a) Preparation of primer of the β-tubulin base sequence region of other fungi including Hamigera abernea and Cladosporium described in SEQ ID NO: 34 Alignment analysis (DNAsis Pro) was performed to identify sites with significant base differences. Among the sites, from the region where the specificity of Hamigella Avellanea is particularly high on the 3 ′ end side, 1) there are about several bases unique to the genus, and 2) the GC content is approximately 30% to 80%. The partial regions satisfying the four conditions of 3) low possibility of self-annealing and 4) Tm value of about 55 to 65 ° C. were examined. Seven primer pairs were designed on the basis of this base sequence, and the effectiveness of the method for distinguishing between H. genus and Cladosporium cladosporodes by PCR reaction using DNA extracted from various cells as a template was examined. . In other words, we examined that DNA amplification reaction was observed in the size expected from the designed primer pair in the reaction using Hemigella DNA, and no amplification product was observed in the reaction using other mold DNA as a template. It was. As a result, specific amplification of DNA was observed in the genus Hemigera including Hamiguella abernea with the two primer pairs, and amplification products were not found in reactions using other mold genomic DNA as a template. Based on this result, it was confirmed that the genus Hamigera and Cladosporium cladosporides can be distinguished. The primer pair whose effectiveness was confirmed was a primer pair consisting of an oligonucleotide represented by the nucleotide sequences set forth in SEQ ID NOs: 18 and 19 and a primer consisting of an oligonucleotide represented by the nucleotide sequences set forth in SEQ ID NOs: 20 and 21 It is a pair. The primer used was purchased from Sigma Aldrich Japan (salt desalted product, 0.02 μmol scale).

(B) Preparation of Specimens The fungi listed in Table 14 were used as fungi used for evaluating the effectiveness of the designed primer, that is, the genus Hamigera and other heat-resistant fungi and general fungi. These fungi were stored and used by Chiba University School of Medicine, Center for Fungal Medicine, and managed by IFM numbers.
Each cell 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 at the optimum temperature, that is, general mold was cultured at 25 ° C. and heat-resistant mold was cultured at 30 ° C. for 7 days.

(C) Preparation of genomic DNA Each bacterial cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (trade name: PrepMan ultra, manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 50 ng / μl.

(D) PCR reaction As a DNA template, 1 μl of the above-prepared Hamigera abernea and Cladosporium genomic DNA solution, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water are mixed. In addition, 0.5 μl of a primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 18 and 0.5 μl of a primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 19 were added, and 25 μl of PCR was added. A reaction solution was prepared.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were 30 cycles with (i) a heat denaturation reaction at 97 ° C. for 10 seconds, (ii) an annealing reaction at 60 ° C. for 1 minute, and (iii) an extension reaction at 72 ° C. for 1 minute in one cycle. Cycled.

(E) Confirmation of amplified gene fragment After PCR reaction, 2 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. The electropherogram of the agarose gel is shown in FIG. The numbers in the figure indicate samples that have been reacted using DNA extracted from the samples with the corresponding sample numbers listed in Table 14.
As a result, amplification of a gene fragment of about 200 bp was confirmed in the sample containing the genomic DNA of fungi belonging to the fungus belonging to the genus Hamigera (sample numbers 1 to 3). On the other hand, amplification of the gene fragment was not confirmed in the sample containing the genomic DNA of the fungus belonging to the genus Cladosporium (sample No. 17) and the sample not containing the genomic DNA of the other fungus belonging to the genus Hamigera. From the above results, by using the oligonucleotides (r) and (s), it is possible to identify whether the fungus contained in the sample is a fungus belonging to the genus Hamigera or a fungus belonging to the genus Cladosporium. it can.

(D-4) Detection and identification of fungi belonging to the genus Hamigera (a) Preparation of primers Comprising oligonucleotides represented by the nucleotide sequences of SEQ ID NOs: 18 to 21 designed in Example 1 (D-3) Primers were used.

(B) Preparation of Specimen In order to confirm the specificity of the oligonucleotides (r) to (u) for the β-tubulin gene of fungi belonging to the genus Hamigera, Each strain of Horigera sriata was used.
Each bacterial cell was cultured in the same manner as (D-1) described above.

(C) Preparation of genomic DNA A genomic DNA solution was prepared in the same manner as in the above (D-1). The concentration of the DNA solution was adjusted to 50 ng / μl.

(D) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 18. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 19 were added to prepare a 25 μl PCR reaction solution. Similarly, the primer was changed to 0.5 μl of the primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 20 and 0.5 μl of the primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 21 A PCR reaction solution was prepared in the same manner as described above except that.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were (i) 95 ° C for 1 minute heat denaturation reaction, (ii) 61-59 ° C for 1 minute annealing reaction, and (iii) 72 ° C for 1 minute extension reaction for one cycle. For 35 cycles.

(E) Confirmation of amplified gene fragment After PCR reaction, 4 μl was taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and stained with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen). The presence or absence of the amplified DNA fragment was confirmed. An electropherogram of the agarose gel is shown in FIG.
As a result, the reaction system using the primer represented by the base sequence described in SEQ ID NO: 18 and 19 and the reaction system using the primer represented by the base sequence described in SEQ ID NO: 20 and 21 are used. Specific amplified DNA fragments were confirmed in all the strains of Hamigella striata (9 to 16 lanes). Further, the detected band was clearer in the reaction system using the primers represented by the nucleotide sequences described in SEQ ID NOs: 20 and 21 (9 to 12 lanes). Therefore, it can be seen that by using the oligonucleotide of the present invention, fungi belonging to the genus Hemigera can be specifically detected with high accuracy regardless of the type of strain.

(D-5) Detection and identification of fungi belonging to the genus Hamigera (a) Preparation of primers Consisting of oligonucleotides represented by the nucleotide sequences of SEQ ID NOs: 18 to 21 designed in Example 1 (D-3) Primers were used.

(B) Preparation of Specimen In order to confirm the specificity of the oligonucleotides (r) to (u) for the β-tubulin gene of fungi belonging to the genus Hamigera, Each strain of Avernea ( Hamigera avellanea ) was used.
Each bacterial cell was cultured in the same manner as (D-1) described above.

(C) Preparation of genomic DNA A genomic DNA solution was prepared in the same manner as in the above (D-1). The concentration of the DNA solution was adjusted to 50 ng / μl.

(D) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 18. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 19 were added to prepare a 25 μl PCR reaction solution. Similarly, the primer was changed to 0.5 μl of the primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 20 and 0.5 μl of the primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 21 A PCR reaction solution was prepared in the same manner as described above except that.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 59 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute. Cycled.

(E) Confirmation of amplified gene fragment After PCR reaction, 2 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. The electropherogram of the agarose gel is shown in FIG.
As a result, the reaction system using the primer represented by the base sequence described in SEQ ID NO: 18 and 19 and the reaction system using the primer represented by the base sequence described in SEQ ID NO: 20 and 21 are used. Specific amplified DNA fragments were confirmed in all the strains of Hamagera abernea (1 to 8 lanes each). Therefore, it can be seen that by using the oligonucleotide of the present invention, fungi belonging to the genus Hemigera can be specifically detected with high accuracy regardless of the type of strain.

(D-6) Detection and identification of fungi belonging to the genus Hamigera (a) Primer A primer composed of an oligonucleotide represented by the nucleotide sequence of SEQ ID NOS: 18 to 21 designed in Example 1 (D-3) used.

(B) Preparation of Specimen In order to confirm the specificity of the oligonucleotides (r) to (u) with respect to the genus Hamigera, fungus belonging to the genus Bisocramis, Each strain of Bisoclamis fulva was used. In addition, these fungi were obtained by using those managed by NBRC numbers by the National Institute of Technology and Product Evaluation Technology and those managed by CBS numbers by The Centraalbureau voor Schimmelcultures.
Each cell was cultured in the same manner as in Example 1 (D-1).

(C) Preparation of genomic DNA A genomic DNA solution was prepared in the same manner as in Example 1 (D-1). The concentration of the DNA solution was adjusted to 50 ng / μl.

(D) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, 13 μl of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μl of sterile distilled water were mixed and represented by the nucleotide sequence set forth in SEQ ID NO: 18. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 19 were added to prepare a 25 μl PCR reaction solution. Similarly, the primer was changed to 0.5 μl of the primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 20 and 0.5 μl of the primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 21 A PCR reaction solution was prepared in the same manner as described above except that.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 59 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute. Cycled.

(E) Confirmation of amplified gene fragment After PCR reaction, 2 μl is taken from the PCR reaction solution, electrophoresed on a 2% agarose gel, and after DNA staining with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen) The presence or absence of the amplified DNA fragment was confirmed. Electropherograms of the agarose gel are shown in FIGS.
As a result, as shown in FIG. 26, in the reaction system using the primers represented by the nucleotide sequences shown in SEQ ID NOs: 18 and 19, some strains NBRC31877 and NBRC31878 of Bisoclamis fulva other than the positive control Hamigella abernea. In the strains (lanes 9 and 10), gene amplification was observed at a size of 200 bp, but no gene amplification was observed for other fungi belonging to the genus Bisoclamis. The reason why gene amplification was observed in the NBRC 31877 and 31878 strains is presumed to be because these strains are genetically related to the genus Hamigera compared to other strains.
On the other hand, as shown in FIG. 27, in the reaction system using the primers represented by the nucleotide sequences shown in SEQ ID NOs: 20 and 21, no gene amplification was observed in the B. miracle strain NBRC31877 and NBRC31878. Therefore, it can be seen that by using the oligonucleotides described in SEQ ID NOs: 20 and 21, only fungi belonging to the genus Hemigera can be specifically detected with high accuracy without detecting fungi belonging to the genus Bisoclamis.

  As described above, it was found that heat-resistant fungi can be detected by using the oligonucleotides defined in the present invention. That is, fungi belonging to the genus Bisoclamis can be identified by using the oligonucleotides of SEQ ID NOs: 1 and 2. By using the oligonucleotides of SEQ ID NOs: 3 to 11, fungi belonging to the genus Talalomyces can be identified. Fungi belonging to the genus Neosartoria and Aspergillus fumigatus can be detected by using the oligonucleotides of SEQ ID NOs: 12 to 15. In addition, according to this invention, the fungi which belong to Neosartoria genus and Aspergillus fumigatus can also be distinguished. Fungi belonging to the genus Hamigera can be detected by using the oligonucleotides of SEQ ID NOs: 16 to 21. Therefore, the heat-resistant fungi can be identified by performing at least two or more, preferably all of the steps of detecting the heat-resistant fungi using the specific oligonucleotide in the present invention.

Claims (13)

Byssochlamys (Byssochlamys) genus Talaromyces (Talaromyces) genus Neosartorya (Neosartorya) genus, in a method of detecting at least two fungi selected from the group consisting of Aspergillus fumigatus (Aspergillus fumigatus) and Hamigera (Hamigera) genus, the following 1- A method for detecting heat-resistant fungi, comprising at least two steps selected from the group consisting of 2) to 4-2).
1-2) below (a) and / or conducting the identification of Byssochlamys (Byssochlamys) belonging to the genus fungi using oligonucleotides as nucleic acid primer (b),
2-2) The following oligonucleotides (c) and / or (d), the following oligonucleotides (g) and / or (h), the following oligonucleotides (i) and / or (h), the following (e) and / or oligonucleotides (f), and / or the following (j) and / or conducting the identification of Talaromyces (Talaromyces) belonging to the genus fungi using oligonucleotides as nucleic acid primers (k),
3-2) below (l) and / or (oligonucleotide m), or the following (n) and / or (o Neosartorya using oligonucleotides as nuclei acid primers) (Neosartorya) fungi belonging to the genus and / Or a step of identifying Aspergillus fumigatus ( Aspergillus fumigatus ),
4-2) below (p) and / or (oligonucleotide q), the following (r) and / or (s oligonucleotides), and / or the following (t) and / or (u) Nuclear oligonucleotides A step of identifying fungi belonging to the genus Hamigera using as an acid primer

(A) nucleotide sequence according to SEQ ID NO: 1, or 1 bases in the nucleotide sequence represented by deletion, insertion or a base sequence which can be used as a base sequence substitution and the detector oligonucleotide Table with a base sequence, or the one base deletion in the nucleotide sequence, insertion or a base sequence which can be used as a substituted nucleotide sequence and detecting oligonucleotide according to the oligonucleotide (b) SEQ ID NO: 2 nucleotide sequence according to the oligonucleotides (c) SEQ ID NO: 3 which is, or one nucleotide in the nucleotide sequence are deleted, inserted or a substituted nucleotide sequence and nucleotide sequence can be used as a detector oligonucleotide in nucleotide sequence according to the oligonucleotide (d) SEQ ID NO: 4, represented, or one base deletion in the nucleotide sequence, inserted or substituted salt Nucleotide sequence according to the oligonucleotides (e) SEQ ID NO: 5 represented by the nucleotide sequence which can be used as a a by and the detector oligonucleotide sequence, or a single base in the nucleotide sequence are deleted, inserted or substituted one base deletion in the nucleotide sequence, or the nucleotide sequence of the nucleotide sequence in a by and oligonucleotides represented by the nucleotide sequence which can be used as a detection oligonucleotide (f) SEQ ID NO: 6, inserted or one base deletion in the nucleotide sequence, or the nucleotide sequence of the oligonucleotide (g) SEQ ID NO: 7 represented by the nucleotide sequence which can be used as a substituted nucleotide sequence and the detector oligonucleotide, inserted or substituted nucleotide sequence is a by and oligonucleotides represented by the nucleotide sequence which can be used as a detection oligonucleotide ( ) Nucleotide sequence according to SEQ ID NO: 8, or an oligonucleotide in which one base in the nucleotide sequence represented by deletion, insertion or a base sequence which can be used as a substituted nucleotide sequence and the detector oligonucleotide (i) set forth in SEQ ID NO: 9 nucleotide sequence or a single base in the nucleotide sequence represented by deletion, insertion or a base sequence which can be used as a substituted nucleotide sequence and the detector oligonucleotide Table with a base sequence which can be used as an oligonucleotide (j) sequences nucleotide sequence set forth in ID NO: 10, or the base one base deletion in the sequence, inserted or a substituted nucleotide sequence and the detector oligonucleotide oligonucleotides (k) nucleotide sequence of SEQ ID NO: 11, or one base deletion in the nucleotide sequence, inserted or substituted salts One base deletion in the nucleotide sequence, or the nucleotide sequence of the oligonucleotide (l) SEQ ID NO: 12 represented by the nucleotide sequence which can be used as a by and the detector oligonucleotide group sequence, insertion or substitution base sequence in a by and according to an oligonucleotide (m) SEQ ID NO: 13 represented by the nucleotide sequence which can be used as a detection oligonucleotide nucleotide sequence, or one base deletion in the nucleotide sequence, insertion or substituted nucleotide sequence is a by and nucleotide sequence according to the oligonucleotides (n) SEQ ID NO: 14 represented by the nucleotide sequence which can be used as a detection oligonucleotide, or one base deletion in the nucleotide sequence , Origonukureo represented by the nucleotide sequence which can be used as an insert or a substituted nucleotide sequence and the detector oligonucleotide Nucleotide sequence according to de (o) SEQ ID NO: 15, or a single base in the nucleotide sequence represented by deletion, insertion or base sequence which can be used as a substituted nucleotide sequence and the detector oligonucleotide that nucleotide sequence according to the oligonucleotides (p) SEQ ID NO: 16, or a single base in the nucleotide sequence are deleted, an insertion or a substituted nucleotide sequence and the nucleotide sequence which can be used as a detection oligonucleotide base nucleotide sequence according to the oligonucleotides (q) SEQ ID NO: 17, or a single base in the nucleotide sequence are deleted, can be used as an insert or substituted by a nucleotide sequence and detecting an oligonucleotide represented one base deletion in the nucleotide sequence, or the nucleotide sequence of the oligonucleotide (r) SEQ ID NO: 18 of SEQ, insertion or One base deletion in the nucleotide sequence, or the nucleotide sequence of the oligonucleotide (s) SEQ ID NO: 19 represented by the nucleotide sequence which can be used as conversion has been a nucleotide sequence and detecting oligonucleotide, inserted or substituted nucleotide sequence according to nucleotide sequence in a by and oligonucleotides represented by the nucleotide sequence which can be used as a detection oligonucleotide (t) SEQ ID NO: 20, or a single base is deleted in the nucleotide sequence loss, insertion or substituted nucleotide sequence is a by and nucleotide sequence according to the oligonucleotides (u) SEQ ID NO: 21 represented by the nucleotide sequence which can be used as a detection oligonucleotide, or a single base in the nucleotide sequence Is a nucleotide sequence that has been deleted, inserted or substituted and can be used as a detection oligonucleotide. Gonucleotide In order to perform identification, a gene amplification reaction is performed using any one of the following oligonucleotide pairs (1-a) to (4-a) as a nucleic acid primer pair, and the presence or absence of an amplification product is confirmed. The method for detecting a heat-resistant fungus according to claim 1.

(1-a) the oligonucleotide pair of (a) and (b) above,
(2-a) the oligonucleotide pair of (c) and (d), the oligonucleotide pair of (g) and (h), the oligonucleotide pair of (i) and (h), (e) and ( f) the oligonucleotide pair and the oligonucleotide pairs (j) and (k) above,
(3-a) the oligonucleotide pair of (l) and (m), and the oligonucleotide pair of (n) and (o),
(4-a) the oligonucleotide pair (p) and (q), the oligonucleotide pair (r) and (s), and the oligonucleotide pair (t) and (u)   The method for detecting a thermostable fungus according to claim 2, wherein the amplification reaction is performed by a polymerase chain reaction (PCR) method. In the detection method of the heat-resistant fungi, characterized in that it further comprises a step of identifying whether growth can temperature zones Neosartorya by utilizing a difference in (Neosartorya) belonging to the genus fungi or Aspergillus fumigatus (Aspergillus fumigatus), claim The method for detecting a heat-resistant fungus according to any one of 1 to 3 . In the detection method of the heat-resistant fungi, further (v) below and / or (w) of Neosartorya using oligonucleotides as nucleic acid primers (Neosartorya) fungi or Aspergillus fumigatus belonging to the genus (Aspergillus fumigatus) or identifying a The method for detecting heat-resistant fungi according to any one of claims 1 to 4 , further comprising:
(V) a base sequence set forth in SEQ ID NO: 22, or a single base in the nucleotide sequence represented by deletion, insertion or a base sequence which can be used as a substituted nucleotide sequence and the detector oligonucleotide Table with a base sequence which can be used as an oligonucleotide (w) sequences nucleotide sequence set forth in ID NO: 23, or the base one base deletion in the sequence, inserted or a base sequence substitution and the detector oligonucleotide Oligonucleotide In the step of detecting a fungus belonging to the genus Talaromyces, the oligonucleotide pair (c) and (d), the oligonucleotide pair (g) and (h), or the above (i) and (h) The gene amplification treatment step is performed using the oligonucleotide pair and the oligonucleotide pair (e) and (f) or the oligonucleotide pair (j) and (k) at the same time. 3. A method for detecting a heat-resistant fungus according to 3. Gene amplification treatment using either the oligonucleotide pair (p) and (q), the oligonucleotide pair (r) and (s) or the oligonucleotide pair (t) and (u) The heat resistance according to claim 2 or 3, wherein the sample confirmed to be amplified by a gene is subjected to a gene amplification treatment using the other oligonucleotide pair to detect a fungus belonging to the genus Hamigera . Method for detecting fungi. A thermostable fungus detection kit comprising at least two oligonucleotide pairs selected from the following groups (1) to (4) as oligonucleotide pairs selected from different groups.
(1) was a base sequence to which the oligonucleotide pair (a) nucleotide sequence according to SEQ ID NO: 1, or 1 bases in the nucleotide sequence are deleted, inserted or substituted in the following (a) and (b) Te and an oligonucleotide represented by the nucleotide sequence which can be used as a detection oligonucleotide (b) nucleotide sequence according to SEQ ID NO: 2, or a single base in the nucleotide sequence are deleted, inserted or substituted nucleotide sequence And an oligonucleotide represented by a base sequence that can be used as a detection oligonucleotide (2) an oligonucleotide pair (c) and (d) below, an oligonucleotide pair (g) and (h) below, i) and (h) oligonucleotide pairs, (e) and (f) oligonucleotide pairs, and (j) and (k) oligonucleotide pairs below. Is represented by a base sequence, or one base deletion in the nucleotide sequence, insertion or a base sequence which can be used as a substituted nucleotide sequence and detecting oligonucleotide according to the group (c) SEQ ID NO: 3 that oligonucleotides (d) as set forth in SEQ ID NO: 4 nucleotide sequence, or a single base in the nucleotide sequence are deleted, an insertion or a substituted nucleotide sequence and the nucleotide sequence which can be used as a detection oligonucleotide base nucleotide sequence according to the oligonucleotides (e) SEQ ID NO: 5, or 1 bases in the nucleotide sequence are deleted, it can be used as an insert or substituted by a nucleotide sequence and detecting an oligonucleotide represented one base deletion in the nucleotide sequence, or the nucleotide sequence of the oligonucleotide (f) SEQ ID NO: 6 as shown in SEQ, insertion or is substituted One base deletion in the nucleotide sequence, or the nucleotide sequence of the nucleotide sequence in a by and oligonucleotides represented by the nucleotide sequence which can be used as a detection oligonucleotide (g) SEQ ID NO: 7, inserting or one base deletion in the nucleotide sequence, or the nucleotide sequence of the oligonucleotide (h) SEQ ID NO: 8 represented by the nucleotide sequence which can be used as a substituted nucleotide sequence and the detector oligonucleotide, inserted or substituted nucleotide sequence according to nucleotide sequence in a by and oligonucleotides represented by the nucleotide sequence which can be used as a detection oligonucleotide (i) SEQ ID NO: 9, or one base deletion in the nucleotide sequence Oligonucleotide represented by a nucleotide sequence that has been deleted, inserted or substituted and that can be used as a detection oligonucleotide Plastid (j) according to SEQ ID NO: 10 nucleotide sequence or a single base in the nucleotide sequence represented by deletion, insertion or base sequence which can be used as a substituted nucleotide sequence and the detector oligonucleotide that oligonucleotides (k) SEQ ID NO: 11 nucleotide sequence set forth in, or a single base in the nucleotide sequence are deleted, an insertion or a substituted nucleotide sequence and the nucleotide sequence which can be used as a detection oligonucleotide (3) a group consisting of the following (l) and (m) oligonucleotide pairs, the following (n) and (o) oligonucleotide pairs, and the following (v) and (w) oligonucleotide pairs: (l) SEQ ID NO: 12 nucleotide sequence set forth in, or the base one base deletion in the sequence, inserted or substituted nucleotide sequence is a by and Oh detection Nucleotide sequence according to the oligonucleotides (m) SEQ ID NO: 13 represented by the nucleotide sequence which can be used as Gore-nucleotides, or one nucleotide in the nucleotide sequence are deleted, Katsu an insertion or substituted nucleotide sequence there the nucleotide sequence nucleotide sequence according to the oligonucleotides (n) SEQ ID NO: 14 represented by the nucleotide sequence which may be used, or a single base in the nucleotide sequence are deleted, inserted or substituted as a detection oligonucleotide Te and an oligonucleotide represented by the nucleotide sequence which can be used as a detection oligonucleotide (o) nucleotide sequence of SEQ ID NO: 15, or a single base in the nucleotide sequence are deleted, inserted or substituted nucleotide sequence a is and the oligonucleotides (v) SEQ ID NO: 22 represented by the nucleotide sequence which can be used as a detection oligonucleotide Placing the nucleotide sequence, or the base one base deletion in the sequence, inserted or a substituted nucleotide sequence and an oligonucleotide represented by the nucleotide sequence which can be used as a detection oligonucleotide (w) SEQ ID NO: nucleotide sequence according to 23, or an oligonucleotide in which one base in the nucleotide sequence represented by deletion, insertion or a base sequence which can be used as a substituted nucleotide sequence and the detector oligonucleotide (4) A group consisting of the following (p) and (q) oligonucleotide pairs, the following (r) and (s) oligonucleotide pairs, and the following (t) and (u) oligonucleotide pairs (p) described in SEQ ID NO: 16 nucleotide sequence of, or use a single base in the nucleotide sequence are deleted, as an insertion or substituted nucleotide sequence and the detector oligonucleotide Can oligonucleotide represented by the nucleotide sequence (q) SEQ ID NO: 17 nucleotide sequence set forth in, or the base one base deletion in the sequence, inserted or substituted by a nucleotide sequence and detecting oligonucleotide for base nucleotide sequence according to the oligonucleotides (r) SEQ ID NO: 18 represented by sequence or the single nucleotide deletion in the nucleotide sequence, insertion or a substituted nucleotide sequence and detecting that can be used as nucleotide sequence according to the oligonucleotide (s) SEQ ID NO: 19 represented by the nucleotide sequence which can be used as an oligonucleotide, or a single base in the nucleotide sequence are deleted, Katsu an insertion or substituted nucleotide sequence nucleotide sequence according to the oligonucleotides (t) SEQ ID NO: 20 represented by the nucleotide sequence which can be used as a detection oligonucleotide, or the salt One base deletion in the group sequence, inserted or substituted nucleotide sequence is a by and oligonucleotides represented by the nucleotide sequence which can be used as a detection oligonucleotide (u) nucleotide sequence according to SEQ ID NO: 21, Alternatively, an oligonucleotide represented by a base sequence in which one base is deleted, inserted or substituted in the base sequence and can be used as a detection oligonucleotide 9. The heat-resistant fungus detection kit according to claim 8 , wherein each group of (1) to (4) includes at least one pair of oligonucleotide pairs of each group. The oligonucleotide pair (a) and (b), the oligonucleotide pair (i) and (h), the oligonucleotide pair (j) and (k), the oligonucleotide (n) and (o) The kit for detecting a thermostable fungus according to claim 8 or 9 , comprising a pair, the oligonucleotide pair (t) and (u), and the oligonucleotide pair (v) and (w). Byssochlamys (Byssochlamys) genus Talaromyces (Talaromyces) genus Neosartorya (Neosartorya) genus, in a method of detecting at least two fungi selected from the group consisting of Aspergillus fumigatus (Aspergillus fumigatus) and Hamigera (Hamigera) genus, the following 1- 3) A method for detecting heat-resistant fungi comprising at least two steps selected from the group consisting of 4-3).
1-3) following (a ') and / or (b' detecting the Byssochlamys (Byssochlamys) fungi belonging to the genus by using the oligonucleotide as a nucleic acid primer),
2-3) The following oligonucleotides (c ′) and / or (d ′), the following oligonucleotides (g ′) and / or (h ′), the following oligonucleotides (i ′) and / or (h ′) nucleotides, oligonucleotides, and / or Talaromyces (Talaromyces) genus with the detector oligonucleotide of the following (j ') and / or (k') as nucleic acid primers described below (e ') and / or (f') Detecting a fungus belonging to
3-3) following oligonucleotides (l ') and / or (m'), or the oligonucleotide of the following (n ') and / or (o') in Neosartorya (Neosartorya) genus used as nucleic acid primers Detecting the fungus to which it belongs and Aspergillus fumigatus ( Aspergillus fumigatus ),
4-3) The following oligonucleotides (p ′) and / or (q ′), the following oligonucleotides (r ′) and / or (s ′), and / or the following (t ′) and / or (u ′) step of oligonucleotides) for detecting the fungi belonging to Hamigera (Hamigera) genus used as nucleic acid primers

(A ′) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 1 (b ′) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 2 (c ′) represented by the base sequence set forth in SEQ ID NO: 3 Oligonucleotide (d ′) oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 4 oligonucleotide (e ′) oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 (f ′) described in SEQ ID NO: 6 Oligonucleotide represented by the nucleotide sequence (g ′) Oligonucleotide represented by the nucleotide sequence described in SEQ ID NO: 7 (h ′) Oligonucleotide represented by the nucleotide sequence described in SEQ ID NO: 8 (i ′) SEQ ID NO: Oligonucleotide (j ′) represented by the nucleotide sequence described in SEQ ID NO: 10 Oligonucleotide (k ′) represented by the nucleotide sequence represented by SEQ ID NO: 10 Oligonucleotide (n ′) represented by the nucleotide sequence described in SEQ ID NO: 14 and nucleotide (m ′) represented by the nucleotide sequence described in SEQ ID NO: 13 Oligonucleotide (o ′) represented by the base sequence described in SEQ ID NO: 15 Oligonucleotide (p ′) represented by the base sequence described in SEQ ID NO: 16 Oligonucleotide (t ′) sequence represented by the base sequence described in SEQ ID NO: 19 Oligonucleotide (s ′) represented by the base sequence described in SEQ ID NO: 18 Oligonucleotide represented by the nucleotide sequence represented by No. 20 (u ′) Oligonucleotide represented by the nucleotide sequence represented by SEQ ID NO: 21 In the identification method of the heat-resistant fungi, characterized in that it further comprises the step of detecting whether growth can temperature zones Neosartorya by utilizing a difference in (Neosartorya) belonging to the genus fungi or Aspergillus fumigatus (Aspergillus fumigatus), claim 11. A method for detecting a heat-resistant fungus according to 11 . In the identification method of the heat-resistant fungi, further further below (v ') and / or (w') of Neosartorya using oligonucleotides (Neosartorya) fungi or Aspergillus fumigatus belonging to the genus (Aspergillus fumigatus) or detecting a process The method for detecting heat-resistant fungi according to claim 11 , comprising:
(V ′) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 22 (w ′) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 23