JP5548388B2 - Methods for detecting fungi belonging to the genus Neosartoria and Aspergillus fumigatus - Google Patents

Description

The present invention is method for detecting Neosartorya which is a kind of heat-resistant fungi (Neosartorya) fungi and Aspergillus fumigatus belonging to the genus (Aspergillus fumigatus), and the detection DNA used for the same to the detection oligonucleotide.

  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.

One of the main heat-resistant fungi bacteria causing contamination accidents, which may also be detected from the heat sterilization treatment after the food or beverage, Neosartorya (Neosartorya) heat resistance fungi and Aspergillus fumigatus belonging to the genus (Aspergillus fumigatus) is Are known. Detection of heat-resistant fungi belonging to the genus Neosartoria and Aspergillus fumigatus is important for preventing accidents caused by heat-resistant fungi in foods and drinks and their raw materials.

  On the other hand, as a conventional method for detecting heat-resistant fungi, there is a method for detecting a sample by culturing it 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 heat-resistant fungi. The long time required for detection 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. Therefore, establishment of a more rapid method for detecting heat-resistant fungi is required.

  As a rapid detection 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 fungi belonging to the genus Neosartria and Aspergillus fumigatus which are listed as main causative bacteria for food and beverage contamination. Another object of the present invention is to provide a detection DNA and a detection oligonucleotide used in the detection method.

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, in the β-tubulin gene of fungi belonging to the genus Neosartoria and Aspergillus fumigatus ( Aspergillus fumigatus ), a region having a specific base sequence that can be clearly distinguished from those of other fungi (hereinafter referred to as “ It was found that there is a variable region ”. It was also found that by targeting this variable region, the fungi belonging to the above-mentioned Neosartoria genus and Aspergillus fumigatus ( Aspergillus fumigatus ) can be detected specifically and rapidly, and further, these fungi can be identified. The present invention has been completed based on this finding.

The present invention is to identify fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria using a nucleic acid represented by any one of the following base sequences (g) to (l): The present invention relates to a method for detecting fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus.
(G) a fungus belonging to the genus Neosartoria, wherein one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 7 or a complementary sequence thereof, or in the base sequence or the complementary sequence thereof; Or a base sequence that can be used for detection of Aspergillus fumigatus (h) the base sequence set forth in SEQ ID NO: 8 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof is deleted, substituted, or added. A base sequence that can be used to detect fungi and / or Aspergillus fumigatus belonging to the genus Neosartria (i) the base sequence set forth in SEQ ID NO: 9 or its complementary sequence, or one or several of the base sequence or its complementary sequence Base deleted, substituted or added and belongs to the genus Neosartoria (J) the nucleotide sequence of SEQ ID NO: 10 or a complementary sequence thereof, or one or several bases deleted or substituted in the nucleotide sequence or the complementary sequence thereof, which can be used for detecting fungi and / or Aspergillus fumigatus Or a base sequence that can be used to detect fungi and / or Aspergillus fumigatus belonging to the genus Neosartria (k), or a complementary sequence thereof, or 1 in the base sequence or a complementary sequence thereof Alternatively, a base sequence in which several bases are deleted, substituted or added and can be used for detection of fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus (l) the base sequence set forth in SEQ ID NO: 12 or a complementary sequence thereof, Or 1 in the base sequence or its complementary sequence Or a base sequence in which several bases are deleted, substituted or added and can be used to detect fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus

The present invention also relates to a DNA represented by any one of the base sequences (g) to (l) for use in the detection of fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus (Aspergillus fumigatus). It is about.
Further, the present invention, the (g) ~ (l) either represented can hybridize to nucleic acids in a nucleotide sequence of, fungi and / or Aspergillus fumigatus (Aspergillus fumigatus belonging to Neosartorya (Neosartorya) genus ) relates to the detection oligonucleotide which specifically fungi belonging to Neosartorya (Neosartorya) genus which can function as a nucleic acid probe or nucleic acid primer for detecting and / or Aspergillus fumigatus (Aspergillus fumigatus) a.

Further, the present invention relates to the following (a) and (b) at the indicated Neosartorya (Neosartorya) belonging to the genus fungi and Aspergillus fumigatus (Aspergillus fumigatus) detector oligonucleotide pair.
(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 oligonucleotides are, the present invention relates to the following (c) and Neosartorya indicated by (d) (Neosartorya) fungi belonging to the genus and Aspergillus fumigatus (Aspergillus fumigatus) detector oligonucleotide pair.
(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 oligonucleotides are, present invention relates to oligonucleotide pair for detecting whether the following (e) and Neosartorya indicated by (f) (Neosartorya) fungi or Aspergillus fumigatus belonging to the genus (Aspergillus fumigatus) is there.
(E) represented by the nucleotide sequence of 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. Oligonucleotide (f) represented by the nucleotide sequence set forth in SEQ ID NO: 6 or a complementary sequence thereof, or a nucleotide sequence having 70% or more homology to the nucleotide sequence or the complementary sequence and usable as a detection oligonucleotide Further, the present invention relates to a kit for detecting fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus comprising the above-mentioned oligonucleotide or oligonucleotide pair.

  ADVANTAGE OF THE INVENTION According to this invention, DNA and oligonucleotide which can detect specifically and rapidly the fungi which belong to Neosartoria genus mentioned as the main causative bacteria of the contamination accident of food-drinks and Aspergillus fumigatus can be provided. Furthermore, according to the present invention, it is possible to provide a method for detecting fungi belonging to the genus Neosartoria and Aspergillus fumigatus using the DNA and oligonucleotide.

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. In Example 1, it is an electrophoretic diagram of the PCR product amplified using said (a) and (b). In Example 2, it is an electrophoretic diagram of the PCR product amplified using said (c) and (d). In Example 3, it is the electrophoretic diagram of the PCR product amplified using said (e) and (f). In Example 3, it is the electrophoretic diagram of the PCR product amplified using said (e) and (f). In Example 4, it is an electrophoretic diagram using each strain of Neosartria fischeri. It is an electrophoretic diagram using each strain of Neosartoria Fischeri glabra in Example 4. It is an electrophoretic diagram using each strain of Neosartria hiratsukae in Example 4. In Example 4, it is an electrophoretic diagram using each strain of Neosartria Paulis tensis. It is an electrophoretic diagram using each strain of Neosartoria Fischeri spinosa in Example 4.

Hereinafter, the present invention will be described in detail.
The present invention, Neosartorya (Neosartorya) belonging to the genus fungi or Aspergillus fumigatus (Aspergillus fumigatus) of β- tubulin gene of partial nucleotide sequence, i.e. the β- tubulin gene region in fungi or Aspergillus fumigatus belonging to Neosartorya genus Identification of fungi and / or Aspergillus fumigatus belonging to the genus Neosartria using a nucleic acid represented by a nucleotide sequence of a region specific to Neosartria and / or Aspergillus fumigatus or a species-specific region (variable region) This is a method for specifically identifying and detecting fungi and / or Aspergillus fumigatus belonging to the genus Sartoria.
The term “fungi belonging to the genus Neosartoria” in the present invention means an irregular cyst fungus belonging to the family Trichocomaceae . Fungi belonging to the genus Neosartoria have viable spore spores even after heat treatment at 75 ° C. for 30 minutes. 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 ).
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, and the base sequence of this region varies greatly between fungal genera. The variable region of β-tubulin gene of fungi belonging to the genus Neosartoria and Aspergillus fumigatus is a base sequence unique to fungi belonging to the genus Neosartoria and Aspergillus fumigatus, so it can be clearly distinguished from those of other normal fungi Is.

The detection method of the present invention comprises a nucleic acid represented by any one of the following base sequences (g) to (l), that is, a specific region (variable region) in a fungus belonging to the genus Neosartoria or Aspergillus fumigatus β-tubulin gene A nucleic acid corresponding to the base sequence of) is used.
(G) a fungus belonging to the genus Neosartoria, wherein one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 7 or a complementary sequence thereof, or in the base sequence or the complementary sequence thereof; Or a base sequence that can be used for detection of Aspergillus fumigatus (h) the base sequence set forth in SEQ ID NO: 8 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof is deleted, substituted, or added. A base sequence that can be used to detect fungi and / or Aspergillus fumigatus belonging to the genus Neosartria (i) the base sequence set forth in SEQ ID NO: 9 or its complementary sequence, or one or several of the base sequence or its complementary sequence Base deleted, substituted or added and belongs to the genus Neosartoria (J) the nucleotide sequence of SEQ ID NO: 10 or a complementary sequence thereof, or one or several bases deleted or substituted in the nucleotide sequence or the complementary sequence thereof, which can be used for detecting fungi and / or Aspergillus fumigatus Or a base sequence that can be used to detect fungi and / or Aspergillus fumigatus belonging to the genus Neosartria (k), or a complementary sequence thereof, or 1 in the base sequence or a complementary sequence thereof Alternatively, a base sequence in which several bases are deleted, substituted or added and can be used for detection of fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus (l) the base sequence set forth in SEQ ID NO: 12 or a complementary sequence thereof, Or 1 in the base sequence or its complementary sequence Or a base sequence that has several bases deleted, substituted or added and that can be used for detection of fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria. 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. The present invention targets the variable region and nucleic acids and oligonucleotides derived from the variable region.

  The base sequences (g) and (i) to (l) used in the detection method of the present invention correspond to the partial base sequence of the β-tubulin gene of fungi belonging to the genus Neosartoria. The base sequence (h) corresponds to a partial base sequence of the β-tubulin gene of Aspergillus fumigatus.

The nucleotide sequence set forth in SEQ ID NO: 7 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: 9 and 10 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: 11 and 12 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 described in SEQ ID NO: 8 and its complementary sequence are 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. In addition, in the base sequence described in any of SEQ ID NOs: 7 to 12 or a complementary sequence thereof, fungi and / or Aspergillus fumigatus belonging to Neosartria genus in which one or several bases are deleted, substituted or added Similarly, it is possible to specifically identify and identify fungi and / or Aspergillus fumigatus belonging to the genus Neosartori ba, 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.
Hereinafter, the nucleotide sequences (g) to (l) are also referred to as “the nucleotide sequence of the variable region of the present invention”.

  The method for identifying fungi and / or Aspergillus fumigatus belonging to the genus Neosartria using the nucleic acid represented by the base sequence of the variable region of the present invention is not particularly limited, and includes a sequencing method, a hybridization method, a PCR method, etc. It can be performed by a commonly used genetic engineering technique.

In the detection method of the present invention, in order to identify fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria using the nucleic acid represented by the base sequence of the variable region of the present invention, the β-tubulin gene of the subject is used. It is preferable to determine the base sequence of the region and confirm whether or not any of the base sequences (g) to (l) 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 β of any one of (g) to (l) of the present invention. -The base sequence of the variable region of the tubulin gene is compared, and fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria are identified based on the match or difference.
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 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 of the variable region of the present invention is used. A sequence for detecting a fungus and / or Aspergillus fumigatus that can hybridize to a nucleic acid represented by any one of the base sequences (g) to (l) and belongs to the genus Neosartoria. Detection oligonucleotides that can function as nucleic acid probes or nucleic acid primers can be used.
The oligonucleotide for detection of the present invention may be any as long as it can be used for detecting fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus. That is, those that can be used as nucleic acid primers or nucleic acid probes for detecting fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria, or fungi belonging to Neosartria genus and / or Aspergillus fumigatus β-tubulin under stringent conditions Any oligonucleotide that can hybridize to the base sequence of the variable region of the gene may be used. Here, examples of the “stringent conditions” include the conditions described later.

As the detector oligonucleotides of the present invention, the a region selected from the variable region of the nucleotide sequence of the β- tubulin gene of the present invention, (1) Neosartorya (Neosartorya) belonging to the genus fungi or Aspergillus fumigatus (2) 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) self-annealing of the oligonucleotide An oligonucleotide that can hybridize to a region that satisfies the four conditions of (4) the Tm value (melting temperature) of the oligonucleotide is approximately 55 ° C. to 65 ° C. is low.
In the above (1), the “region where the base sequence of a gene unique to a fungus belonging to the genus Neosartoria or Aspergillus fumigatus appears continuously about 10 bases” is the variable region of the β-tubulin gene of the present invention, The base sequence conservation between different genera is particularly low (that is, the specificity of fungi or Aspergillus fumigatus belonging to the genus Neosartria is particularly high). It means the area where the sequence appears continuously.
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 oligonucleotide for detecting fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria of the present invention, the following oligonucleotides (a) to (f) are more preferred.
(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 (C) 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 (D) the nucleotide sequence represented by SEQ ID NO: 4 or its complementary sequence, or An oligonucleotide represented by a base sequence having 70% or more homology to the base sequence or its complementary sequence and usable as a detection oligonucleotide (e) the base sequence of SEQ ID NO: 5 or its complementary sequence; 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 (f) the nucleotide sequence of SEQ ID NO: 6 or its complement Oligonucleotides represented by the above sequences (a) to (f), or an oligonucleotide represented by a base sequence that has 70% or more homology with the base sequence or its complementary sequence and can be used as a detection oligonucleotide Among them, fungi belonging to the genus Neosartria and oligonucleotides for detection of Aspergillus fumigatus As an tide, what is shown by either of said (a)-(d) is preferable. As the oligonucleotide for detecting Aspergillus fumigatus, those shown in the above (e) or (f) are preferable.

That is, as the oligonucleotide for detection of the present invention, an oligonucleotide represented by the base sequence described in any of SEQ ID NOs: 1 to 6 or a complementary sequence thereof, and the base sequence described in any of SEQ ID NOs: 1 to 6 Alternatively, an oligonucleotide represented by a base sequence having 70% or more homology to its complementary sequence, which can be used for detecting fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus is preferable. The base sequence that can be used for the detection of fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria has 70% or more homology to the base sequence described in any of SEQ ID NOs: 1 to 6, As a nucleic acid primer or nucleic acid probe for the detection of fungi and / or Aspergillus fumigatus that can be used for the detection of fungi belonging to Neosartoria and / or Aspergillus fumigatus, fungi belonging to Neosartoria under stringent conditions, and Any base sequence can be used as long as it can hybridize to the β-tubulin gene of Aspergillus fumigatus. 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. Further, the homology is more preferably 75% or more, the homology is more preferably 80% or more, the homology is more preferably 85% or more, and the homology is 90% or more. Is more preferable, and it is particularly preferable that it has a homology of 95% or more and can be used for detection of fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus.
In addition, the oligonucleotide for detection of the present invention includes the oligonucleotide represented by the nucleotide sequence of SEQ ID NOs: 1 to 6 as long as it can be used for detection of fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus. On the other hand, mutations such as base deletions, insertions or substitutions, and modified oligonucleotides are also included. In addition, the oligonucleotide for detection of the present invention includes an oligonucleotide represented by a base sequence described in SEQ ID NOs: 1 to 6, wherein the base sequence described in SEQ ID NO: 1 to 6 is a mutation or modification such as deletion, insertion or substitution of a base. Nucleotides that can be used for detection of heat-resistant fungi as nucleic acid primers and nucleic acid probes for detection of fungi and / or Aspergillus fumigatus belonging to the genus Neosartria, fungi belonging to Neosartria under stringent conditions, and / or It may be a base sequence capable of hybridizing to the Aspergillus fumigatus β-tubulin gene. 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 6 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 6. 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 represented by the base sequences described in SEQ ID NOs: 1 to 4 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, a partial base sequence of the β-tubulin gene of Neosartria grabra is represented by SEQ ID NO: 7. 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: 8. 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 is low in conservation and has a unique base sequence between species.
The oligonucleotides shown in (a) and (b) above are the nucleotide sequence shown in SEQ ID NO: 7, regions 84 to 103, positions 169 to 188, and SEQ ID NO: 8. In the base sequence, it corresponds to the region from position 83 to position 102, position 166 to position 186, respectively. The oligonucleotides represented by (c) and (d) above are the nucleotide sequence of SEQ ID NO: 7 from the 144th position to the 163rd position, the region from the 358th position to the 377th position, 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 shown in SEQ ID NO: 5 and SEQ ID NO: 6 are present in the β-tubulin gene region, and are part of a variable region where Aspergillus fumigatus-specific nucleotide sequences, that is, variations in the nucleotide sequence are likely to accumulate between species. Complementary oligonucleotide. These oligonucleotides can specifically hybridize to portions of Aspergillus fumigatus DNA and RNA.

The partial base sequence of the Aspergillus fumigatus β-tubulin gene is represented by SEQ ID NO: 8. Of these, the region from positions 20 to 50 and the region from positions 200 to 230 of the β-tubulin gene of Aspergillus fumigatus has a conserved nucleotide sequence with fungi, particularly fungi belonging to the genus Neosartoria. We have found that it is low.
The oligonucleotides represented by (e) and (f) 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: 8. 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 the β-tubulin genes of Aspergillus fumigatus, Neosartria fischeri and Neosartria spinosa described in SEQ ID NOs: 8-12. As shown in FIG. 1, the region recognized by the oligonucleotides of SEQ ID NOs: 5 and 6 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 shown in (e) and / or (f), it is possible to identify whether the fungus contained in the subject is a fungus belonging to the genus Neosartoria or Aspergillus fumigatus.

In the detection method of the present invention, among the detection oligonucleotides (a) to (f) above, the following oligonucleotides (a1) to (f1) are preferable, and the nucleotide sequences represented by SEQ ID NOs: 1 to 6 are used. More preferably, the oligonucleotide represented is used.
(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 base sequence which has a homology of at least% and can be used as a detection oligonucleotide

The oligonucleotide pair for detecting fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria of the present invention is an oligonucleotide pair comprising the oligonucleotide (a) and the oligonucleotide (b), and the oligonucleotide (c) And an oligonucleotide pair consisting of the oligonucleotide (d) and an oligonucleotide pair consisting of the oligonucleotide (e) and the oligonucleotide (f).
Among them, in order to detect fungi and Aspergillus fumigatus belonging to the genus Neosartoria, an oligonucleotide pair consisting of the oligonucleotides (a) and (b) or an oligonucleotide consisting of the oligonucleotides (c) and (d) It is preferable to use a nucleotide pair, and it is more preferable to use an oligonucleotide pair consisting of the oligonucleotides (a1) and (b1) or an oligonucleotide pair consisting of the oligonucleotides (c1) and (d1). It is more preferable to use the oligonucleotide pair represented by the base sequence described in 1 and SEQ ID NO: 2 or the oligonucleotide pair represented by the base sequence described in SEQ ID NO: 3 and SEQ ID NO: 4. The oligonucleotide pair comprising the oligonucleotide (a) and the oligonucleotide (b) and the oligonucleotide pair comprising the oligonucleotide (c) and the oligonucleotide (d) are Neosartoria Fischeri, Neo It is particularly preferably used for detection of fungi belonging to the genus Neosartria such as Sartoria spinosa, Neosartria grabra, Neosartria hiratsukae, Neosartria Paulistensis, Neosartria pseudofischeri, and Aspergillus fumigatus.
In order to detect Aspergillus fumigatus, it is preferable to use an oligonucleotide pair consisting of the oligonucleotides (e) and (f), and an oligonucleotide pair consisting of the oligonucleotides (e1) and (f1) It is more preferable to use the oligonucleotide pair, and it is more preferable to use the oligonucleotide pair represented by the base sequences described in SEQ ID NO: 5 and SEQ ID NO: 6. The oligonucleotide pair consisting of the oligonucleotide (e) and the oligonucleotide (f) is preferably used for detection of Aspergillus fumigatus.

  As described above, the oligonucleotides (a) to (d) 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 (a) to (d) of the present invention, the fungi and Aspergillus fumigatus belonging to the Neosartoria genus can be detected specifically, rapidly and simply.

  In the present invention, in the sample that showed a positive reaction by the detection method using the oligonucleotides (a) to (d) above, by utilizing the difference in the temperature range where fungi belonging to the genus Neosartoria and Aspergillus fumigatus can grow Alternatively, by using the oligonucleotide (e) or (f), the bacterial species contained in the sample can be identified.

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. By using the difference in the temperature range in which growth is possible, the bacterial species can be identified by performing the following operation. However, the present invention is not limited to this.
About the sample which showed a positive reaction by the detection method using the oligonucleotides (a) to (d) of the present invention, the mycelium was planted from a single colony into a PDA medium, a PDB medium (potato dextrose liquid medium) or the like. 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.

  In the sample that shows a positive reaction by the detection method using the oligonucleotides (a) to (d), the bacterial species contained in the sample is identified by using the oligonucleotide (e) or (f). can do. As described above, the oligonucleotides represented by the above (e) and (f) can specifically hybridize to a part of Aspergillus fumigatus DNA and RNA. However, it cannot hybridize to a part of DNA and RNA of fungi belonging to the genus Neosartoria. Therefore, when the oligonucleotide shown in (e) and / or (f) is hybridized to DNA or RNA contained in the subject, the fungus contained in the subject is Aspergillus fumigatus. The fungus contained in the specimen can be distinguished from the fungus of the genus Neosartoria.

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 detection oligonucleotide of the present invention can be prepared by an ordinary synthesis method such as chemical synthesis using, for example, an automatic DNA synthesizer. In addition, it can be directly excised from the genes of fungi belonging to the genus Neosartoria or Aspergillus fumigatus using restriction enzymes, etc., or after cloning and isolating and purifying the genes, it can also be cut out using restriction enzymes. is there. 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 detection method of the present invention, in order to identify fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria using the nucleic acid represented by the base sequence of the variable region of the present invention, the above (g) to (l) Labeling a detection oligonucleotide that hybridizes with a nucleic acid represented by any of the nucleotide sequences under stringent conditions, and the nucleic acid obtained by extracting the detection oligonucleotide from a test object under stringent conditions It is preferable to measure the label of the hybridized 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 (g) to (l), 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 of the present invention can be used as a nucleic acid probe. The nucleic acid probe can be prepared by labeling the detection 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. 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. By measuring the label, fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria can be detected. Here, examples of the stringent conditions include the conditions described above.
For example, the oligonucleotide represented by (a) and / or (b), the oligonucleotide represented by (c) and / or (d), or the oligonucleotide represented by (e) and / or (f) It can be labeled to form a nucleic acid probe. A nucleic acid probe comprising the oligonucleotide shown in (a) and / or (b) or the oligonucleotide shown in (c) and / or (d) 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. The nucleic acid probe comprising the oligonucleotide shown in (e) and / or (f) 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. 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.

  In addition, the oligonucleotide of 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 fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria using the nucleic acid represented by the base sequence of the variable region of the present invention, the above (g) to (l) It is also a preferred embodiment that a nucleic acid consisting of a part or all of the nucleic acid represented by any one of the nucleotide sequences is subjected to gene amplification and the presence or absence of an 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.
In this case, the region in the nucleic acid represented by any one of the base sequences (g) to (l), wherein (1) the base sequence of the gene unique to fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus 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. It is preferable to use an oligonucleotide that can hybridize to a region that satisfies the four conditions of about 55 ° C. to 65 ° C. as a nucleic acid primer. Among them, it is preferable to use any one of the detection oligonucleotides (a) to (f) as a nucleic acid primer, and any one of the detection oligonucleotides (a1) to (f1) may be used as a nucleic acid primer. Is more preferable, and it is more preferable to use the oligonucleotide represented by the nucleotide sequence of SEQ ID NOs: 1 to 6 as a nucleic acid primer. In addition, the oligonucleotide pair shown in (a) and (b), the oligonucleotide pair shown in (c) and (d), or the oligonucleotide pair shown in (e) and (f) Is preferably used as a nucleic acid primer pair, the oligonucleotide pair represented by (a1) and (b1), the oligonucleotide pair represented by (c1) and (d1), or the above (e1) and (f1 It is more preferable to use the oligonucleotide pair represented by (2) as the nucleic acid primer pair, the oligonucleotide pair represented by the nucleotide sequence described in SEQ ID NO: 1 and SEQ ID NO: 2, and the base described in SEQ ID NO: 3 and SEQ ID NO: 4. The oligonucleotide pair represented by the sequence and the oligonucleotide pair represented by the nucleotide sequence set forth in SEQ ID NO: 5 and SEQ ID NO: 6 are used as nucleic acid primer pairs. It is more preferably used.
In particular, when detecting fungi and Aspergillus fumigatus belonging to the genus Neosartoria, the oligonucleotide pairs shown in (c) and (d) above are used as nucleic acid primer pairs from the viewpoint of detection specificity and detection sensitivity. It is more preferable that the oligonucleotide pairs shown in the above (c1) and (d1) are used as the nucleic acid primer pair, and the oligonucleotide pair represented by the nucleotide sequence described in SEQ ID NO: 3 and SEQ ID NO: 4 is used. 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 (e) and (f) are preferably used as nucleic acid primer pairs, and (e1) and The oligonucleotide pair represented by (f1) is more preferably used as the nucleic acid primer pair, and the oligonucleotide pair represented by the nucleotide sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6 is more preferably used as the 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 the PCR reaction conditions, for example, in the PCR reaction using the oligonucleotides (a) and (b) or the oligonucleotides (c) and (d), 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 carried out at about 72 ° C. for about 60 seconds, and these are defined as one cycle for 30 to 35 cycles. In the PCR reaction using the oligonucleotides (e) and (f), 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, and the primer pair is made into a single strand An annealing reaction for hybridizing to DNA is carried out at 59-61 ° C. for about 60 seconds, an extension reaction for allowing DNA polymerase to act is carried out at about 72 ° C. for about 60 seconds, and one cycle of these is carried out for 30-35 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 an amplification reaction, a method of confirming the presence or absence of a band corresponding to the size of the amplified gene by electrophoresis on the PCR reaction product, and the base sequence of the PCR reaction product Examples include a method of decoding and a method of allowing a fluorescent substance to enter between 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.
For example, when the specimen contains fungi belonging to the genus Neosartoria and / or Aspergillus fumigatus, PCR reaction is performed using the oligonucleotide pairs of (a) and (b) as a primer set, and the obtained PCR reaction When the product is electrophoresed, an amplification of about 100 bp DNA fragment specific to these fungi is observed. Moreover, when PCR reaction was performed using the oligonucleotide pair of (c) and (d) as a primer set, and the obtained PCR reaction product was subjected to electrophoresis, DNA of about 200 bp specific to these fungi was obtained. Fragment amplification is observed. By performing this operation, fungi and / or Aspergillus fumigatus belonging to the genus Neosartoria can be detected.
When Aspergillus fumigatus is included in the specimen, PCR reaction is performed using the oligonucleotide pair of (e) and (f) as a primer set, and the obtained PCR reaction product is subjected to electrophoresis. Amplification of a DNA fragment of about 200 bp specific to is observed. By performing this operation, Aspergillus fumigatus can be detected.

In the detection method of the present invention, the detection method using the oligonucleotide pair shown in (a) and (b) and / or the oligonucleotide pair shown in (c) and (d), It is also a preferred embodiment that the detection method using the oligonucleotide pair shown in e) and (f) is used in combination. As described above, the detection method using the oligonucleotide pair shown in (a) and (b) and the oligonucleotide pair shown in (c) and (d) are fungi and Aspergillus belonging to the genus Neosartria. Since the detection method using the oligonucleotide pairs shown in (e) and (f) 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 (a1) and (b1) and / or the oligonucleotide pair shown by the above (c1) and (d1), and the above (e1) and (f1 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 of SEQ ID NOs: 1 and 2 and / or SEQ ID NOs: 3 and 4 It is more preferable to use a combination of 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 of SEQ ID NOs: 5 and 6. .
Further, from the viewpoint of detection sensitivity, it is preferable to use the oligonucleotide pair shown in (c) and (d) in combination with the oligonucleotide pair shown in (e) and (f). More preferably, the oligonucleotide pair represented by c1) and (d1) and the oligonucleotide pair represented by (e1) and (f1) are used in combination, and the nucleotide sequences described in SEQ ID NOs: 3 and 4 are used. More preferably, the detection method using the represented oligonucleotide pair and the detection method using the oligonucleotide pair represented by the nucleotide sequences set forth in SEQ ID NOs: 5 and 6 are used in combination.

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 fungi belonging to the genus Neosartria and Aspergillus fumigatus according to the present invention, it is possible to perform the steps from the preparation of the specimen to the fungus detection process in a short time of about 5 to 12 hours.

  The fungus belonging to the genus Neosartria and the Aspergillus fumigatus detection / identification kit of the present invention contain the detection oligonucleotide of the present invention as a nucleic acid probe or nucleic acid primer. This kit can be used in a method for detecting fungi belonging to the genus Neosartoria and Aspergillus fumigatus. In addition to the nucleic acid probe or nucleic acid primer, the kit of the present invention includes a label detection substance, a buffer, a nucleic acid synthesizing enzyme (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), an enzyme substrate (dNTP, rNTP, etc.) depending on the purpose. ) And other substances commonly used for detecting fungi. The kit of the present invention may include a positive control (positive control) for confirming that a detection reaction is possible with the detection oligonucleotide 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
1. 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: 13), Bt2b (5 '-ACCCTCAGTGTAGTGACCCTTGGC-3': SEQ ID NO: 14) (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 gene of Neosartria grabra, Neosartria fischeri, Neosartria spinosa and Aspergillus fumigatus determined by sequencing method and the base sequence information of known β-tubulin genes of various fungi, Alignment analysis is performed using DNA analysis software (trade name: DNAsis pro, manufactured by Hitachi Software Co., Ltd.), and β-tubulin gene containing a base sequence specific to fungi belonging to the genus Neosartoria and Aspergillus fumigatus Specific regions (SEQ ID NOs: 7-12) were 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: 1 and 2, and a primer consisting of an oligonucleotide represented by the base sequences described in SEQ ID NOs: 3 and 4 It is a pair. The primer used was purchased from Sigma Aldrich Japan (salt desalted product, 0.02 μmol scale).

(2) Preparation of specimens As fungi belonging to the genus Neosartoria and Aspergillus fumigatus, the fungi listed in Table 1 were used. In order to confirm the specificity of the oligonucleotides (a) and (b) for the β-tubulin gene of these fungi, the fungi shown in Table 1 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: 1. The primer (20 pmol / μl) 0.5 μl and the primer (20 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 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. The 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 oligonucleotide of the present invention.

(6) Discrimination between fungi belonging to the genus Neosartoria and Aspergillus fumigatus For the specimen in which the amplification of the gene fragment was confirmed in the above (5), the mycelium was obtained from a single colony in PDA medium (trade name: potato dextrose medium, Eiken Chemical Co. Inoculated into the company), 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.

Example 2
(1) Primer The primer which consists of the oligonucleotide represented by the base sequence of sequence number 3 and 4 which was designed in Example 1 was used.

(2) Preparation of specimens The fungi listed in Table 2 and Table 3 were used as fungi belonging to the genus Neosartoria and Aspergillus fumigatus. In order to confirm the specificity of the oligonucleotides (c) and (d) for fungi belonging to the genus Neosartria and β-tubulin gene of Aspergillus fumigatus, other fungi shown in Tables 2 and 3 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: 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) 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. 3 (a) and 3 (b). 3A shows an electrophoretic diagram for the fungal sample shown in Table 2, and FIG. 3B shows an electrophoretic diagram for the fungal sample shown in Table 3. 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 oligonucleotide of the present invention.

(6) Discrimination between fungi belonging to the genus Neosartoria and Aspergillus fumigatus For specimens 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 agar medium, Eiken Chemical Co., Ltd.) Manufactured), cultured at 50 ° C. for 1 day, and observed for hyphal elongation by 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.

Example 3
(1) Design of primers for detecting Aspergillus fumigatus Using the alignment analysis (DNAsis Pro) of the base sequences of β-tubulin genes of Neosartria grabra, Aspergillus fumigatus, Neosartria fischeri and Neosartria spinosa shown in SEQ ID NOs: 7-12 A region where a difference between the base sequences of both species exists was determined. 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 described in SEQ ID NOs: 5 and 6. 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 4 and 5 were used as fungi belonging to the genus Neosartoria and Aspergillus fumigatus. For reference, fungi other than those belonging to the genus Neosartria shown in Tables 4 and 5 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: 5. The primer (20 pmol / μl) 0.5 μl and the primer (20 pmol / μl) represented by the nucleotide sequence shown in SEQ ID NO: 6 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. The electropherograms of the agarose gel are shown in FIGS. 4 shows an electrophoretic diagram for the fungal sample shown in Table 4, and FIG. 5 shows an electrophoretic diagram for the fungal sample shown in Table 5. 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. 4 and 5, 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, it can be seen that Aspergillus fumigatus can be specifically detected by performing gene amplification using the oligonucleotides (e) and (f) and confirming the presence or absence of the amplification product.

Example 4
(1) Primer The primer which consists of the oligonucleotide represented by the base sequence of sequence number 3-6 designed in Example 1 and 3 was used.

(2) Preparation of specimen Confirm the detection specificity of the oligonucleotides (c) and (d) to fungi belonging to the genus Neosartoria, and the detection specificity of the oligonucleotides (e) and (f) to Aspergillus fumigatus. Therefore, each strain of Neosartria fischeri shown in FIG. 6, each strain of Neosartria gravula shown in FIG. 7, each strain of Neosartria hiratsukae shown in FIG. 8, each strain of Neosartria paulistensis shown in FIG. Each strain of Neosartria spinosa shown in FIG. 10 was used. In addition, Aspergillus fumigatus was used as a positive control for the reaction system using the oligonucleotides (e) and (f). 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: 3. 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: 4 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: 5 and 0.5 μl of the primer (20 pmol / μl) represented by the base sequence described in SEQ ID NO: 6. 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. 6 shows an electrophoretic diagram for samples of each strain of Neosartria fischeri, FIG. 7 shows an electrophoretic diagram for samples of each strain of Neosartria grabra, and FIG. 8 shows each of the strains of Neosartria hiratsukae. FIG. 9 shows an electrophoretic diagram for a sample of each strain of Neosartoria Paulistensis, and FIG. 10 shows an electrophoretic diagram for a sample of each strain of Neosartoria spinosa.
As a result, in the reaction system using the primer represented by the base sequence described in SEQ ID NOs: 3 and 4, the Neosartoria Fischeri, Neosartoria grubra, Neosartoria hiratsukae, Neosartoria Paulistensis and Neosartoria spinosa used Specific amplified DNA fragments were confirmed in all strains (FIGS. 6 (a), 7 (a), 8 (a), 9 (a), and 10 (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: 5 and 6, 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 Neosartoria (FIGS. 6 (b), 7 (b), 8 (b), 9 (b), FIG. 10 (b)). Therefore, it can be seen that Aspergillus fumigatus can be specifically detected by performing gene amplification using the oligonucleotides (e) and (f) and confirming the presence or absence of the amplification product.

Claims (13)

Following (a) carrying out the identification of the ~ fungi belonging at least one of the Neosartorya (Neosartorya) genus used as nucleic acid primers selected from the group consisting of an oligonucleotide (f) and / or Aspergillus fumigatus (Aspergillus fumigatus) detection method Neosartorya characterized by (Neosartorya) belonging to the genus fungi and / or Aspergillus fumigatus (Aspergillus fumigatus).
(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 Oligonucleotides having a substituted base sequence and a base sequence that can be used as a detection oligonucleotide In order to perform identification, a gene amplification reaction is performed using the oligonucleotides (a) and (b) and / or the oligonucleotides (c) and (d) as a nucleic acid primer pair, and the presence or absence of an amplification product is confirmed. detection method for Neosartorya of claim 1 wherein (Neosartorya) fungi belonging to the genus and / or Aspergillus fumigatus (Aspergillus fumigatus), characterized by. The Aspergillus fumigatus (1) according to claim 1, wherein a gene amplification reaction is performed using the oligonucleotides (e) and (f) as a nucleic acid primer pair to confirm the presence or absence of an amplification product. Aspergillus fumigatus ). Further below (A) and / or detection methods described by the use of (i) in claim 2 identifies whether Neosartorya (Neosartorya) belonging to the genus fungi or Aspergillus fumigatus (Aspergillus fumigatus).
(A) by using the difference of the development possible temperature zones or fungi belonging to the Neosartorya (Neosartorya) genus, a method for detecting whether Aspergillus fumigatus (Aspergillus fumigatus).
(A) A method for detecting Aspergillus fumigatus, which comprises using the oligonucleotides (e) and (f) as a nucleic acid primer pair. The detection of the amplification reaction Neosartorya of any one of claims 2-4, characterized in that performing the polymerase chain reaction (PCR) method (Neosartorya) fungi belonging to the genus and / or Aspergillus fumigatus (Aspergillus fumigatus) Method. Following (a ') and (b') of the oligonucleotide or the following (c ') and (d') of Neosartorya (Neosartorya), which comprises using an oligonucleotide as a nucleic acid primer fungi belonging to the genus and Aspergillus fumigatus ( Aspergillus fumigatus ).
(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 ′) represented by the nucleotide sequence set forth in SEQ ID NO: 4 Further below (A) and / or (b ') detecting method according to claim 6, wherein identifying whether Neosartorya (Neosartorya) belonging to the genus fungi or Aspergillus fumigatus (Aspergillus fumigatus) By using.
(A) a method for detecting whether the development possible temperature range Neosartorya by using the difference of (Neosartorya) fungi Aspergillus fumigatus belonging to the genus (Aspergillus fumigatus).
(A ′) A method for detecting Aspergillus fumigatus characterized by using the following oligonucleotides (e ′) and (f ′) as nucleic acid primers.
(E ′) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 5 (f ′) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 6 Following (a) ~ (f) of the selected from the group consisting of an oligonucleotide, the detector nucleic acid primer Neosartorya (Neosartorya) fungi belonging to the genus and / or Aspergillus fumigatus (Aspergillus fumigatus).
(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 Oligonucleotides having a substituted base sequence and a base sequence that can be used as a detection oligonucleotide Following (a) and (b) or (c) below and (d) at the indicated Neosartorya (Neosartorya) fungi and Aspergillus fumigatus (Aspergillus fumigatus) belonging to the genus detector oligonucleotide pair.
(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 An oligonucleotide represented by the nucleotide sequence which can be used as a a by and the detector oligonucleotide sequence Aspergillus fumigatus detection oligonucleotide pairs shown in (e) and (f) below.
(E) nucleotide sequence according to SEQ ID NO: 5, 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 which can be used as an oligonucleotide (f) SEQ ID NO: 6 nucleotide sequence according to, or the base one base deletion in the sequence, inserted or a substituted nucleotide sequence and the detector oligonucleotide Oligonucleotide Following (a) and (b) an oligonucleotide and / or the following (c) and (d) the oligonucleotide Neosartorya (Neosartorya) fungi and Aspergillus fumigatus belonging to the genus (Aspergillus fumigatus) detection kit comprising a nucleic acid primer for.
(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 An oligonucleotide represented by the nucleotide sequence which can be used as a a by and the detector oligonucleotide sequence The detection kit according to claim 11 , further comprising the following oligonucleotides (e) and (f) as nucleic acid primers.
(E) nucleotide sequence according to SEQ ID NO: 5, 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 which can be used as an oligonucleotide (f) SEQ ID NO: 6 nucleotide sequence according to, or the base one base deletion in the sequence, inserted or a substituted nucleotide sequence and the detector oligonucleotide Oligonucleotide A kit for detecting Aspergillus fumigatus comprising the following oligonucleotides (e) and (f) as nucleic acid primers:
(E) nucleotide sequence according to SEQ ID NO: 5, 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 which can be used as an oligonucleotide (f) SEQ ID NO: 6 nucleotide sequence according to, or the base one base deletion in the sequence, inserted or a substituted nucleotide sequence and the detector oligonucleotide Oligonucleotide