JP5820658B2 - Method for detecting Neosartoria fungi - Google Patents

Description

The present invention relates to a method for detecting a fungus belonging to the genus Neosartorya.

Heat-resistant fungi (fungi) are widely distributed in nature and propagate in agricultural products such as vegetables and fruits, and contaminate foods and drinks made from these agricultural products. Moreover, heat-resistant fungi form ascospores having higher heat resistance in the life cycle than other normal fungi. For example, even when an acid beverage is heat sterilized, the heat-resistant fungi can survive and multiply, causing mold generation.
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 glabra (Neosartorya glabra), Neosartorya-Hiratsukae (Neosartorya hiratsukae), Neo Sarutoria pseudotuberculosis fischeri (Neosartorya pseudofischeri), is known Neosartorya-fischeri (Neosartorya fischeri) and Neosartorya-spinosa (Neosartorya spinosa) Neosartorya (Neosartorya) refractory fungus belonging to the genus or the like. In order to prevent accidents caused by heat-resistant fungi in foods and drinks and these raw materials, detection of heat-resistant fungi belonging to the genus Neosartoria is important.

  Conventional methods for detection and identification of fungi belonging to the genus Neosartoria are mainly morphological species classification by culture. This method requires a long period of at least 14 days since culture must be continued until morphological characteristics occur. In addition, since morphological identification requires extremely high expertise, it is impossible to deny the risk that identification results differ depending on the judge. In addition, there are cases in which microbial cells that are damaged by heating or chemicals lose their morphogenic ability, and these microbial cells do not form a characteristic morphology even after long-term culture, so the reliability of the identification results is a problem. was there. In this way, morphological detection and identification methods that require a long time to detect fungi and have problems with the reliability of identification results are from the viewpoints of hygiene management of food and drink, ensuring freshness of raw materials, restrictions on distribution, etc. It ’s hard to say that it ’s always satisfactory. Accordingly, it has been required to establish a detection / identification method that solves the problems of rapidity and reliability.

Patent Document 1 describes a method for detecting a Neosartria fungus by amplifying a specific region of the β-tubulin gene in the genomic DNA of the Neosartria fungus. However, the method described in Patent Document 1 identifies Neosartria fungi at the genus level, and the fungus detected by these methods can only be identified as Neosartorya sp. Further studies are needed to identify Sartoria fungi at the species level.

JP 2010-4879 A

As a feature of Neosartoria fungi having heat resistance, it is known that heat resistance varies depending on the species. For example, various D values of neosartoria fungi (one of the indices indicating the heat resistance of heat-resistant bacteria, the sterilization unit required to reduce the initial number of bacteria to 1/10) and the Z value (heat resistance) As a measure of the heat resistance of fungi, the temperature change necessary to reduce or increase the sterilization unit to 1/10 or 10 times) was measured. Is D85 ° C. = 96 minutes (where “D85 ° C. is the time required to reduce the initial number of bacteria to 1/10 when treated at 85 ° C.), Z = 14 ° C. (ie 85 ° C. In order to reduce the initial number of bacteria to 1/10, it must be treated for 96 minutes, so the initial number of bacteria is reduced to 1/10 in 9.6 minutes (1/10/96). To do this, the treatment at (85 + 14) = 99 ° C is necessary, and the treatment time is 960 minutes (10 times 96 times). To reduce the number of bacteria to 1/10, (85-14) = treatment at 71 ° C is necessary.) Neosartria Fiselli has a D85 ° C value of 10 minutes and a Z value of 10 ° C. is there. Thus, even if it is the same Neosartoria genus fungus, heat resistance differs, Therefore In the food-and-beverage industry etc., the disinfection and sterilization process conditions of the heat-resistant Neosartria genus fungus which change with bacterial species are required.
Furthermore, Neosartoria australensis ( Neosartorya australensis ) etc. exist as a fungus of the genus Neosartoria. These bacterial species have high heat resistance like other Neosartria fungi, but unlike the Neosartria grabra, there are few reports of food and beverage contamination accidents.
Therefore, in order to perform accurate risk assessment of harmful bacteria in the food and beverage industry and the like, it is important to identify Neosartria fungi not only at the genus level but also at the species level.

  An object of the present invention is to provide a method capable of quickly detecting, at the species level, a neosartria fungus that is one of the main causative bacteria of food and beverage contamination. Another object of the present invention is to provide oligonucleotides, oligonucleotide pairs and detection kits applicable to this method.

  In view of such a problem, the present inventors have intensively studied to search for a new DNA region that can specifically detect and identify Neosartria fungi at the species level. As a result, a site having a specific base sequence that can be clearly distinguished from those of other fungi in the β-tubulin gene and / or calmodulin gene of Neosartoria fungi (hereinafter referred to as “variable site”). ")" Was found. In addition, it has been found that by targeting these variable sites, Neosararia fungi can be specifically and rapidly detected at the species level. The present invention has been completed based on this finding.

The present invention includes an oligonucleotide pair consisting of the following oligonucleotides (a) and (b), an oligonucleotide pair consisting of the following oligonucleotides (c) and (d), and the following oligonucleotides (e) and (f): At least one oligo selected from the group consisting of an oligonucleotide pair consisting of the following (g) and (h) oligonucleotide pairs, and an oligonucleotide pair consisting of the following (i) and (j) oligonucleotide pairs: The present invention relates to a method for detecting Neosartria fungi, which comprises the step of performing identification at the species level of Neosartria fungi using nucleotide pairs.
(A) An oligonucleotide represented by the base sequence set forth in SEQ ID NO: 1, or one or several bases deleted, substituted, inserted or added in the base sequence set forth in SEQ ID NO: 1 and Neosartoria Oligonucleotide that can be used for detection at the fungal species level (b) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 2, or one or several bases deleted in the nucleotide sequence set forth in SEQ ID NO: 2, Oligonucleotide which is substituted, inserted or added and can be used for detection at the species level of Neosartoria fungus (c) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3, or the base set forth in SEQ ID NO: 3 Detection at the species level of Neosartria fungi with one or several bases deleted, substituted, inserted or added in the sequence Usable oligonucleotide (d) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 4 or one or several bases deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 4 And (e) an oligonucleotide represented by the nucleotide sequence represented by SEQ ID NO: 5, or one or several bases in the nucleotide sequence represented by SEQ ID NO: 5 Is deleted, substituted, inserted or added and can be used for detection at the species level of Neosartoria fungi (f) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 6, or SEQ ID NO: 6 1 or several bases are deleted, substituted, inserted or added in the base sequence described in 1. Oligonucleotides that can be used for detection at the species level of the genus Rhizoma (g) The oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7 or the nucleotide sequence set forth in SEQ ID NO: 7 lacks one or several bases Oligonucleotide which is deleted, substituted, inserted or added and can be used for detection at the species level of Neosartoria fungi (h) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8, or SEQ ID NO: 8 An oligonucleotide in which one or several bases are deleted, substituted, inserted or added in the nucleotide sequence of the nucleotide sequence and can be used for detection at the species level of Neosartria fungus (i) In the oligonucleotide represented or the nucleotide sequence set forth in SEQ ID NO: 9, one or several bases are deleted, substituted or inserted. Is added and can be used for detection at the species level of Neosartoria fungi (j) 1 in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 10 or the nucleotide sequence of SEQ ID NO: 10 Or an oligonucleotide that has several bases deleted, substituted, inserted or added and can be used for detection at the species level of Neosartoria fungi

The present invention also provides an oligonucleotide pair comprising the oligonucleotides (a) and (b), an oligonucleotide pair comprising the oligonucleotides (c) and (d), and the oligonucleotides (e) and (f). At least one selected from the group consisting of an oligonucleotide pair consisting of nucleotides, an oligonucleotide pair consisting of oligonucleotides (g) and (h) above, and an oligonucleotide pair consisting of oligonucleotides (i) and (j) above The present invention relates to a method for detecting Neosartria fungi, wherein the presence of a nucleic acid represented by any one of the following base sequences (k) to (t) is confirmed using the oligonucleotide pair:
(K) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 11 or its complementary sequence (l) one or several bases are deleted, substituted, inserted or added in the base sequence described in SEQ ID NO: 11 And a base sequence that can be used for detection at the species level of Neosartoria grubra, or its partial sequence (m) the partial base sequence of the β-tubulin gene described in SEQ ID NO: 12 or its complementary sequence (n) 12. A nucleotide sequence in which one or several bases are deleted, substituted, inserted or added in the nucleotide sequence according to 12, and can be used for detection at the species level of Neosartoria hiratsukae, or its complementary sequence (o) sequence The partial base sequence of the calmodulin gene shown in No. 13 or its complementary sequence (p) In the base sequence shown in SEQ ID No. 13, one or several bases are deleted, substituted, inserted or Is a base sequence that can be used for detection at the species level of Neosartoria pseudofisheri, or its complementary sequence (q), a partial base sequence of the calmodulin gene described in SEQ ID NO: 14, or its complementary sequence (r) A nucleotide sequence in which one or several bases are deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 14 and can be used for detection at the species level of Neosartria fischeri, or its complementary sequence (s ) A partial base sequence of the calmodulin gene shown in SEQ ID NO: 15 or a complementary sequence thereof (t) one or several bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 15, and Neosartria・ Base sequences that can be used for detection at the spinosa species level, or their complementary sequences

  The present invention also provides the oligonucleotides (a) and (b), the oligonucleotides (c) and (d), the oligonucleotides (e) and (f), the (g) and (h) Or an oligonucleotide pair consisting of the oligonucleotides (i) and (j) above.

  The present invention also relates to an oligonucleotide selected from the group consisting of the oligonucleotides (a) to (j).

  The present invention also relates to a Neosartria fungus detection kit comprising the oligonucleotide or the oligonucleotide pair.

  The detection method of the present invention can rapidly detect Neosartria spp., One of the main causative bacteria of food and beverage contamination accidents, at the species level. Moreover, the oligonucleotide, oligonucleotide pair and detection kit of the present invention can be suitably applied to the above method.

Part of the β-tubulin gene of Neosartoria grubura and other Neosartria fungi (including Aspergillus fumigatus ), which is morphologically very similar to the anamorphs (asexual generation) of Neosartria fischeri It is a figure which shows the positional relationship of the base sequence which the oligonucleotide of said (a) and (b) in the base sequence and the base sequence of the β-tubulin gene of Neosartoria grubura recognizes. A partial base sequence of the β-tubulin gene of Neosartria hiratsukae and other Neosartria fungi (including Aspergillus fumigatus, which is morphologically very similar to the anamorph of the Neosartria fischeri), and It is a figure which shows the positional relationship of the base sequence which the oligonucleotide of said (c) and (d) recognizes in the base sequence of the beta-tubulin gene of Neosartoria hiratsukae. Partial base sequence of the calmodulin gene of Neosartria pseudofischeri and other Neosartria fungi (including Aspergillus fumigatus, which is morphologically very similar to the anamorphs of the neosartria fischeri), and neo It is a figure which shows the positional relationship of the base sequence which the oligonucleotide of said (e) and (f) recognizes in the base sequence of the calmodulin gene of Sartoria pseudofiseri. A partial base sequence of the calmodulin gene of Neosartoria fischeri and other Neosartria fungi (including Aspergillus fumigatus, which is morphologically very similar to the anamorphs of the neosartria fischeri), It is a figure which shows the positional relationship of the base sequence which the oligonucleotide of said (g) and (h) recognizes in the base sequence of a Fischery's calmodulin gene. Partial base sequence of the calmodulin gene of Neosartoria spinosa and other Neosartria fungi (including Aspergillus fumigatus and its relatives that are morphologically very similar to the anamorphs (asexual generation) of Neosartria fischeri), It is also a diagram showing the positional relationship of the base sequences recognized by the oligonucleotides (i) and (j) in the base sequence of the neosartoria spinosa calmodulin gene. FIG. 2 is an electrophoretogram of PCR products amplified using the oligonucleotide pairs (a) and (b) in the examples. FIG. 3 is an electrophoretogram of PCR products amplified using the oligonucleotide pairs (c) and (d) in the examples. FIG. 3 is an electrophoretogram of PCR products amplified using the oligonucleotide pairs (e) and (f) in the examples. FIG. 3 is an electrophoretogram of PCR products amplified using the oligonucleotide pairs (g) and (h) in Examples. FIG. 3 is an electrophoretogram of PCR products amplified using the oligonucleotide pairs (i) and (j) in the examples. FIG. 3 is an electrophoretogram of PCR products amplified using the oligonucleotide pairs (i) and (j) in the examples. FIG. 3 is an electrophoretogram of PCR products amplified using the oligonucleotide pairs (i) and (j) in the examples.

The present invention, Neosartorya (Neosartorya) genus fungi β- tubulin gene or specific partial nucleotide sequence of the calmodulin gene, i.e. each specific base sequence sites species Neosartorya spp fungi present in the gene (the variable region ) Using a pair of oligonucleotides that can hybridize under stringent conditions to detect Neosartria fungi and specifically identify and detect Neosartria fungi at the species level. Here, the “variable site” is a site where base mutations tend to accumulate in the β-tubulin gene or calmodulin gene, and the base sequence of this site varies greatly among Neosartoria species.
In the present specification, “β-tubulin” is a protein constituting a microtubule, and “β-tubulin gene” is a gene encoding β-tubulin. In addition, “calmodulin” is one of calcium binding proteins widely distributed in eukaryotes, and is a protein that regulates the activity of enzymes such as calmodulin kinase and calcineurin by binding calcium to calmodulin. Is a gene encoding calmodulin.

The term “Neosartoria fungus” in the present invention means an irregular aspergillus belonging to the family Trichocomaceae . Neosartoria fungi are heat-resistant fungi that form ascospores that can survive even after heat treatment at 75 ° C. for 30 minutes.
“Neosartria grabra” is a kind of Neosartria fungus, which has fine protrusions on the lens surface of ascospores, and two equatorial belt-like ridges separated from each other, with a minute in the groove between them. It is a bacterial species having a form in which two protrusions are arranged in parallel. Neosartria grabra is known as a causative agent of contamination accidents such as fruit juice drinks and baked goods.
“Neosartria hiratsukae” is a type of fungus belonging to the genus Neosartria, with large ascospores, a fine lens surface, and a narrow band of two equatorial planes. It is a bacterial species having a form that is in close contact with each other. Neosartria hiratsukae is known as a causative agent of contamination accidents such as fruit juice jelly and vegetable drinks.
“Neosartoria Pseudofischeri” is a type of fungus belonging to the genus Neosartoria, with large ascospores, a triangular and short streak on the lens surface, and two equatorial belts. It is a bacterial species having a form with a bump. Neosartoria Pseudofischeri is known as a causative agent of pollution accidents such as beverages and canned foods.
In addition, “Neosartoria Fischeri” is a type of fungus belonging to the genus Neosartoria, and the ascospore lens surface has a network structure, and the two equator plane ridges are in close contact with each other. It is. Neosartria Fischeri is known as a causative agent of contamination accidents such as canned foods and jellies.
In addition, “Neosartoria spinosa” is a type of Neosartoria fungus, which is a species of fungus that has a stab-like projection on the lens surface of the ascospore and has two equatorial belt-like ridges. . Neosartoria spinosa is known as a causative agent of contamination accidents such as fruit juice and canned foods.

In general, fungi as a whole, not limited to Neosartoria fungi, may be difficult to identify at the gene level even though they can be classified morphologically into different species. For example, a very related bacterial strains to the Neosartorya-spinosa, Neosartorya-Koreana (Neosartorya coreana), Neosartorya-Rashiniosa (Neosartorya lacinosa) and Neosartorya Pauli Sten cis (Neosartorya paulistensis) is present. Although these species may be reported as separate species, there is almost no morphological difference. Furthermore, the homology of the genes used for the index of phylogeny is extremely high, and it is difficult to distinguish these bacterial species at the gene level. First, fungi are classified according to their morphological characteristics. Therefore, when the form changes depending on the culture conditions or the like, the original bacterial species that are originally the same bacterial species may be proposed as new species. Furthermore, it is known that fungi have many synonyms (one bacterial species has multiple scientific names) compared to bacteria. Therefore, there are cases where Neosartria fungi are actually the same species, even among the bacterial species proposed as different species. Therefore, these bacterial species are considered to have the same harm in foods as heat resistance, and the possibility that they are synonymous with each other cannot be denied.
From the above viewpoint, bacterial species having the same gene level are treated as the same species in the present invention even though they are classified as different species from the morphological viewpoint. To explain with specific examples, Neosartria spinosa, Neosartria colana, Neosartria lasiniosa and Neosartria Paulistensis may be proposed as different species. As shown in FIG. 2, since they are genetically very similar, they are treated as the same species in the present invention. That is, in the present specification, “Neosartoria spinosa” includes Neosartoria Correana, Neosartoria Lasiniosa, and Neosartoria Paulistensis, which have the same gene level. In the present specification, “gene level is the same” means that the homology of base sequences of a plurality of genes (for example, β-tubulin gene, calmodulin gene, actin gene, etc.) having a high genetic evolution rate is compared. , 97% or more (preferably 98% or more, more preferably 99% or more). The base sequence homology is calculated by the Lipman-Pearson method (see Science, 227, 1435, 1985, etc.) and the like. Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the parameter Unit size to compare (ktup) is set to 2 and the calculation is performed. Can do.

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

The detection method of the present invention is characterized by using an oligonucleotide pair represented by a nucleic acid corresponding to a variable site in a β-tubulin gene or calmodulin gene of a Neosartoria fungus.
As shown in FIGS. 1 to 5, the inventors determined the base sequences of β-tubulin gene or calmodulin gene of various fungi including Neosartoria genus, and determined the genetic distance and base between fungal species. Sequence homology analysis was performed. That is, the base sequence of β-tubulin gene or calmodulin gene of each fungus was determined by sequencing method, and the matching base region was examined by alignment analysis. As a result, in the β-tubulin gene or the calmodulin gene, a conserved region was found in the same species, but the conserved property of the base sequence was low between different species, and a variable site having a unique base sequence depending on the species was found. At this variable site, Neosararia fungi has a species-specific base sequence. Therefore, this region is useful as a genetic indicator for identifying and identifying Neosartoria fungi at the species level.

The detection method of the present invention comprises an oligonucleotide pair comprising the following oligonucleotides (a) and (b), an oligonucleotide pair comprising the following oligonucleotides (c) and (d), and the following (e) and (f): At least one selected from the group consisting of an oligonucleotide pair consisting of oligonucleotides, an oligonucleotide pair consisting of oligonucleotides (g) and (h) below, and an oligonucleotide pair consisting of oligonucleotides (i) and (j) below Species oligonucleotide pairs are used.
(A) An oligonucleotide represented by the base sequence set forth in SEQ ID NO: 1, or one or several bases deleted, substituted, inserted or added in the base sequence set forth in SEQ ID NO: 1 and Neosartoria Oligonucleotide that can be used for detection at the fungal species level (b) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 2, or one or several bases deleted in the nucleotide sequence set forth in SEQ ID NO: 2, Oligonucleotide which is substituted, inserted or added and can be used for detection at the species level of Neosartoria fungus (c) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3, or the base set forth in SEQ ID NO: 3 Detection at the species level of Neosartria fungi with one or several bases deleted, substituted, inserted or added in the sequence Usable oligonucleotide (d) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 4 or one or several bases deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 4 And (e) an oligonucleotide represented by the nucleotide sequence represented by SEQ ID NO: 5, or one or several bases in the nucleotide sequence represented by SEQ ID NO: 5 Is deleted, substituted, inserted or added and can be used for detection at the species level of Neosartoria fungi (f) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 6, or SEQ ID NO: 6 1 or several bases are deleted, substituted, inserted or added in the base sequence described in 1. Oligonucleotides that can be used for detection at the species level of the genus Rhizoma (g) The oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7 or the nucleotide sequence set forth in SEQ ID NO: 7 lacks one or several bases Oligonucleotide which is deleted, substituted, inserted or added and can be used for detection at the species level of Neosartoria fungi (h) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8, or SEQ ID NO: 8 An oligonucleotide in which one or several bases are deleted, substituted, inserted or added in the nucleotide sequence of the nucleotide sequence and can be used for detection at the species level of Neosartria fungus (i) In the oligonucleotide represented or the nucleotide sequence set forth in SEQ ID NO: 9, one or several bases are deleted, substituted or inserted. Is added and can be used for detection at the species level of Neosartoria fungi (j) 1 in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 10 or the nucleotide sequence of SEQ ID NO: 10 Or an oligonucleotide that has several bases deleted, substituted, inserted or added and can be used for detection at the species level of Neosartoria fungi

The variable site of the β-tubulin gene of Neosartoria grubura, a kind of Neosartoria fungus, will be described based on the base sequence described in FIG. 1 and SEQ ID NO: 11. In addition, the base sequence described in SEQ ID NO: 11 represents a partial base sequence of the β-tubulin gene of Neosartria glabra CBS 111.55T strain. In addition, Figure 1 shows Neosartria glabra CBS 111.55T and other Neosartria fungi (Neosartoria Nishimurae CBS 116047, Neosartria Australensis CBS 112.55T, Neosartria Feneriae CBS 598.74T , Neosartria hiratsukae NHL 3008T, Neosartria Udagawae CBM-FA-0702T, Neosartria Correana KACC 41659T, Neosartria Paulistensis CBM-FA-0690T, Neosartria Raciniosa KACC 41657T, Neo Sartoria spinosa CBS 483.65T, Neosartoria Fischeri CBS 544.65T, Aspergillus fumigatus IAM 13869 ex type and Neosartoria pseudofischeri CBS 208.92T ) Partial base sequence of β-tubulin gene As well as the positional relationship of the oligonucleotides to recognize the base sequence of said at nucleotide sequence of the β- tubulin gene of Neosartorya glabra (a) and (b).
The oligonucleotide (a) (also referred to herein as Ngl_F1) and the oligonucleotide (b) (also referred to herein as Ngl_R1) are each described in SEQ ID NO: 11, as shown in FIG. The oligonucleotide is hybridizable under stringent conditions to the region from position 81 to position 102 and the region from position 318 to position 340. These regions are variable regions with low conserved base sequences between species, and regions with particularly low conserved base sequences between Neosartria grabra and other species, and the base sequences of these regions The present inventors have found that is a base sequence inherently possessed by Neosartria grabra. Therefore, Neosartria grabra can be specifically identified and identified at the species level using the oligonucleotides (a) and (b). Specifically, if this region is designed so that 5 bases or more of the 3 ′ end of the primer, which is the direction of DNA extension by polymerase, hybridize, it can be used as a primer specific for Neosartria grabra. Identification by the polymerase chain reaction (PCR) method using these primers becomes possible. In addition, probes designed to hybridize to this region for 10 bases or more specifically hybridize to the β-tubulin gene of Neosartria glabra. Therefore, identify Neosartria glabra by the presence or absence of hybridization. Is possible.

In the present invention, using the oligonucleotides (a) and (b), the presence of a nucleic acid (preferably, the variable site) represented by the base sequence (k) and / or (l) below is confirmed. It is preferable.
(K) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 11 or a complementary sequence thereof (l) one or several (preferably 1 to 25, more preferably 1) in the base sequence described in SEQ ID NO: 11 ~ 20, more preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases deleted, substituted, inserted or added and at the species level of Neosartria grabra Nucleotide sequences that can be used for detection in DNA, or their complementary sequences

The variable site of the β-tubulin gene of Neosartoria hiratsukae, which is a type of Neosartoria fungus, will be described based on the base sequence described in FIG. 2 and SEQ ID NO: 12. In addition, the base sequence described in SEQ ID NO: 12 represents a partial base sequence of the β-tubulin gene of Neosartria hiratsukae NHL 3008T strain. Figure 2 also shows Neosartria hiratsukae NHL 3008T and other Neosartria fungi (Neosartria australens CBS 112.55T, Neosartria feneliae CBS 598.74T, Neosartria glabra CBS 111.55T , Neosartoria Nishimurae CBS 116047, Neosartoria Udagawae CBM-FA-0702T, Neosartoria Correana KACC 41659T, Neosartoria Paulistensis CBM-FA-0690T, Neosartoria Raciniosa KACC 41657T, Neo Sartoria spinosa CBS 483.65T, Neosartoria Fischeri CBS 544.65T, Aspergillus fumigatus IAM 13869 ex type and Neosartoria pseudofischeri CBS 208.92T ) Partial base sequence of β-tubulin gene , And the positional relationship of the nucleotide sequences recognized by the oligonucleotides (c) and (d) in the nucleotide sequence of the β-tubulin gene of Neosartoria hiratsukae.
The oligonucleotide (c) (also referred to herein as Nhi_F1) and the oligonucleotide (d) (also referred to herein as Nhi_R1) are each represented by SEQ ID NO: 12, as shown in FIG. The oligonucleotide is hybridizable under stringent conditions to the region from position 28 to position 51 and the region from position 311 to position 336. These regions are variable regions with low conserved base sequences between species, and regions with particularly low conserved base sequences between Neosartria hiratsukae and other species, and the base sequences of these regions The present inventors have found that is a base sequence inherently possessed by Neosartria hiratsukae. Therefore, Neosartria hiratsukae can be specifically identified and identified at the species level using the oligonucleotides (c) and (d). Specifically, it can be used as a primer specific for Neosartria hiratsukae if it is designed such that 5 'or more of the 3' end of the primer, which is the direction of DNA extension by polymerase, hybridizes to this region, Identification by PCR using these primers becomes possible. In addition, since the probe designed to hybridize to this region more than 10 bases hybridizes specifically with the β-tubulin gene of Neosartria hiratsukae, it is necessary to identify Neosartria hiratsukae by the presence or absence of hybridization. Is possible.

In the present invention, using the oligonucleotides (c) and (d), the presence of a nucleic acid (preferably the variable site) represented by the base sequence (m) and / or (n) below is confirmed. It is preferable.
(M) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 12 or a complementary sequence thereof (n) one or several (preferably 1-25, more preferably 1) in the base sequence described in SEQ ID NO: 12 -20, more preferably 1-15, particularly preferably 1-10, most preferably 1-5 bases are deleted, substituted, inserted or added and the species level of Neosartria hiratsukae Nucleotide sequences that can be used for detection in DNA, or their complementary sequences

The variable site of the calmodulin gene of Neosartoria pseudofischeri, which is a type of Neosartoria fungus, will be described based on the base sequence described in FIG. 3 and SEQ ID NO: 13. The base sequence described in SEQ ID NO: 13 is a partial base sequence of the calmodulin gene of Neosartoria pseudofischeri CBS 208.92T strain. In addition, FIG. 3 shows Neosartria pseudofiseri CBS 208.92T and other Neosartria fungi (Neosartoria Australensis CBS 112.55T, Neosartria hiratsukae NHL 3008T, Neosartria Feneriae CBS 598.74 T-strain, Neosartria grabra CBS 111.55T, Neosartria Nishimurae CBS 116047, Neosartria Udagawae CBM-FA-0702T, Neosartria Koreana KACC 41659T, Neosartria Paulistensis CBM-FA-0690T , Neosartoria Lasiniosa KACC 41657T, Neosartoria Spinosa CBS 483.65T, Aspergillus fumigatus IAM 13869 ex type strain, which is morphologically very similar to Neosartoria Fischeri anamorph, and Neosartria Fischeri CBS 544.65T Partial base sequence of calmodulin gene And wherein in Neosartorya pseudotuberculosis fischeri nucleotide sequence of calmodulin gene of oligonucleotides (e) and (f) shows the positional relationship recognized nucleotide sequence.
The oligonucleotide (e) (also referred to herein as Npf_F2) and the oligonucleotide (f) (also referred to herein as Npf_R2) are each described in SEQ ID NO: 13, as shown in FIG. The oligonucleotide is hybridizable under stringent conditions to the region from position 138 to position 158 and the region from position 382 to position 405. These regions are variable regions with low conserved base sequences between species, and regions with particularly low conserved base sequences between Neosartria pseudofischeri and other species. The present inventors have found that the base sequence is a base sequence inherently possessed by Neosartria pseudofischeri. Therefore, using the oligonucleotides (e) and (f), Neosartria pseudofischeri can be specifically identified and identified at the species level. Specifically, if this region is designed so that 5 bases or more of the 3 ′ end of the primer, which is the direction of DNA extension by polymerase, hybridize, it can be used as a primer specific for Neosartria pseudofischeri. Yes, identification by PCR using these primers becomes possible. In addition, since the probe designed to hybridize to this region for 10 bases or more specifically hybridizes with the calmodulin gene of Neosartria pseudofischeri, it identifies Neosartria pseudofischeri by the presence or absence of hybridization. It becomes possible.

In the present invention, using the oligonucleotides (e) and (f), the presence of a nucleic acid (preferably the variable site) represented by the base sequence (o) and / or (p) below is confirmed. It is preferable.
(O) a partial base sequence of the calmodulin gene shown in SEQ ID NO: 13 or its complementary sequence (p) one or several (preferably 1-25, more preferably 1-20) in the base sequence shown in SEQ ID NO: 13 More preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases have been deleted, substituted, inserted or added and at the Neosartoria Pseudofischeri species level. Sequence that can be used for detection of DNA, or its complementary sequence

The variable site of the calmodulin gene of Neosartoria fischeri, which is one of Neosartoria fungi, will be described based on the base sequence described in FIG. 4 and SEQ ID NO: 14. In addition, the base sequence described in SEQ ID NO: 14 represents a partial base sequence of the calmodulin gene of Neosartria fischeri CBS 544.65T strain. Figure 4 also shows Neosartria fischeri CBS 544.65T and other neosartria fungi (Aspergillus fumigatus IAM 13869 ex type, Neosartoria austra Rencis CBS 112.55T, Neosartria hiratsukae NHL 3008T, Neosartria feneriae CBS 598.74T, Neosartria grabra CBS 111.55T, Neosartria Nishimurae CBS 116047, Neosartria Udagawae CBM-FA-0702T , Neosartoria Correana KACC 41659T, Neosartoria Paulistensis CBM-FA-0690T, Neosartoria Raciniosa KACC 41657T, Neosartoria Spinosa CBS 483.65T, and Neosartoria Pseudofischeri CBS 208.92T Partial base sequence of calmodulin gene And wherein the nucleotide sequence of the calmodulin gene Neosartorya-fischeri oligonucleotides (g) and (h) showing the positional relationship recognized nucleotide sequence.
The oligonucleotide (g) (also referred to herein as Nfi_F3) and the oligonucleotide (h) (also referred to herein as Nfi_R3) are each described in SEQ ID NO: 14, as shown in FIG. The oligonucleotide is hybridizable under stringent conditions to the region from position 24 to position 46 and the region from position 363 to position 385. These regions are variable regions with low conserved base sequences between species, and regions with particularly low conserved base sequences between Neosartoria Fischeri and other species, and the base sequences of these regions The present inventors have found that is a base sequence inherently possessed by Neosartria Fischeri. Therefore, Neosartria fischeri can be specifically identified and identified at the species level using the oligonucleotides (g) and (h). Specifically, it can be used as a primer specific for Neosartria fischeri if it is designed so that 5 bases or more of the 3 ′ end of the primer, which is the direction of DNA extension by polymerase, hybridizes to this region, Identification by PCR using these primers becomes possible. In addition, a probe designed to hybridize to this region for 10 bases or more specifically hybridizes with the neosartoria fischerry calmodulin gene. Become.

In the present invention, using the oligonucleotides (g) and (h), the presence of a nucleic acid (preferably the variable site) represented by the base sequence (q) and / or (r) below is confirmed. It is preferable.
(Q) a partial base sequence of the calmodulin gene described in SEQ ID NO: 14 or a complementary sequence thereof (r) 1 or several (preferably 1-25, more preferably 1-20) in the base sequence described in SEQ ID NO: 14 And more preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases are deleted, substituted, inserted or added and are detected at the species level of Neosartria fischeri. Base sequence that can be used for the above, or its complementary sequence

The variable site of the calmodulin gene of Neosartoria spinosa, which is one of Neosartoria fungi, will be described based on the base sequence described in FIG. 5 and SEQ ID NO: 15. The base sequence described in SEQ ID NO: 15 represents the partial base sequence of the calmodulin gene of Neosartoria spinosa CBS 483.65T strain. Further, FIG. 5 shows Neosartria spinosa CBS 483.65T strain, Neosartoria Correana (KACC 41659T strain), Neosartoria Lasiniosa (KACC 41657T strain) and Neosartria treated as the same strain as Neosartria spinosa in the present invention. Pauli Sten cis (CBM-FA-0690T, Ltd.), as well as other Neosartorya the genus fungi (Neosartorya-Aureora (Neosartorya aureola) CBS 105.55T Co., Neosartorya-Udagawae CBM-FA-0702T strain and Neosartorya-fischeri CBS Aspergillus fumigatus IAM 13869 ex type strain and its relatives ( Aspergillus viridinutans ) CBS 127.56T strain, Aspergillus nobohumimitas (Aspergillus novofumigatus) ITB 16806T strain, Ass Rugirusu-Fumishinematasu (Aspergillus fumisynnematus) IFM 42277T strain, Aspergillus lentus (Aspergillus lentulus) FH5T Ltd.)) partial nucleotide sequence of the calmodulin gene as well as the in the nucleotide sequence of the calmodulin gene Neosartorya-spinosa (i) and (j) The positional relationship of the base sequence recognized by the oligonucleotide is shown.
The oligonucleotide (i) (also referred to herein as Nspc_1F) and the oligonucleotide (j) (also referred to herein as Nspc_1R) are each described in SEQ ID NO: 15, as shown in FIG. The oligonucleotide is hybridizable under stringent conditions to the region from the 105th position to the 124th position and the region from the 336th position to the 355th position. These regions are variable regions with low conserved base sequences between species, and regions with particularly low conserved base sequences between Neosartria spinosa and other species, and the base sequences of these regions The present inventors have found that is a base sequence inherently possessed by Neosartria spinosa. Therefore, Neosartria spinosa can be specifically identified and identified at the species level using the oligonucleotides (i) and (j). Specifically, it can be used as a primer specific to Neosartria spinosa if it is designed so that 5 bases or more of the 3 ′ end of the primer, which is the direction of DNA extension by polymerase, hybridize to this region, Identification by PCR using these primers becomes possible. In addition, since the probe designed to hybridize more than 10 bases in this region specifically hybridizes with the neosartoria spinosa calmodulin gene, it is possible to identify Neneasartoria spinosa by the presence or absence of hybridization. It becomes.

In the present invention, using the oligonucleotides (i) and (j), the presence of a nucleic acid (preferably, the variable site) represented by the following base sequence (s) and / or (t) is confirmed. It is preferable.
(S) a partial base sequence of the calmodulin gene described in SEQ ID NO: 15 or a complementary sequence thereof (t) one or several (preferably 1-25, more preferably 1-20) in the base sequence described in SEQ ID NO: 15 And more preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases deleted, substituted, inserted or added and at the species level of Neosartria spinosa Base sequence that can be used for the above, or its complementary sequence

The oligonucleotides (a) to (j) used in the detection method of the present invention are preferably oligonucleotides represented by the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 10. The oligonucleotide used in the detection method of the present invention is an oligonucleotide represented by a base sequence having 70% or more homology to the base sequence described in any of SEQ ID NOs: 1 to 10 or a complementary sequence thereof. The homology is more preferably 80% or more, the homology is more preferably 85% or more, the homology is more preferably 90% or more, and the homology is 95% or more. It is particularly preferred that In addition, the oligonucleotide that can be used in the present invention has one or several, preferably 1 to 5, more preferably 1 to 4, more preferably 1 in the base sequence described in any one of SEQ ID NOs: 1 to 10. Are oligos that have 1 to 3, more preferably 1 to 2, particularly preferably 1 base deletions, substitutions, insertions or additions and can be used for detection at the species level of Neosartoria fungi Nucleotides are also included. Moreover, you may add an appropriate base sequence to the base sequence in any one of sequence number 1-10.
The base sequence homology is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985) or the like. Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the parameter Unit size to compare (ktup) is set to 2 and the calculation is performed. Can do.

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

  The oligonucleotide can be prepared by a usual synthesis method such as chemical synthesis using, for example, an automatic DNA synthesizer. It is also possible to cut out directly from a gene of Neosartoria fungi using a restriction enzyme or the like, or after cloning and isolating and purifying the gene, cutting out 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.

  There is no particular limitation as a method for confirming the presence of the partial base sequence of the β-tubulin gene and / or calmodulin gene of the Neosartoria fungus using the oligonucleotide pair, and identifying the Neosartoria fungus at the species level. It can be performed by a commonly used genetic engineering technique such as a singing method, a hybridization method, a PCR method, or a LAMP method.

  The oligonucleotide for detection of the present invention can be used as a nucleic acid primer and 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. 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. 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 oligonucleotide labeled in this way is hybridized with the nucleic acid extracted from the test object by a conventional method under stringent conditions, and then the label of the hybridized detection oligonucleotide is measured. Can detect Neosartria fungi. 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.
The oligonucleotide can also be used as a capture probe by binding to a solid support. 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 detect Neosartria spp. At the species level, the oligonucleotide pair comprising the oligonucleotides (a) and (b) and the oligonucleotides (c) and (d) An oligonucleotide pair, an oligonucleotide pair consisting of the oligonucleotides (e) and (f), an oligonucleotide pair consisting of the oligonucleotides (g) and (h), and / or the above (i) and (j) It is preferable to perform hybridization using an oligonucleotide pair consisting of oligonucleotides as a nucleic acid probe. In order to detect Neosartria grabra, it is preferable to use an oligonucleotide pair consisting of the oligonucleotides (a) and (b) as a nucleic acid probe. In order to detect Neosartria hiratsukae, it is preferable to use an oligonucleotide pair consisting of the oligonucleotides (c) and (d) as a nucleic acid probe. In order to detect Neosartria pseudofischer, it is preferable to use an oligonucleotide pair consisting of the oligonucleotides (e) and (f) as a nucleic acid probe. In order to detect Neosartria fischeri, it is preferable to use an oligonucleotide pair consisting of the oligonucleotides (g) and (h) as a nucleic acid probe. In order to detect Neosartria spinosa, it is preferable to use an oligonucleotide pair consisting of the oligonucleotides (i) and (j) as a nucleic acid probe.

  In order to detect Neosartria fungi in a specimen at the species level, an oligonucleotide pair comprising the oligonucleotides (a) and (b), and an oligonucleotide comprising the oligonucleotides (c) and (d) A pair of oligonucleotides comprising the oligonucleotides (e) and (f), an oligonucleotide pair comprising the oligonucleotides (g) and (h), and an oligonucleotide comprising the oligonucleotides (i) and (j) At least one oligonucleotide pair selected from the group consisting of nucleotide pairs is labeled to form a nucleic acid probe, the resulting nucleic acid probe is hybridized with DNA or RNA, and the label of the hybridized probe is detected by an appropriate detection method do it. Since the above-mentioned nucleic acid probe specifically hybridizes with the variable site of the β-tubulin gene or calmodulin gene of the Neosartoria fungus, the Neosartoria fungus in the specimen can be detected quickly and easily at the species level. . As a method for measuring the label of the nucleic acid probe hybridized with DNA or RNA, a usual method (FISH method, dot blot method, Southern blot method, Northern blot method, etc.) can be used.

In the detection method of the present invention, in order to detect / identify Neosartoria fungi, it is preferable to confirm the presence or absence of an amplification product using the at least one oligonucleotide pair. The method for amplifying a DNA fragment is not particularly limited. The polymerase chain reaction (PCR) method, LCR (Ligase Chain Reaction) method, SDA (Strand Displacement Amplification) method, NASBA (Nucleic Acid Sequence-based Amplification) method, RCA (Rolling) Conventional methods such as -circle amplification) and LAMP (Loop mediated isothermal amplification) can be used. In the present invention, the PCR method is preferably used.
In the present invention, a case where an amplification reaction is carried out by the PCR method to detect Neosartoria fungi will be described in detail.

In the present invention, in order to detect Neosartria glabra, the oligonucleotides (a) and (b) (preferably the oligonucleotide represented by the base sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 PCR is carried out using each of the oligonucleotides represented by the nucleotide sequence of N) as a nucleic acid primer, and a partial nucleotide sequence of the β-tubulin gene of Neosartoria grubura (preferably the base of (k) or (l) above) It is preferable to amplify a part of the sequence and confirm the presence or absence of the amplification product.
Moreover, the oligonucleotide pair which consists of the oligonucleotide of said (a) and (b) can be made into the oligonucleotide pair for Neosartoria genus fungi (specifically Neosartria grabra) detection.

In the present invention, in order to detect Neosartria hiratsukae, the oligonucleotides (c) and (d) (preferably, the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 and the sequence set forth in SEQ ID NO: 4 PCR is carried out using each of the oligonucleotides represented by the nucleotide sequence of N) as a nucleic acid primer, and a partial nucleotide sequence of the β-tubulin gene of Neosartoria hiratsukae (preferably the base of (m) or (n) above It is preferable to amplify a part of the sequence and confirm the presence or absence of the amplification product.
Moreover, the oligonucleotide pair consisting of the oligonucleotides of (c) and (d) can be used as an oligonucleotide pair for detecting Neosartria fungi (specifically, Neosartria hiratsukae).

In the present invention, in order to detect Neosartria pseudofischeri, the oligonucleotides (e) and (f) (preferably the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 and SEQ ID NO: 6 PCR is carried out using each of the oligonucleotides represented by the base sequence described in 1) as a nucleic acid primer, and a partial base sequence of the calmodulin gene of Neosartoria pseudofischerii (preferably the above (o) or (p) It is preferable to amplify a part of the base sequence and confirm the presence or absence of the amplified product.
The oligonucleotide pair consisting of the oligonucleotides of (e) and (f) can be used as an oligonucleotide pair for detecting Neosartria fungi (specifically, Neosartria pseudofischer).

In the present invention, in order to detect Neosartoria fischeri, the oligonucleotides (g) and (h) (preferably the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7 and SEQ ID NO: 8 PCR is carried out using each of the oligonucleotides represented by the nucleotide sequence of (N) as a nucleic acid primer, and a partial nucleotide sequence (preferably one of the nucleotide sequences of (q) or (r) above) of the neosartoria fischerry calmodulin gene. Part) is preferably amplified and the presence or absence of the amplified product is confirmed.
The oligonucleotide pair consisting of the oligonucleotides (g) and (h) can be used as an oligonucleotide pair for detecting Neosartria fungi (specifically, Neosartria fischeri).

In the present invention, in order to detect Neosartria spinosa, the oligonucleotides (i) and (j) (preferably the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 9 and SEQ ID NO: 10 PCR is carried out using each of the oligonucleotides represented by the nucleotide sequence of (N) as a nucleic acid primer, and the partial nucleotide sequence of the calmodulin gene of Neosartoria spinosa (preferably one of the nucleotide sequences of (s) or (t) above) Part) is preferably amplified and the presence or absence of the amplified product is confirmed.
The oligonucleotide pair consisting of the oligonucleotides (i) and (j) can be used as an oligonucleotide pair for detecting Neosartria fungi (specifically, Neosartria spinosa).

The PCR conditions are not particularly limited as long as the target DNA fragment can be amplified to a detectable level.
For example, when PCR is performed using the oligonucleotides (a) and (b) as nucleic acid primers, a heat denaturation reaction in which a double-stranded DNA is made into a single strand is carried out at 95 to 98 ° C. for about 5 to 60 seconds. An annealing reaction for hybridizing the primer pair to the single-stranded DNA is performed at 57 to 63 ° C. for about 5 to 60 seconds, and an extension reaction for allowing the DNA polymerase to act is performed at about 72 ° C. for about 10 to 120 seconds. About 30 to 35 cycles are performed. When performing the PCR method using the oligonucleotides (c) and (d) as nucleic acid primers, a heat denaturation reaction in which a double-stranded DNA is made into a single strand is carried out at 95 to 98 ° C. for about 5 to 60 seconds. An annealing reaction for hybridizing the primer pair to the single-stranded DNA is carried out at 56 to 63 ° C. for about 5 to 60 seconds, and an extension reaction for allowing the DNA polymerase to act is carried out at about 72 ° C. for about 10 to 60 seconds. About 30 to 35 cycles are performed. When performing the PCR method using the oligonucleotide pair (e) and (f) as a nucleic acid primer, a heat denaturation reaction in which a double-stranded DNA is made into a single strand is carried out at 95 to 98 ° C. for about 5 to 60 seconds. An annealing reaction for hybridizing the primer pair to the single-stranded DNA is performed at 57 to 63 ° C. for about 5 to 60 seconds, and an extension reaction for allowing DNA polymerase to act is performed at about 72 ° C. for about 10 to 60 seconds. About 30 to 35 cycles are performed. When performing the PCR method using the oligonucleotide pairs (g) and (h) as nucleic acid primers, a heat denaturation reaction in which a double-stranded DNA is made into a single strand is carried out at 95 to 98 ° C. for about 5 to 60 seconds. An annealing reaction for hybridizing the primer pair to the single-stranded DNA is performed at 57 to 63 ° C. for about 5 to 60 seconds, and an extension reaction for allowing DNA polymerase to act is performed at about 72 ° C. for about 10 to 60 seconds. About 30 to 35 cycles are performed. When performing the PCR method using the oligonucleotide pairs (i) and (j) as nucleic acid primers, a heat denaturation reaction in which a double-stranded DNA is converted to a single strand is performed at 95 to 98 ° C. for about 5 to 60 seconds. An annealing reaction for hybridizing the primer pair to the single-stranded DNA is performed at 55 to 61 ° C. for about 5 to 60 seconds, an extension reaction for allowing the DNA polymerase to act is performed for about 10 to 60 seconds at about 72 ° C. About 30 to 35 cycles are performed.

In the present invention, confirmation of gene fragments can be performed by a usual method. Examples include a method for confirming the presence or absence of a band corresponding to the size of the amplified gene by electrophoresis on the amplified product, a method for measuring the amount of amplified product over time, and a method for decoding the base sequence of the amplified product. The present invention is not limited to these methods. In the present invention, a method of performing electrophoresis after gene amplification treatment and confirming the presence or absence of a band corresponding to the size of the amplified gene is preferable. In the present invention, the amplification product can be detected by a usual method. For example, a method of incorporating nucleotides labeled with a radioactive substance during an amplification reaction, a method of using a primer labeled with a fluorescent substance, or the like, and fluorescence intensity is increased by binding to DNA such as ethidium bromide between two amplified DNA strands. Although the method etc. which enter the fluorescent substance which becomes strong are mentioned, this invention is not limited to these methods. In the present invention, a method in which a fluorescent substance whose fluorescence intensity is increased by binding to DNA between the amplified DNA double strands is preferable.
When a specimen contains a fungus to be detected, PCR is performed using the oligonucleotide pair of the present invention as a primer set, and when the obtained PCR product is electrophoresed, a DNA fragment of a specific size is confirmed. Specifically, when the sample contains Neosartoria grubras, electrophoresis of the PCR product obtained using the oligonucleotides (a) and (b) as nucleic acid primers results in a size of about 250 bp. A DNA fragment is identified. When the sample contains Neosartria hiratsukae, electrophoresis of the PCR product obtained using the oligonucleotides (c) and (d) as nucleic acid primers confirmed a DNA fragment with a size of about 300 bp. The When the sample contains Neosartoria pseudofischeri, electrophoresis of the PCR product obtained using the oligonucleotide pair (e) and (f) as a nucleic acid primer yields a DNA fragment of about 250 bp in size. Is confirmed. When the sample contains Neosartoria Fischeri, a DNA fragment is confirmed at a size of about 350 bp by electrophoresis of the PCR product obtained by using the oligonucleotide pair (g) and (h) as a nucleic acid primer. Is done. When the sample contains Neosartoria spinosa, electrophoresis of the PCR product obtained using the oligonucleotide pair (i) and (j) as a nucleic acid primer confirms a DNA fragment with a size of about 250 bp. Is done.
By performing such an operation, it can be confirmed whether or not the detection target fungus is contained in the specimen.

In the present invention, in order to identify a neosartoria fungus, a partial base sequence of a subject β-tubulin gene or calmodulin gene is determined using the oligonucleotide pair, and the partial base sequence of the gene is the above ( k) to (t) by confirming whether or not the nucleic acid is represented by any one of the nucleotide sequences (k) to (t) Genus fungi can also be detected at the species level. That is, the detection method of the present invention analyzes and determines the partial base sequence of the β-tubulin gene or calmodulin gene possessed by the subject, and determines the base sequence and any of the base sequences (k) to (t). And the presence of the nucleic acid represented by any one of the base sequences (k) to (t) is confirmed based on the coincidence or difference, and the species level of Neosartoria fungi is identified. . For example, an oligonucleotide pair consisting of the oligonucleotides (a) and (b), an oligonucleotide pair consisting of the oligonucleotides (c) and (d), and an oligonucleotide consisting of the oligonucleotides (e) and (f) At least one oligonucleotide pair selected from the group consisting of a nucleotide pair, an oligonucleotide pair comprising the oligonucleotides (g) and (h), and an oligonucleotide pair comprising the oligonucleotides (i) and (j) Is used as a primer, and the base sequence of the amplification product obtained is compared with any one of the base sequences (k) to (t). If the base sequence of the amplification product is included as part of the nucleic acid represented by the base sequence (k) or (l), the microorganism contained in the sample can be identified as Neosartoria grubra, If it is contained as part of the nucleic acid represented by the base sequence of (m) or (n), the microorganism contained in the specimen can be identified as Neosartria hiratsukae, (o) or (p) If it is contained as part of the nucleic acid represented by the base sequence, the microorganism contained in the specimen can be identified as Neosartria pseudofischeri and represented by the base sequence (q) or (r). If it is contained as part of the nucleic acid, the microorganism contained in the specimen can be identified as Neosartria fischeri, and it is contained as part of the nucleic acid represented by the base sequence (s) or (t). Included in the sample. Microorganisms can be identified as Neosartorya-spinosa to be.
The method for analyzing and determining the base sequence is not particularly limited, and a commonly used technique of RNA or DNA sequencing can be used.
Specific examples include electrophoresis such as Maxam-Gilbert method and Sanger method, mass spectrometry, hybridization method and the like. Examples of the Sanger method include a method of labeling a primer or a terminator by a radiation labeling method, a fluorescence labeling method, or the like.

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

There is no restriction | limiting in particular as a test substance used in this invention, Food-drinks itself, the raw material of food-drinks, an isolated microbial cell, a cultured microbial cell, etc. can be used.
The method for preparing DNA from a sample is not particularly limited as long as a DNA having a degree of purification and an amount sufficient to detect Neosartria fungi can be obtained, and can be used in an unpurified state. It can also be used after pretreatment such as extraction, concentration and purification. For example, it can be purified by extraction with phenol and chloroform, or purified using a commercially available extraction kit to increase the purity of the nucleic acid. In addition, DNA obtained by reverse transcription of RNA in a subject can also be used.

  The kit for detecting Neosartria fungi of the present invention contains the detection oligonucleotide or the detection oligonucleotide pair of the present invention as a nucleic acid probe or a nucleic acid primer. 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 or the detection oligonucleotide pair of the present invention. Examples of the positive control include DNA containing a region amplified by the method 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.

(1) Analysis of partial base sequence of β-tubulin gene specific to Neosartoria fungi Various fungi belonging to the Neosartoria genus (Neosartoria grubra, Neosartoria / Nishimurae, Neosartoria / Australen) Cis, Neosartria Feneliae, Neosartria Hiratsukae, Neosartria Udagawae, Neosartria Colleana, Neosartria Paulistensis, Neosartria Lasiniosa, Neosartria Spinosa, Neosartria Fischeri and Neosartria Pseudofiseli And the base sequence of the β-tubulin gene of Aspergillus fumigatus, an anamorph of Neosartoria fischeri.
DNA was extracted from bacterial cells cultured in the dark for 7 days at 30 ° C. on a PDA (potato dextrose agar) slant medium using Gen Torukun ™ (Takara Bio Inc.). PCR amplification of the target site was carried out using PuRe Taq ™ Ready-To-Go PCR Beads (GE Health Care UK LTD), with Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3 ′: SEQ ID NO: 16) and Bt2b (5 ′ -ACCCTCAGTGTAGTGACCCTTGGC-3 ': SEQ ID NO: 17) (see Glass and Donaldson, Appl., Environ., Microbiol., 61, p. 1233-1330, 1995). The amplification conditions were a denaturation temperature of 95 ° C., an annealing temperature of 59 ° C., an extension temperature of 72 ° C., and 35 cycles. The PCR product was purified using Auto Seg ™ G-50 (Amersham Pharmacia Biotech). The PCR product is BigDye terminator Ver. 3.1 (trade name, manufactured by Applied Biosystems) was used for labeling, and electrophoresis was performed using ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). For determination of the base sequence from the fluorescence signal during electrophoresis, the software 'ATGC Ver. 4 ′ (Genetyx) was used.
Clustal W (http://clustalw.ddbj.nig.ac.jp/top) based on the base sequence information of various β-tubulin genes of various Neosararia fungi and various fungi determined by sequencing methods. -j.html) was used to perform alignment analysis to determine specific sites in the β-tubulin gene containing nucleotide sequences specific to Neosartoria grubra and Neosartoria hiratsukae, respectively.

(2) Analysis of partial base sequence of calmodulin gene specific to Neosartoria fungi Various fungi belonging to the Neosartoria genus (Neosartoria Grubra, Neosartoria Nishimurae, Neosartoria Australensis, Neo (Sartria Fenegliae, Neosartria Hiratkae, Neosartria Udagawae, Neosartria Colleana, Neosartria Paulistensis, Neosartria Raciniosa, Neosartria Spinosa, Neosartria Fischeri and Neosartria Pseudofiseli) and Neo The base sequences of the calmodulin gene of Aspergillus fumigatus, which is an anamorph of Sartoria fischeri, and its related bacteria were determined.
DNA was extracted from cells obtained by culturing in a dark place at 30 ° C. for 3 to 5 days in a PDA (potato dextrose agar) slant medium 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 (GE Health Care UK LTD) as primers, cmd5 (5′-CCGAGTACAAGGAGGCCTTC-3 ′: SEQ ID NO: 18), cmd6 (5 ′ -CCGATAGAGGTCATAACGTGG-3 ': SEQ ID NO: 19) was used. The amplification conditions were a denaturation temperature of 95 ° C, an annealing temperature of 55 ° C, an extension temperature of 72 ° C, and 35 cycles. The PCR product was purified using Auto Seg ™ G-50 (Amersham Pharmacia Biotech). The PCR product is BigDye terminator Ver. 3.1 (trade name, manufactured by Applied Biosystems) was used for labeling, and electrophoresis was performed using ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). For determination of the base sequence from the fluorescence signal during electrophoresis, the software 'ATGC Ver. 4 ′ (Genetyx) was used.
Clustal W (http://clustalw.ddbj.nig.ac.jp/top-j.) Based on the base sequence information of various neosararia fungi determined by sequencing methods and known calmodulin genes of various fungi. html) was used to perform alignment analysis, and specific sites in the calmodulin gene containing base sequences specific to Neosartria pseudofiseri, Neosartria fischeri, and Neosartria spinosa were determined.

(3) Primer design Based on the base sequence site specific to each of the Neosartoria fungi determined above, a primer (Ngl_F1 primer) consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 1, SEQ ID NO: 2 From a primer (Ngl_R1 primer) comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3, a primer comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 (Nhi_F1 primer), and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 Primer (Nhi_R1 primer), a primer composed of an oligonucleotide represented by the base sequence of SEQ ID NO: 5 (Npf_F2 primer), a primer composed of an oligonucleotide represented by the base sequence of SEQ ID NO: 6 (Npf_R2 primer), SEQ ID NO: 7 Consisting of oligonucleotides represented by the nucleotide sequence of A primer (Nfi_F3 primer), a primer comprising the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 8 (Nfi_R3 primer), a primer comprising the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 (Nspc_1F primer), and SEQ ID NO: 10 A primer (Nspc_1R primer) composed of an oligonucleotide represented by the nucleotide sequence of the above was designed, and synthesis was requested from Sigma-Aldrich Japan (desalted product, 0.02 μmol scale) and purchased.
As shown in FIGS. 1 to 5, each of the above-mentioned primers has a specific region of the β-tubulin gene of Neosartria glabra and Neosartria hiratsukae, Neosartria pseudofischeri, Neosartria fischeri and Neosartria It is designed based on the base sequence of each specific region of the spinosa calmodulin gene.

(4) Detection of Neosartria grabra The strains listed in Table 2 were used as fungi used for evaluating the effectiveness of the designed Ngl_F1 primer and Ngl_R1 primer, that is, Neosartria grabra and other fungi. These strains are stored by Chiba University School of Medicine, Center for Fungal Medicine and controlled by the Control Number, The Centraalbureau voor Schimmelcultures, stored by the CBS Number and controlled by the Fermentation Research Institute. Stocks managed by IFO number were obtained and used.
Each strain was cultured under optimal conditions. The culture conditions were cultured at 25-30 ° C. for 3-5 days using a potato dextrose medium (trade name: Pearl Core Potato Dextrose Agar Medium, manufactured by Eiken Chemical Co., Ltd.).

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

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 0.5 μL of Ngl_F1 primer (20 pmol / μL) and Ngl_R1 primer (20 pmol / 20 μmol / μL) were mixed. (μL) 0.5 μL was added to prepare a 25 μL PCR solution.
The PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR conditions were (i) 98 ° C for 5 seconds heat denaturation reaction, (ii) 61 ° C for 5 seconds annealing reaction, and (iii) 72 ° C for 10 seconds extension reaction for 1 cycle, 35 cycles. went.

After PCR, 2 μL was taken from the PCR solution, mixed well with the loading buffer, and subjected to electrophoresis (135 V, 25 minutes) using a 2% agarose gel. After completion of electrophoresis, the agarose gel was stained with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen), and the presence or absence of the amplified DNA fragment was confirmed. The result is shown in FIG. 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 molecular weight marker, EZ Load ^™ 100 bp Molecular Ruler (trade name, manufactured by BIO RAD) was used.

  As a result, the gene fragment was confirmed at a size of about 250 bp in the sample containing genomic DNA of Neosartria grabra. On the other hand, gene fragments were not confirmed in samples containing genomic DNA of fungi other than Neosartria grabra. Therefore, by using a specific oligonucleotide pair, Neosartria glabra can be specifically detected and identified at the species level.

(5) Detection of Neosartria hiratsukae The fungi used for the evaluation of the effectiveness of the designed Nhi_F1 primer and Nhi_R1 primer, that is, Neosartria hiratsukae and other fungi, the strains listed in Table 3 were used. About these strains, stocks managed by the National Institute of Technology and Evaluation managed by the NBRC number, strains maintained by the Fungal Medicine Research Center of Chiba University School of Medicine and managed by the IFM number, The Centraalbureau voor Schimmelcultures Stocks stored and managed by the CBS number, and strains managed by the Fermentation Research Institute and managed by the IFO number were obtained and used.
Each strain was cultured under optimal conditions. The culture conditions were cultured at 25-30 ° C. for 3-5 days using a potato dextrose medium (trade name: Pearlcore potato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.).

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

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 0.5 μL of Nhi_F1 primer (20 pmol / μL) and Nhi_R1 primer (20 pmol / μL) were mixed. (μL) 0.5 μL was added to prepare a 25 μL PCR solution.
The PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR conditions are 30 cycles of (i) heat denaturation reaction at 98 ° C for 5 seconds, (ii) annealing reaction at 61 ° C for 5 seconds, and (iii) extension reaction at 72 ° C for 10 seconds for 1 cycle. went.

After PCR, 2 μL was taken from the PCR solution, mixed well with the loading buffer, and subjected to electrophoresis (135 V, 25 minutes) using a 2% agarose gel. After completion of electrophoresis, the agarose gel was stained with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen), and the presence or absence of the amplified DNA fragment was confirmed. The result is shown in FIG. 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 molecular weight marker, EZ Load ^™ 100 bp Molecular Ruler (trade name, manufactured by BIO RAD) was used.

  As a result, in the sample containing the genomic DNA of Neosartria hiratsukae, a gene fragment was confirmed with a size of about 300 bp. On the other hand, no gene fragment was confirmed in samples containing genomic DNA of fungi other than Neosartoria hiratsukae. Therefore, by using a specific oligonucleotide pair, Neosartria hiratsukae can be specifically detected and identified at the species level.

(6) Detection of Neosartria pseudofischeri Fungi used for evaluating the effectiveness of the designed Npf_F2 primer and Npf_R2 primer, that is, Neosartria pseudofischeri and other fungi include the strains described in Table 3 above. It was used.
Each strain was cultured under optimal conditions. The culture conditions were cultured at 25-30 ° C. for 3-5 days using a potato dextrose medium (trade name: Pearlcore potato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.).

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

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μL of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.) and 10 μL of sterile distilled water were mixed, 0.5 μL of Npf_F2 primer (20 pmol / μL) and Npf_R2 primer (20 pmol / μL) (μL) 0.5 μL was added to prepare a 25 μL PCR solution.
The PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR conditions were (i) 98 ° C for 5 seconds heat denaturation reaction, (ii) 61 ° C for 5 seconds annealing reaction, and (iii) 72 ° C for 10 seconds extension reaction for 1 cycle, 35 cycles. went.

After PCR, 2 μL was taken from the PCR solution, mixed well with the loading buffer, and subjected to electrophoresis (135 V, 25 minutes) using a 2% agarose gel. After completion of electrophoresis, the agarose gel was stained with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen), and the presence or absence of the amplified DNA fragment was confirmed. The result is shown in FIG. 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 molecular weight marker, EZ Load ^™ 100 bp Molecular Ruler (trade name, manufactured by BIO RAD) was used.

  As a result, the gene fragment was confirmed at a size of about 250 bp in the sample containing the genomic DNA of Neosartria pseudofischeri. On the other hand, gene fragments were not confirmed in samples containing genomic DNA of fungi other than Neosartoria pseudofischeri. Therefore, by using a specific oligonucleotide pair, Neosartria pseudofischer can be specifically detected and identified at the species level.

(7) Detection of Neosartria fischeri The strains listed in Table 2 above are used as fungi used for evaluating the effectiveness of the designed Nfi_F3 primer and Nfi_R3 primer, that is, Neosartria pseudofischeri and other fungi did.
Each strain was cultured under optimal conditions. The culture conditions were cultured at 25-30 ° C. for 3-5 days using a potato dextrose medium (trade name: Pearlcore potato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.).

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

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 0.5 μL of Nfi_F3 primer (20 pmol / μL) and Nfi_R3 primer (20 pmol / 20 μmol / μL) were mixed. (μL) 0.5 μL was added to prepare a 25 μL PCR solution.
The PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR conditions were (i) 98 ° C for 5 seconds heat denaturation reaction, (ii) 61 ° C for 5 seconds annealing reaction, and (iii) 72 ° C for 10 seconds extension reaction for 1 cycle, 35 cycles. went.

After PCR, 2 μL was taken from the PCR solution, mixed well with the loading buffer, and subjected to electrophoresis (135 V, 25 minutes) using a 2% agarose gel. After completion of electrophoresis, the agarose gel was stained with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen), and the presence or absence of the amplified DNA fragment was confirmed. The result is shown in FIG. 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 molecular weight marker, EZ Load ^™ 100 bp Molecular Ruler (trade name, manufactured by BIO RAD) was used.

  As a result, the gene fragment was confirmed to be about 350 bp in the sample containing genomic DNA of Neosartria fischeri. On the other hand, gene fragments were not confirmed in samples containing genomic DNA of fungi other than Neosartoria fischeri. Therefore, by using a specific oligonucleotide pair, Neosartria fischeri can be specifically detected and identified at the species level.

(8) Detection of Neosartria spinosa Fungi used to evaluate the effectiveness of the designed Nspc_1F primer and Nspc_1R primer, namely Neosartria spinosa (including Neosartria colana, Neosartria lasinosa and Neosartria Paulistensis) ) And other fungi, the strains listed in Tables 4 to 6 were used. These strains are stored by Chiba University School of Medicine, Center for Fungal Medicine and controlled by the Control Number, The Centraalbureau voor Schimmelcultures, stored by the CBS Number and controlled by the Fermentation Research Institute. Stocks managed by IFO number were obtained and used.
Each strain was cultured under optimal conditions. The culture conditions were cultured at 25-30 ° C. for 3-5 days using a potato dextrose medium (trade name: Pearlcore potato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.).

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

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 0.5 μL of Nspc_1F primer (20 pmol / μL) and Ngl_R1 primer (20 pmol / 20 μmol / μL) were mixed. (μL) 0.5 μL was added to prepare a 25 μL PCR solution.
The PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR conditions were (i) 97 ° C for 1 minute heat denaturation reaction, (ii) 60 ° C for 1 minute annealing reaction, and (iii) 72 ° C for 1 minute extension reaction for 1 cycle, 35 cycles. went.

After PCR, 2 μL was taken from the PCR solution, mixed well with the loading buffer, and subjected to electrophoresis (135 V, 25 minutes) using a 2% agarose gel. After completion of electrophoresis, the agarose gel was stained with SYBR Safe DNA gel stain in 1 × TAE (Invitrogen), and the presence or absence of the amplified DNA fragment was confirmed. The results are shown in FIGS. 10 shows an electrophoretic diagram for the fungal sample shown in Table 4, FIG. 11 shows an electrophoretic diagram for the fungal sample shown in Table 5, and FIG. 12 shows the fungal sample shown in Table 6. An electropherogram is shown. 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 molecular weight marker, EZ Load ^™ 100 bp Molecular Ruler (trade name, manufactured by BIO RAD) was used.

  As a result, a gene fragment having a size of about 250 bp was confirmed in a sample containing genomic DNA of Neosartoria spinosa, Neosartoria correana, Neosartoria lasiniosa, or Neosartoria Paulistensis. On the other hand, gene fragments were not confirmed in samples containing genomic DNA of fungi other than Neosartoria spinosa. Therefore, by using a specific oligonucleotide pair, Neosartria spinosa (including Neosartria coleana, Neosartria lasinosa, and Neosartria Paulistensis) can be specifically detected and identified at the species level it can.

  From the above results, by using the oligonucleotide of the present invention, it is possible to confirm the presence of a partial base sequence of the β-tubulin gene or calmodulin gene of the Neosartoria fungus, and the Neosartoria fungus is specific at the species level. It can be seen that it can be detected automatically. Therefore, according to the method of the present invention, it is possible to detect Neosartria fungi simply, quickly and specifically at the species level.

Claims (29)

An oligonucleotide pair consisting of the following oligonucleotides (a) and (b), an oligonucleotide pair consisting of the following (c) and (d) oligonucleotides, and an oligonucleotide pair consisting of the following (e) and (f) oligonucleotides: And at least one oligonucleotide pair selected from the group consisting of oligonucleotide pairs consisting of the following oligonucleotides (g) and (h) and oligonucleotide pairs consisting of the following oligonucleotides (i) and (j): Neosartorya (Neosartorya) genus comprises conducting the identification of fungal species level, Neosartorya (Neosartorya) genus detection method of a fungal Te.
(A) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, or one base is deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 1, and Neosartria grabra ( (Neosartorya glabra) can be used for detection at the species level (b) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 2 or a single base deleted in the nucleotide sequence set forth in SEQ ID NO: 2, substituted, inserted or added in and and Neosartorya glabra (Neosartorya glabra) oligonucleotides that can be used for detection at the species level (c) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3, or SEQ ID NO: 3 one base deletion in the nucleotide sequence set forth in replacement has been inserted or added and Neosartorya-Hiratsukae Neosartorya hiratsukae) 1 bases are deleted in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 4, represented by the nucleotide sequence set forth in the oligonucleotide (d) SEQ ID NO: 4 can be used to detect at the species level, substituted, inserted or added in and and Neosartorya-Hiratsukae (Neosartorya hiratsukae) oligonucleotides that can be used for detection at the species level (e) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5, or SEQ ID NO: 5 one base deletion in the nucleotide sequence set forth in substitution, insertion or addition has been provided and Neosartorya pseudotuberculosis fischeri (Neosartorya pseudofischeri) can be used to detect at the species level of oligonucleotide (f) SEQ ID NO: 6 To the oligonucleotide represented by the base sequence described in SEQ ID NO: 6, or One base deletion you are, substitutions, nucleotide sequence according to the oligonucleotides (g) SEQ ID NO: 7 that can be used to detect at the species level of inserted or added to and and Neosartorya pseudotuberculosis fischeri (Neosartorya pseudofischeri) in one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 7 is represented, used for the detection of at the species level of substitution, insertion or addition it has been provided and Neosartorya-fischeri (Neosartorya fischeri) Oligonucleotide (h) oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8, or one base is deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 8, and Neosartria · fischeri (Neosartorya fischeri) oligonucleotides (i) SEQ ID that can be used to detect at the species level Oligonucleotides represented by the nucleotide sequence set forth in issue 9, or one base deletion in the nucleotide sequence set forth in SEQ ID NO: 9, substitution, insertion or addition has been provided and Neosartorya-spinosa for (Neosartorya spinosa) Oligonucleotide that can be used for detection at the species level (j) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 10, or one base in the nucleotide sequence set forth in SEQ ID NO: 10 is deleted, substituted, inserted, or oligonucleotides can be used to detect at the species level of added to and and Neosartorya-spinosa (Neosartorya spinosa) The Neosartorya spp fungi is a Neosartorya Glove la, Neosartorya-Hiratsuka et, Neosartorya-Shudofishe Li, Neosartorya-Fishe Li及 beauty Neosartorya, Supino least one kind of service, claim 1 Symbol placement of the detection method.   The Neosartria spinosa includes Neosartorya spinosa, Neosartorya coreana, Neosartorya lacinosa and Neosartorya paulistensis as the same species, Item 3. The detection method according to Item 1 or 2. In order to perform identification, the partial base sequence of the β-tubulin gene and / or calmodulin gene of the Neosartoria fungus is amplified using the at least one oligonucleotide pair, and the presence or absence of an amplification product is confirmed. The detection method of any one of 1-3 . The detection method according to claim 4 , wherein the amplification reaction of the partial base sequence is performed by a polymerase chain reaction (PCR) method. The detection method according to claim 5 , wherein the polymerase chain reaction method is performed using the oligonucleotides (a) and (b) as nucleic acid primers to amplify a partial base sequence of the β-tubulin gene of Neosartoria grubura. . 6. The detection method according to claim 5 , wherein the polymerase chain reaction method is performed using the oligonucleotides (c) and (d) as nucleic acid primers to amplify a partial base sequence of the β-tubulin gene of Neosartoria hiratsukae. . The detection method according to claim 5 , wherein the polymerase chain reaction method is performed using the oligonucleotides (e) and (f) as nucleic acid primers to amplify a partial base sequence of a calmodulin gene of Neosartoria pseudofischerry. The detection method according to claim 5 , wherein the polymerase chain reaction method is performed using the oligonucleotides (g) and (h) as nucleic acid primers to amplify a partial base sequence of a calmodulin gene of Neosartria fischeri. The detection method according to claim 5 , wherein the polymerase chain reaction method is performed using the oligonucleotides (i) and (j) as nucleic acid primers to amplify a partial base sequence of a calmodulin gene of Neosartria spinosa. An oligonucleotide pair consisting of the following oligonucleotides (a) and (b), an oligonucleotide pair consisting of the following (c) and (d) oligonucleotides, and an oligonucleotide pair consisting of the following (e) and (f) oligonucleotides: And at least one oligonucleotide pair selected from the group consisting of oligonucleotide pairs consisting of the following oligonucleotides (g) and (h) and oligonucleotide pairs consisting of the following oligonucleotides (i) and (j): Te, the following (k) ~ to confirm the presence of a nucleic acid represented by any one of the nucleotide sequences of (t), Neosartorya (Neosartorya) genus detection methods fungi.
(A) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, or one base is deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 1, and Neosartria grabra ( (Neosartorya glabra) can be used for detection at the species level (b) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 2 or a single base deleted in the nucleotide sequence set forth in SEQ ID NO: 2, substituted, inserted or added in and and Neosartorya glabra (Neosartorya glabra) oligonucleotides that can be used for detection at the species level (c) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3, or SEQ ID NO: 3 one base deletion in the nucleotide sequence set forth in replacement has been inserted or added and Neosartorya-Hiratsukae Neosartorya hiratsukae) 1 bases are deleted in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 4, represented by the nucleotide sequence set forth in the oligonucleotide (d) SEQ ID NO: 4 can be used to detect at the species level, substituted, inserted or added in and and Neosartorya-Hiratsukae (Neosartorya hiratsukae) oligonucleotides that can be used for detection at the species level (e) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5, or SEQ ID NO: 5 one base deletion in the nucleotide sequence set forth in substitution, insertion or addition has been provided and Neosartorya pseudotuberculosis fischeri (Neosartorya pseudofischeri) can be used to detect at the species level of oligonucleotide (f) SEQ ID NO: 6 To the oligonucleotide represented by the base sequence described in SEQ ID NO: 6, or One base deletion you are, substitutions, nucleotide sequence according to the oligonucleotides (g) SEQ ID NO: 7 that can be used to detect at the species level of inserted or added to and and Neosartorya pseudotuberculosis fischeri (Neosartorya pseudofischeri) in one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 7 is represented, used for the detection of at the species level of substitution, insertion or addition it has been provided and Neosartorya-fischeri (Neosartorya fischeri) Oligonucleotide (h) oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8, or one base is deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 8, and Neosartria · fischeri (Neosartorya fischeri) oligonucleotides (i) SEQ ID that can be used to detect at the species level Oligonucleotides represented by the nucleotide sequence set forth in issue 9, or one base deletion in the nucleotide sequence set forth in SEQ ID NO: 9, substitution, insertion or addition has been provided and Neosartorya-spinosa for (Neosartorya spinosa) Oligonucleotide that can be used for detection at the species level (j) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 10, or one base in the nucleotide sequence set forth in SEQ ID NO: 10 is deleted, substituted, inserted, or detection β- tubulin gene partial nucleotide sequence or its complementary sequence according to an oligonucleotide (k) SEQ ID NO: 11 that can be used at the species level of added and are Neosartorya-spinosa (Neosartorya spinosa) (l) sequence 1 or several nucleotides are deleted in the nucleotide sequence set forth in ID NO: 11, substitution, insertion or addition has been provided and the (a)及(B) oligonucleotide pair consisting of an oligonucleotide of is hybridizable under stringent conditions, the nucleotide sequence can be used to detect at the species level of Neosartorya glabra (Neosartorya glabra), or its complementary sequence (m) A partial base sequence of the β-tubulin gene shown in SEQ ID NO: 12 or its complementary sequence (n), wherein one or several bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 12; (c) above and (d) an oligonucleotide pair consisting of an oligonucleotide of is hybridizable under stringent conditions, the nucleotide sequence can be used to detect at the species level of Neosartorya-Hiratsukae (Neosartorya hiratsukae), or Complementary sequence (o) Partial base sequence of calmodulin gene shown in SEQ ID NO: 13 or Complementary sequence (p) 1 or several nucleotides are deleted in the nucleotide sequence set forth in SEQ ID NO: 13, substitution, are inserted or added and the (e) and the oligonucleotide pair consisting of an oligonucleotide of (f) There are hybridizable under stringent conditions, calmodulin gene described in nucleotide sequence, or its complementary sequence (q) SEQ ID NO: 14 that can be used to detect at the species level of Neosartorya pseudotuberculosis fischeri (Neosartorya pseudofischeri) A partial base sequence or its complementary sequence (r) wherein one or several bases are deleted, substituted, inserted or added in the base sequence set forth in SEQ ID NO: 14 and from the oligonucleotides (g) and (h) above Can be hybridized under stringent conditions, and Neosartria Fischeri ( Neo sartorya fischeri ), a base sequence that can be used for detection at the species level, a complementary sequence (s) thereof, a partial base sequence of the calmodulin gene described in SEQ ID NO: 15 or a complementary sequence thereof (t) in the base sequence described in SEQ ID NO: 15 One or several bases are deleted, substituted, inserted or added and the oligonucleotide pair consisting of the oligonucleotides (i) and (j) above can hybridize under stringent conditions. Nucleotide sequence that can be used for detection at the species level of Spinosa ( Neosartorya spinosa ) or its complementary sequence The Neosartorya spp fungi is a Neosartorya Glove la, Neosartorya-Hiratsuka et, Neosartorya-Shudofishe Li, Neosartorya-Fishe Li及 beauty Neosartorya, Supino least one kind of service, claim 11. The detection method according to 11 .   The Neosartria spinosa includes Neosartorya spinosa, Neosartorya coreana, Neosartorya lacinosa and Neosartorya paulistensis as the same species, Item 13. The detection method according to Item 11 or 12. A part of the base sequence of any one of (k) to (t) is amplified using the at least one oligonucleotide pair, the presence or absence of an amplification product is confirmed, and any of the above (k) to (t) The detection method of any one of Claims 11-13 which confirms presence of the nucleic acid represented by the said base sequence. The detection method according to claim 14 , wherein an amplification reaction of a part of the base sequence is performed by a polymerase chain reaction (PCR) method. Performing the polymerase chain reaction using the oligonucleotides (a) and (b) as nucleic acid primers, amplifying a part of the base sequence (k) or (l), and identifying Neosartria grabra The detection method according to claim 15 , which is performed. Performing the polymerase chain reaction using the oligonucleotides (c) and (d) as nucleic acid primers, amplifying a part of the base sequence (m) or (n), and identifying Neosartria hiratsukae The detection method according to claim 15 , which is performed. The polymerase chain reaction method is carried out using the oligonucleotides (e) and (f) as nucleic acid primers, a part of the base sequence (o) or (p) is amplified, and Neosartoria pseudofischer The detection method according to claim 15 , wherein identification is performed. Performing the polymerase chain reaction using the oligonucleotides (g) and (h) as nucleic acid primers, amplifying a part of the base sequence (q) or (r), and identifying Neosartria fischeri The detection method according to claim 15 , which is performed. Performing the polymerase chain reaction using the oligonucleotides (i) and (j) as nucleic acid primers, amplifying a part of the base sequence (s) or (t), and identifying Neosartria spinosa The detection method according to claim 15 , which is performed. The following (a) and (b) oligonucleotides, the following (c) and (d) oligonucleotides, the following (e) and (f) oligonucleotides, the following (g) and (h) oligonucleotides, or An oligonucleotide pair consisting of the oligonucleotides (i) and (j).
(A) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, or one base is deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 1, and Neosartria grabra ( (Neosartorya glabra) can be used for detection at the species level (b) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 2 or a single base deleted in the nucleotide sequence set forth in SEQ ID NO: 2, substituted, inserted or added in and and Neosartorya glabra (Neosartorya glabra) oligonucleotides that can be used for detection at the species level (c) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3, or SEQ ID NO: 3 one base deletion in the nucleotide sequence set forth in replacement has been inserted or added and Neosartorya-Hiratsukae Neosartorya hiratsukae) 1 bases are deleted in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 4, represented by the nucleotide sequence set forth in the oligonucleotide (d) SEQ ID NO: 4 can be used to detect at the species level, substituted, inserted or added in and and Neosartorya-Hiratsukae (Neosartorya hiratsukae) oligonucleotides that can be used for detection at the species level (e) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5, or SEQ ID NO: 5 one base deletion in the nucleotide sequence set forth in substitution, insertion or addition has been provided and Neosartorya pseudotuberculosis fischeri (Neosartorya pseudofischeri) can be used to detect at the species level of oligonucleotide (f) SEQ ID NO: 6 To the oligonucleotide represented by the base sequence described in SEQ ID NO: 6, or One base deletion you are, substitutions, nucleotide sequence according to the oligonucleotides (g) SEQ ID NO: 7 that can be used to detect at the species level of inserted or added to and and Neosartorya pseudotuberculosis fischeri (Neosartorya pseudofischeri) in one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 7 is represented, used for the detection of at the species level of substitution, insertion or addition it has been provided and Neosartorya-fischeri (Neosartorya fischeri) Oligonucleotide (h) oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8, or one base is deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 8, and Neosartria · fischeri (Neosartorya fischeri) oligonucleotides (i) SEQ ID that can be used to detect at the species level Oligonucleotides represented by the nucleotide sequence set forth in issue 9, or one base deletion in the nucleotide sequence set forth in SEQ ID NO: 9, substitution, insertion or addition has been provided and Neosartorya-spinosa for (Neosartorya spinosa) Oligonucleotide that can be used for detection at the species level (j) Oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 10, or one base in the nucleotide sequence set forth in SEQ ID NO: 10 is deleted, substituted, inserted, or oligonucleotides can be used to detect at the species level of added to and and Neosartorya-spinosa (Neosartorya spinosa) 23. The oligonucleotide pair of claim 21 , wherein the oligonucleotide is a nucleic acid primer. Said (a) and oligonucleotide (b) is, by polymerase chain reaction (PCR) to amplify a portion of the nucleotide sequence of the following (k) and / or (l), Neosartorya glabra (Neosartorya glabra) 23. The oligonucleotide pair of claim 21 or 22 , which can function as a nucleic acid primer for specific detection at the species level.
(K) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 11 or its complementary sequence (l) one or several bases are deleted, substituted, inserted or added in the base sequence described in SEQ ID NO: 11 And a nucleotide sequence that can be hybridized under stringent conditions and can be used for detection at the species level of Neosartorya glabra. Or its complementary sequence Oligonucleotides of the (c) and (d), part of the nucleotide sequence of the polymerase chain reaction following by (PCR) method (m) and / or (n) can amplify the Neosartorya-Hiratsukae (Neosartorya hiratsukae) 23. An oligonucleotide pair according to claim 21 or 22 , which can function as a nucleic acid primer for specific detection at the species level.
(M) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 12 or its complementary sequence (n) one or several bases are deleted, substituted, inserted or added in the base sequence described in SEQ ID NO: 12 and which and (c) above and (d) an oligonucleotide pair consisting of an oligonucleotide of is hybridizable under stringent conditions, the nucleotide sequence can be used to detect at the species level of Neosartorya-Hiratsukae (Neosartorya hiratsukae) Or its complementary sequence Wherein (e) and oligonucleotide (f) is, by polymerase chain reaction (PCR) to amplify a portion of the nucleotide sequence of the following (o) and / or (p), Neosartorya pseudotuberculosis fischeri (Neosartorya pseudofischeri 23. The oligonucleotide pair according to claim 21 or 22 , which can function as a nucleic acid primer for specifically detecting at a species level.
(O) a partial base sequence of the calmodulin gene described in SEQ ID NO: 13 or a complementary sequence thereof (p) one or several bases are deleted, substituted, inserted or added in the base sequence described in SEQ ID NO: 13; (e) above and the oligonucleotide pair consisting of an oligonucleotide of (f) is capable of hybridizing under stringent conditions, the nucleotide sequence can be used to detect at the species level of Neosartorya pseudotuberculosis fischeri (Neosartorya pseudofischeri), Or its complementary sequence The oligonucleotide (g) and (h), a part of the following nucleotide sequence by polymerase chain reaction (PCR) (q) and / or (r) can amplify the Neosartorya-fischeri (Neosartorya fischeri) 23. An oligonucleotide pair according to claim 21 or 22 , which can function as a nucleic acid primer for specific detection at the species level.
(Q) a partial base sequence of the calmodulin gene described in SEQ ID NO: 14 or a complementary sequence thereof (r) one or several bases are deleted, substituted, inserted or added in the base sequence described in SEQ ID NO: 14; An oligonucleotide pair comprising the oligonucleotides (g) and (h) above can be hybridized under stringent conditions, and can be used for detection at the species level of Neosartorya fischeri , or a sequence thereof Complementary sequence The oligonucleotides (i) and (j) can amplify a part of the base sequence (s) and / or (t) below by the polymerase chain reaction (PCR) method, and Neosartorya spinosa ( Neosartorya spinosa ) 23. An oligonucleotide pair according to claim 21 or 22 , which can function as a nucleic acid primer for specific detection at the species level.
(S) a partial base sequence of the calmodulin gene shown in SEQ ID NO: 15 or a complementary sequence thereof (t) one or several bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 15; An oligonucleotide pair comprising the oligonucleotides (i) and (j) above can be hybridized under stringent conditions, and can be used for detection at the species level of Neosartorya spinosa , or a sequence thereof Complementary sequence   The Neosartria spinosa includes Neosartorya spinosa, Neosartorya coreana, Neosartorya lacinosa and Neosartorya paulistensis as the same species, Item 28. The oligonucleotide pair according to any one of Items 21, 22, and 27. Neosartorya (Neosartorya) genus fungi detection kit comprising oligonucleotide pair of any one of claims 21-28.