JP5654265B2 - Method for detecting fungi belonging to the genus Bisoclamis - Google Patents

Description

The present invention relates to a method for detecting fungi belonging to the genus Byssochlamys .

Heat-resistant fungi (fungi) are widely distributed in nature and propagate on agricultural products such as vegetables and fruits, and contaminate foods and drinks made from these agricultural products. Moreover, heat-resistant fungi form ascospores having higher heat resistance in the life cycle than other normal fungi. For example, even when an acid beverage is heat sterilized, 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, Byssochlamys Faruba (Byssochlamys fulva), Byssochlamys nivea (Byssochlamys nivea), Byssochlamys Ragankurarie (Byssochlamys lagunculariae ) and heat fungi are known belonging to Byssochlamys (Byssochlamys) genus such as Byssochlamys Zorerunia (Byssochlamys zollernia). In order to prevent accidents caused by heat-resistant fungi in foods and drinks and their raw materials, it is important to detect heat-resistant fungi belonging to the genus Bisoclamis. In addition, B. clamis species have lower oxygen demand than other fungi, can grow even under low oxygen partial pressure of beverages and the like, and synthesize pectin-degrading enzymes to cause changes in food properties. For this reason, Bisoclamis species are wary of important harmful bacteria in the food industry.

  A conventional method for detecting heat-resistant fungi is performed by observing morphological characteristics after cell culture. However, in this method, it is necessary to continue the culture until morphological characteristics are generated, and therefore a long period of about 14 days or more is required at the shortest. In addition, since morphological identification requires extremely high expertise, it is impossible to deny the risk that identification results differ depending on the judge. Furthermore, there are cases where microbial cells damaged by heating, chemicals, etc. lose their morphogenic ability, and these microbial cells do not form characteristic morphologies even after long-term culture, so the reliability of the identification results is problematic. there were. The long time required for detection of heat-resistant fungi is not always satisfactory from the viewpoints of hygiene management of food and drink, ensuring freshness of raw materials, restrictions on distribution, and the like. Therefore, establishment of a detection / identification method that solves the problems of rapidity and reliability is required.

As a rapid and reliable detection method for heat-resistant fungi belonging to the genus Bisoclamis, an amplification method (for example, PCR method or LAMP method) targeting a specific base sequence of a gene is known (for example, Patent Document 1). To 3). However, these methods identify fungi belonging to the genus Bisoclamis at the genus level, and the fungi detected by these methods can only be identified as Byssochlamys sp. Further studies are needed to identify fungi belonging to the species at the species level.
Therefore, in the field of hygiene management of foods and beverages that require quick and reliable detection of heat-resistant fungi belonging to the genus Bisocramis, it is required to quickly detect fungi belonging to the genus Bisoclamis at the species level. Furthermore, fungi belonging to the genus Bisoclamis differ in their ability to produce mold venoms depending on the species, and for example, it is known that patulin, which is a type of mold venom produced by fungi, is produced only by Bisocramis nivea ( Non-patent document 1 etc.). For this reason, it is necessary to identify the fungi belonging to the genus Bisoclamis at the species level.

JP 2009-284832 A JP 2010-004876 A JP 2010-004880 A

Samson et al., Persoonia 22, 2009, p. 14-27

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

  In view of the above problems, the present inventors have intensively studied a method for specifically detecting and identifying fungi belonging to the genus Bisoclamis at the species level. As a result, it was found that fungi belonging to the genus Bisocramis can be specifically and rapidly detected at the species level by using a specific oligonucleotide capable of hybridizing to the β-tubulin gene of fungi belonging to the genus Bisoclamis. . The present invention has been completed based on these findings.

The present invention includes the following oligonucleotide pairs (a) and (b), oligonucleotide pairs (c) and (d) below, oligonucleotide pairs (e) and (f) below, and (g) and ( h) detection of fungi belonging to the genus Bisoclamis, comprising the step of identifying at the species level the fungi belonging to the genus Bisoclamis using at least one oligonucleotide pair selected from the group consisting of the oligonucleotide pairs of h) Regarding the method.
(A) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, or one or several bases deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 1 and belonging to the genus Bisoclamis Oligonucleotide that can be used for detection at the species level of the fungus to which it belongs (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 , An oligonucleotide which is substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Bisoclamis (c) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3, or the sequence set forth in SEQ ID NO: 3 Fungi belonging to the genus Bisoclamis with one or several bases deleted, substituted, inserted or added in the base sequence of Oligonucleotide that can be used for detection at the species level (d) An oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 4, or one or several bases deleted or substituted in the nucleotide sequence set forth in SEQ ID NO: 4, Oligonucleotide inserted or added and usable for detection at the species level of fungi belonging to the genus Bisoclamis (e) oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5, or the nucleotide sequence set forth in SEQ ID NO: 5 In which one or several bases are deleted, substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Bisoclamis (f) represented by the base sequence set forth in SEQ ID NO: 6 Or one or several bases in the nucleotide sequence set forth in SEQ ID NO: 6 is deleted, substituted, or inserted Is an oligonucleotide that can be used for detection at the species level of fungi belonging to the genus Bisoclamis (g) in the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7, or in the nucleotide sequence set forth in SEQ ID NO: 7 Oligonucleotide which has one or several bases deleted, substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Bisoclamis (h) represented by the base sequence set forth in SEQ ID NO: 8 Oligonucleotide or oligonucleotide in which one or several bases are deleted, substituted, inserted or added in the base sequence described in SEQ ID NO: 8 and can be used for detection at the species level of fungi belonging to the genus Bisoclamis

The present invention also provides the oligonucleotide pairs (a) and (b), the oligonucleotide pairs (c) and (d), the oligonucleotide pairs (e) and (f), and the (g) And (h) using at least one oligonucleotide pair selected from the group consisting of oligonucleotide pairs, confirming the presence of the nucleic acid represented by any one of the following base sequences (i) to (p) The present invention relates to a method for detecting a fungus belonging to the genus Bisoclamis.
(I) a partial base sequence of the β-tubulin gene shown in SEQ ID NO: 9 or its complementary sequence (j) one or several bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 9 And a base sequence that can be used for detecting Byssochlamys fulva , or a complementary sequence (k) thereof, a partial base sequence of the β-tubulin gene described in SEQ ID NO: 10 or its complementary sequence (l) in SEQ ID NO: 10 A nucleotide sequence in which one or several bases are deleted, substituted, inserted or added in the described nucleotide sequence and can be used for detection of Byssochlamys nivea , or its complementary sequence (m) described in SEQ ID NO: 11 A partial base sequence of the β-tubulin gene or its complementary sequence (n) in the base sequence set forth in SEQ ID NO: 11, wherein one or several bases are deleted, substituted, inserted or Nucleotide sequences can be used to detect pressure has been provided and Byssochlamys Ragankurarie (Byssochlamys lagunculariae), or its complementary sequence (o) SEQ ID NO: 12 beta-tubulin gene partial nucleotide sequence or its complementary sequence according to (p) SEQ ID NO: 12. A nucleotide sequence in which one or several bases are deleted, substituted, inserted or added in the nucleotide sequence described in 12, and can be used for detecting Byssochlamys zollernia , or a complementary sequence thereof

  The present invention also relates to the oligonucleotide for detecting a fungus belonging to the genus Bisoclamis, which is any one of the oligonucleotides (a) to (h) and can be used as a detection oligonucleotide at the species level of the fungus belonging to the genus Bisoclamis. Relates to nucleotides.

  The present invention also provides the oligonucleotide pair (a) and (b), the oligonucleotide pair (c) and (d), the oligonucleotide pair (e) and (f), or the above (g). And (h) an oligonucleotide pair for detecting fungi belonging to the genus Bisoclamis, represented by the oligonucleotide pair.

  The present invention also relates to a fungus detection kit belonging to the genus Bisoclamis comprising the oligonucleotide pair.

  The detection method of the present invention can quickly detect fungi belonging to the genus Bisoclamis, which is 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.

Positional relationship of the base sequence recognized by the oligonucleotide of the present invention in the base sequence of β-tubulin gene of fungi belonging to the genus Bisoclamis and other heat-resistant fungi, and the base sequence of β-tubulin gene of fungi belonging to the genus Bisoclamis FIG. Positional relationship of the base sequence recognized by the oligonucleotide of the present invention in the base sequence of β-tubulin gene of fungi belonging to the genus Bisoclamis and other heat-resistant fungi, and the base sequence of β-tubulin gene of fungi belonging to the genus Bisoclamis (A continuation of FIG. 1). In the Example, it is an electrophoretic diagram of the PCR product amplified using the oligonucleotide of (a) and (b) of this invention. In the Example, it is an electrophoretic diagram of the PCR product amplified using the oligonucleotide of (a) and (b) of this invention. In the Example, it is an electrophoretic diagram of the PCR product amplified using the oligonucleotide of (c) and (d) of this invention. In the Example, it is an electrophoretic diagram of the PCR product amplified using the oligonucleotide of (c) and (d) of this invention. FIG. 2 is an electrophoretogram of PCR products amplified using the oligonucleotides (e) and (f) of the present invention in Examples. FIG. 3 is an electrophoretogram of PCR products amplified using the oligonucleotides (g) and (h) of the present invention in Examples.

  The present invention relates to a specific partial base sequence of a β-tubulin gene of a fungus belonging to the genus Bisoclamis, that is, each bacterial species of a fungus belonging to the genus Bisoclamis present in the β-tubulin gene sequence of a fungus belonging to the genus Bisoclamis. A method for identifying and detecting fungi belonging to the genus Bisoclamis at the species level by identifying fungi belonging to the genus Bisoclamis using oligonucleotides that can hybridize to specific regions (variable regions) under stringent conditions It is. Here, the “variable region” is a region in which base mutations tend to accumulate in the β-tubulin gene, and the base sequence of this region varies greatly among other fungal species. “Β-tubulin” is a protein constituting a microtubule, and “β-tubulin gene” is a gene encoding β-tubulin. Here, “stringent conditions” include, for example, the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell., Cold Spring Harbor Laboratory Press], for example, 6 × SSC (1 × SSC composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. And the conditions of constant temperature for 8 to 16 hours and hybridization.

The term “fungi belonging to the genus Bisoclamis” in the present invention means an irregular cyst fungus belonging to the family Trichocomaceae . Fungi belonging to the genus Bisoclamis are heat-resistant fungi that form viable ascospores even after heat treatment at 75 ° C. for 30 minutes. Examples of fungi belonging to the genus Bisoclamis include bisoclamis falba, bisoclamis nivea, bisoclamis laganclarie and bisoclamis zorrenia.

The detection method of the present invention is characterized by using an oligonucleotide pair represented by a nucleic acid corresponding to a base sequence of a specific (variable) region in a β-tubulin gene of a fungus belonging to the genus Bisoclamis.
As shown in FIGS. 1 and 2, the inventors determined the base sequences of β-tubulin genes of various fungi including the genus Bisoclamis, and the genetic distance between the fungal species and the homology of the base sequences. Was analyzed. That is, the base sequence of β-tubulin 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, a region (variable region) having a unique base sequence depending on the species was found, which is highly conserved within the same species but low in conservation of the base sequence between different species. In this variable region, fungi belonging to the genus Bisoclamis have a species-specific base sequence. Therefore, this region is useful as a genetic index for identifying and identifying fungi belonging to the genus Bisoclamis at the species level.

The detection method of the present invention comprises the following oligonucleotide pairs (a) and (b), oligonucleotide pairs (c) and (d) below, oligonucleotide pairs (e) and (f) below, and (g ) And (h) at least one oligonucleotide pair selected from the group consisting of oligonucleotide pairs is used.
(A) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, or one or several bases deleted, substituted, inserted or added in the nucleotide sequence set forth in SEQ ID NO: 1 and belonging to the genus Bisoclamis Oligonucleotide that can be used for detection at the species level of the fungus to which it belongs (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 , An oligonucleotide which is substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Bisoclamis (c), an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 3, or Fungi belonging to the genus Bisoclamis with one or several bases deleted, substituted, inserted or added in the base sequence of Oligonucleotide that can be used for detection at the species level (d) An oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 4, or one or several bases deleted or substituted in the nucleotide sequence set forth in SEQ ID NO: 4, Oligonucleotide inserted or added and usable for detection at the species level of fungi belonging to the genus Bisoclamis (e) oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5, or the nucleotide sequence set forth in SEQ ID NO: 5 In which one or several bases are deleted, substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Bisoclamis (f) represented by the base sequence set forth in SEQ ID NO: 6 Or one or several bases in the nucleotide sequence set forth in SEQ ID NO: 6 is deleted, substituted, or inserted Is an oligonucleotide that can be used for detection at the species level of fungi belonging to the genus Bisoclamis (g) in the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7, or in the nucleotide sequence set forth in SEQ ID NO: 7 Oligonucleotide which has one or several bases deleted, substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Bisoclamis (h) represented by the base sequence set forth in SEQ ID NO: 8 Oligonucleotide or oligonucleotide in which one or several bases are deleted, substituted, inserted or added in the base sequence described in SEQ ID NO: 8 and can be used for detection at the species level of fungi belonging to the genus Bisoclamis

Of the fungi belonging to the genus Bisoclamis, the variable region of the β-tubulin gene of Bisoclamis falba will be described based on the base sequence described in FIGS. In addition, the base sequence described in SEQ ID NO: 9 shows a partial base sequence of the β-tubulin gene of Bisoclamis farva CBS146.48 strain.
The oligonucleotide (a) (also referred to herein as B.fulva1F) and the oligonucleotide (b) (also referred to herein as B.fulva1R) are as shown in FIGS. The oligonucleotide is capable of hybridizing under stringent conditions to the region from position 116 to position 135 and the region from position 330 to position 348 of the base sequence set forth in SEQ ID NO: 9. These regions are variable regions with low conserved base sequences between fungal species, and regions with particularly low conserved base sequences between B. clamis falba and other fungal species, and the base sequences of these regions The present inventors have found that is a base sequence inherently possessed by Bisoclamis falba. Therefore, using the oligonucleotides (a) and (b), Bisoclamis falba 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 to bisoclamis falba. Identification by PCR reaction using primers is possible. In addition, a probe designed to hybridize in this region with 10 or more bases specifically hybridizes with bisoclamis farba, so that bisoclamis farba can be identified by the presence or absence of hybridization.

In the present invention, the presence of a nucleic acid (preferably the variable region) represented by the following base sequence (i) or (j) is confirmed using the oligonucleotide pair (a) and (b). Is preferred.
(I) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 9 or a complementary sequence thereof (j) one or several (preferably 1-25, more preferably 1) in the base sequence described in SEQ ID NO: 9 -20, more preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases are deleted, substituted, inserted or added and can be used for the detection of bisoclamis falba. Base sequence or its complementary sequence

Among the fungi belonging to the genus Bisoclamis, the variable region of the β-tubulin gene of Bisocramis nivea will be described based on the base sequence described in FIGS. In addition, the base sequence described in SEQ ID NO: 10 represents a partial base sequence of the β-tubulin gene of B. clamis nivea CBS100.11 strain.
The oligonucleotide (c) (also referred to herein as B.nivea1F) and the oligonucleotide (d) (also referred to herein as B.nivea1R) are respectively as shown in FIGS. The oligonucleotide is capable of hybridizing under stringent conditions to the region from position 43 to position 62 and the region from position 343 to position 362 of the base sequence set forth in SEQ ID NO: 10. These regions are variable regions with low conserved base sequences between fungal species, and regions with particularly low conserved base sequences between B. clamis nivea and other fungal species, and the base sequences of these regions The present inventors have found that is a base sequence inherently possessed by Bisocramis nivea. Therefore, using the oligonucleotides (c) and (d), B. clamis nivea 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 bisocramis nivea. Identification by PCR reaction using primers is possible. In addition, a probe designed to hybridize in this region with 10 or more bases specifically hybridizes with bisoclamis nivea, so that bisoclamis nivea can be identified by the presence or absence of hybridization.

In the present invention, using the oligonucleotide pair (c) and (d), the presence of a nucleic acid (preferably the variable region) represented by the base sequence (k) or (l) below is confirmed. Is preferred.
(K) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 10 or a complementary sequence thereof (1) one or several (preferably 1-25, more preferably 1) in the base sequence described in SEQ ID NO: 10 -20, more preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases have been deleted, substituted, inserted or added and can be used to detect Bisoclamis nivea. Base sequence or its complementary sequence

Among the fungi belonging to the genus Bisoclamis, the variable region of the β-tubulin gene of Bisoclamis laganclarie will be described based on the base sequence described in FIGS. In addition, the base sequence described in SEQ ID NO: 11 represents a partial base sequence of the β-tubulin gene of Bisoclamis laganclarie strain CBS373.70.
The oligonucleotide (e) (also referred to herein as B.lag1F) and the oligonucleotide (f) (also referred to herein as B.lag1R) are as shown in FIGS. The oligonucleotide is capable of hybridizing under stringent conditions to the region from position 90 to position 109 and the region from position 468 to position 487 in the base sequence set forth in SEQ ID NO: 11. These regions are variable regions with low conserved base sequences between fungal species, and are regions with particularly low conserved base sequences between B. clam laganclarie and other fungal species, and the base sequences of these regions The present inventors have found that is a base sequence inherently possessed by Bisoclamis Laganclarie. Therefore, using the oligonucleotides (e) and (f), B. clamislaganclari 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 to B. clamis laganclare. Identification by PCR reaction using primers is possible. In addition, a probe designed to hybridize in this region with 10 or more bases specifically hybridizes with B. clamis laganclare, so that it is possible to identify B. clamis laganclarie by the presence or absence of hybridization.

In the present invention, the presence of the nucleic acid (preferably the variable region) represented by the base sequence (m) or (n) below is confirmed using the oligonucleotide pair (e) and (f) above. Is preferred.
(M) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 11 or its complementary sequence (n) one or several (preferably 1 to 25, more preferably 1 to 25) base sequences described in SEQ ID NO: 11 20 bases, more preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases that have been deleted, substituted, inserted or added and can be used for the detection of Bisoclamis laganclarie Sequence or its complementary sequence

Among the fungi belonging to the genus Bisoclamis, the variable region of the β-tubulin gene of Bisocramis zolernia will be described based on the base sequence described in FIGS. In addition, the base sequence described in SEQ ID NO: 12 shows a partial base sequence of the β-tubulin gene of Bisochloris zolernia CBS345.70 strain.
The oligonucleotide (g) (also referred to herein as B.zol1F) and the oligonucleotide (h) (also referred to herein as B.zol1R) are as shown in FIGS. The oligonucleotide is capable of hybridizing under stringent conditions to the region from position 36 to position 55 and the region from position 319 to position 338 of the base sequence set forth in SEQ ID NO: 12. These regions are variable regions with low conserved base sequences between fungal species, and regions with particularly low conserved base sequences between B. clamis zollernia and other fungal species, and the base sequences of these regions The present inventors have found that is a base sequence inherently possessed by Bisoclamis zolernia. Therefore, using the oligonucleotides (g) and (h), Bisochloris zollernia 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 bisochloris zolernia. Identification by PCR reaction using primers is possible. In addition, a probe designed to hybridize in this region with 10 or more bases specifically hybridizes with bisoclamis zorelnia, so that it is possible to identify bisoclamis zorelnia by the presence or absence of hybridization.

In the present invention, the presence of the nucleic acid (preferably the variable region) represented by the base sequence (o) or (p) below is confirmed using the oligonucleotide pair (g) and (h) above. Is preferred.
(O) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 12 or a complementary sequence thereof (p) one or several (preferably 1 to 25, more preferably 1 to 25) base sequences described in SEQ ID NO: 12 20 bases, more preferably 1-15, particularly preferably 1-10, most preferably 1-5) bases are deleted, substituted, inserted or added and can be used for the detection of bisoclamis zolernia Sequence or its complementary sequence

The oligonucleotides (a) to (h) used in the detection method of the present invention are preferably oligonucleotides represented by the base sequences described in SEQ ID NOs: 1 to 8. Further, the detection oligonucleotide of the present invention may be 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 8, The homogeneity is more preferably 80% or more, the homology is more preferably 90% or more, and the homology is particularly preferably 95% or more. In addition, the oligonucleotide used in the detection method of the present invention has one or several oligonucleotides (preferably 1 to 5, more preferably 1 to 4, more preferably 1) in the base sequence described in any of SEQ ID NOs: 1 to 8. 1 to 3, more preferably 1 to 2, particularly preferably 1) of which is deleted, substituted, inserted or added, and can be used for detection at the species level of fungi belonging to the genus Bisoclamis It may be an oligonucleotide. Further, an appropriate base sequence may be added to the oligonucleotides (a) to (h).
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 excise directly from a gene of a fungus belonging to the genus Bisoclamis using a restriction enzyme or the like, or after cloning and isolating and purifying the gene and then excising it 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 on the method of confirming the presence of the base sequence of the β-tubulin gene of fungi belonging to the genus Bisocramis using the oligonucleotide pair, and identifying the fungus belonging to the genus Bisoclamis at the species level. It can be carried out by a commonly used genetic engineering technique such as a digestion method, a PCR method or a LAMP method.

  The oligonucleotides (a) to (h) used in the detection method of the present invention can be used as nucleic acid primers and nucleic acid probes.

The nucleic acid probe can be prepared by labeling the oligonucleotide with a label. The label is not particularly limited, and usual labels such as radioactive substances, enzymes, fluorescent substances, luminescent substances, antigens, haptens, enzyme substrates, insoluble carriers and the like can be used. The labeling method may be end labeling, labeling in the middle of the sequence, or labeling on a sugar, phosphate group, or base moiety. Since the nucleic acid probe specifically hybridizes with a part of the variable region of the β-tubulin gene of a fungus belonging to the genus Bisoclamis, it is possible to quickly and easily detect the fungus belonging to the genus Bisoclamis in the subject. 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. In this way, fungi belonging to the genus Bisoclamis can be detected. 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 fungi belonging to the genus Bisoclamis at the species level, the oligonucleotide pairs (a) and (b), the oligonucleotide pairs (c) and (d), (e It is preferable to perform hybridization using the oligonucleotide pair of () and (f) and / or the oligonucleotide pair of (g) and (h) above as a nucleic acid probe. In order to detect bisoclamis falba, it is preferable to use the oligonucleotide pair (a) and (b) as a nucleic acid probe. In order to detect B. clamis nivea, it is preferable to use the oligonucleotide pair (c) and (d) as a nucleic acid probe. In order to detect B. clamis laganclare, it is preferable to use the oligonucleotide pairs (e) and (f) as nucleic acid probes. In order to detect bisoclamis zolernia, the oligonucleotide pairs (g) and (h) are preferably used as nucleic acid probes.

  In order to detect fungi belonging to the genus Bisoclamis in a specimen at the species level, the oligonucleotide pairs (a) and (b), the oligonucleotide pairs (c) and (d), (e) and The oligonucleotide pair of (f) and / or the oligonucleotide pair of (g) and (h) above is labeled as a nucleic acid probe, and the resulting nucleic acid probe is hybridized with DNA or RNA, and the hybridized probe The label may be detected by an appropriate detection method. Since the above-mentioned nucleic acid probe specifically hybridizes with the variable region of the β-tubulin gene of fungi belonging to the genus Bisocramis, it is possible to quickly and easily detect the fungus belonging to the genus Bisocramis in the specimen 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 fungi belonging to the genus Bisoclamis, 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, it is preferable to use the PCR method from the viewpoint of rapidity and simplicity.
In the present invention, a case where a fungus belonging to the genus Bisoclamis is detected by performing an amplification reaction by PCR will be described.

In the present invention, it is preferable to use the oligonucleotide pair of (a) and (b) as a nucleic acid primer in order to detect bisoclamis farva, and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 and It is more preferable to use an oligonucleotide represented by the base sequence described in SEQ ID NO: 2 as a nucleic acid primer.
The oligonucleotide pair (a) and (b) can be an oligonucleotide pair for detecting a fungus belonging to the genus Bisoclamis (specifically, Bisoclamis falba).

In the present invention, it is preferable to use the oligonucleotide pair (c) and (d) as a nucleic acid primer in order to detect B. clamis nivea, and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 and It is more preferable to use an oligonucleotide represented by the base sequence shown in SEQ ID NO: 4 as a nucleic acid primer.
The oligonucleotide pairs (c) and (d) can be used as oligonucleotide pairs for detecting fungi belonging to the genus Bisocramis (specifically, Bisocramis nivea).

In the present invention, it is preferable to use the oligonucleotide pair of (e) and (f) as a nucleic acid primer in order to detect B. clamis laganclarie, and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 and It is more preferable to use the oligonucleotide represented by the base sequence described in SEQ ID NO: 6 as a nucleic acid primer.
The oligonucleotide pair (e) and (f) can be an oligonucleotide pair for detecting a fungus belonging to the genus Bisoclamis (specifically, Bisocramis laganclarie).

In the present invention, it is preferable to use the oligonucleotide pair of (g) and (h) as a nucleic acid primer in order to detect bisoclamis zolernia, and the oligonucleotide represented by the base sequence described in SEQ ID NO: 7 and It is more preferable to use the oligonucleotide represented by the base sequence shown in SEQ ID NO: 8 as a nucleic acid primer.
The oligonucleotide pairs (g) and (h) can be used as oligonucleotide pairs for detecting fungi belonging to the genus Bisoclamis (specifically, Bisocramis zolernia).

The conditions for the PCR reaction are not particularly limited as long as the target DNA fragment can be amplified to a detectable level.
As a preferable example of the PCR reaction conditions, for example, in the case where the PCR method is performed using the oligonucleotide pairs (a) and (b) as the nucleic acid primers, a heat denaturation reaction in which a double-stranded DNA is converted to a single strand is performed. An annealing reaction for hybridizing the primer pair to the single-stranded DNA is performed at 50 to 63 ° C. for about 60 seconds, and an extension reaction for allowing the DNA polymerase to act is performed at about 72 ° C. for about 60 seconds. These are defined as one cycle for about 30 to 35 cycles. In addition, when performing the PCR method using the oligonucleotide pair (c) and (d) as a nucleic acid primer, a heat denaturation reaction for converting a double-stranded DNA into a single strand is performed at 95 to 98 ° C. for 10 to 60 seconds, An annealing reaction for hybridizing the primer pair to the single-stranded DNA is performed at 50 to 60 ° C. for about 60 seconds, an extension reaction for allowing DNA polymerase to act is performed at about 72 ° C. for about 60 seconds, and these are defined as one cycle. Perform 30-35 cycles. 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 10 to 60 seconds, An annealing reaction for hybridizing the primer pair to the single-stranded DNA is performed at 50 to 63 ° C. for about 60 seconds, an extension reaction for allowing DNA polymerase to act is performed at about 72 ° C. for about 60 seconds, and these are defined as one cycle. Perform 30-35 cycles. When performing the PCR method using the oligonucleotide pairs (g) and (h) as a nucleic acid primer, a heat denaturation reaction for converting double-stranded DNA into a single strand is performed at 95 to 98 ° C. for 10 to 60 seconds, An annealing reaction for hybridizing the primer pair to the single-stranded DNA is performed at 50 to 63 ° C. for about 60 seconds, an extension reaction for allowing DNA polymerase to act is performed at about 72 ° C. for about 60 seconds, and these are defined as one cycle. Perform 30-35 cycles.

When the specimen contains a fungus belonging to the genus Bisoclamis, a DNA fragment having a specific size is obtained by performing a PCR reaction using the oligonucleotide pair of the present invention as a primer set and performing electrophoresis on the obtained PCR reaction product. Amplification is observed.
Specifically, when Bisoclamis phalum is included in the specimen, a DNA fragment of about 250 bp is amplified when PCR reaction is performed using the oligonucleotide pairs (a) and (b) as nucleic acid primers. In the case where Bisoclamis nivea is included in the specimen, a DNA fragment of about 320 bp is amplified when PCR reaction is performed using the oligonucleotide pair (c) and (d) as a nucleic acid primer. In the case where Bisocramis laganclare is included in the specimen, a DNA fragment of about 390 bp is amplified when PCR reaction is performed using the oligonucleotide pairs (e) and (f) as nucleic acid primers. In the case where Bisoclamis zolernia is included in the specimen, a DNA fragment of about 300 bp is amplified by performing a PCR reaction using the oligonucleotide pairs (g) and (h) as nucleic acid primers.
By performing such an operation, it can be confirmed whether the specimen contains fungi belonging to the genus Bisoclamis.

  In the present invention, gene fragment amplification can be confirmed 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.

In the present invention, in order to identify a fungus belonging to the genus Bisoclamis, a partial base sequence of a β-tubulin gene of a subject is determined using the oligonucleotide pair, and the partial base sequence of the gene is the above (i) By confirming whether it is included in any of the base sequences of ~ (p), the presence of the nucleic acid represented by any of the base sequences of (i) ~ (p) is confirmed and belongs to the genus Bisoclamis 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 possessed by the subject, and compares the determined base sequence with any one of the base sequences (i) to (p) above. Then, based on the coincidence or difference, the presence of the nucleic acid represented by any one of the base sequences (i) to (p) is confirmed, and the species level of the fungus belonging to the genus Bisocramis is identified. For example, the oligonucleotide pairs (a) and (b), the oligonucleotide pairs (c) and (d), the oligonucleotide pairs (e) and (f), and the (g) and (h) PCR is performed using at least one oligonucleotide pair selected from the group consisting of the oligonucleotide pairs as a primer, and the base sequence of the obtained amplification product and any one of the base sequences (i) to (p) above Compare. If the base sequence of the amplification product is included as part of the nucleic acid represented by the base sequence of (i) or (j) above, the microorganism contained in the sample can be identified as Bisoclamis falba, ) Or (l) as a part of the nucleic acid represented by the base sequence, the microorganism contained in the specimen can be identified as Bisocramis nivea, and represented by the base sequence of (m) or (n). The microorganism contained in the specimen can be identified as Bisoclamis laganclarie and contained as part of the nucleic acid represented by the base sequence of (o) or (p) above. If so, the microorganism contained in the specimen can be identified as Bisochloris solernia.
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 DNA having a sufficient degree of purification and quantity for detecting fungi belonging to the genus Bisoclamis can be obtained, and can be used in an unpurified state. It can also be used after pretreatment such as separation, 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.

  According to the method for detecting a fungus belonging to the genus Bisoclamis of the present invention, it is possible to carry out the process from the preparation step of the subject to the fungus detection step in a short time of about 5 to 12 hours.

  The fungal detection kit belonging to the genus Bisoclamis of the present invention contains the detection oligonucleotide of the present invention as a nucleic acid probe or a nucleic acid primer. This kit can be used in a method for detecting a fungus belonging to the genus Bisoclamis. In addition to the nucleic acid probe or nucleic acid primer, the kit of the present invention includes a label detection substance, a buffer, a nucleic acid synthesizing enzyme (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), an enzyme substrate (dNTP, rNTP, etc.) depending on the purpose. ) And other substances commonly used for detecting fungi. The kit of the present invention may include a positive control (positive control) for confirming that a detection reaction is possible with the detection oligonucleotide of the present invention. Examples of the positive control include DNA containing a region amplified by the 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 base sequences specific to fungi belonging to the genus Bisocramis The base sequences of various β-tubulin genes of the genus Bisocramis were determined by the following method.
DNA was extracted using Gentorukun TM (manufactured by TAKARA BIO INC.) From fungi belonging to the genus Bisoclamis and various fungi cultured in the dark at 30 ° C. for 7 days on a potato dextrose agar slant medium. . PCR amplification of the target site was performed using PuRe Taq ™ Ready-To-Go PCR Beads (manufactured by GE Health Care UK LTD) as a primer, Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3 ′: SEQ ID NO: 13), Bt2b (5 '-ACCCTCAGTGTAGTGACCCTTGGC-3': SEQ ID NO: 14) (Glass and Donaldson, Appl Environ Microbiol 61: 1323-1330, 1995) was used. The amplification conditions were 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 was labeled using BigDye terminator Ver. 1.1 (trade name: Applied Biosystems) and electrophoresed with ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Software “ATGC Ver. 4” (manufactured by Genetyx) was used to determine the base sequence from the fluorescent signal during electrophoresis.
DNA analysis software (trade name: Clustal W (DNA Data bank of Japan), http: /), based on the base sequence information of known β-tubulin genes of various genus Bisocramis and various fungi determined by sequencing method /clustalw.ddbj.nig.ac.jp/top-j.html), a specific region in the β-tubulin gene containing a base sequence specific to various fungi belonging to the genus Bisoclamis It was determined.

(2) Primer design Primer (B.fulva1F primer) comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1, based on the nucleotide sequence region specific to various fungi belonging to the genus Bisoclamis obtained above, Primer consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 2 (B.fulva1R primer), primer consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 3 (B.nivea1F primer), base sequence of SEQ ID NO: 4 Primer (B.nivea1R primer) consisting of the oligonucleotide represented by the above, primer (B.lag1F primer) consisting of the oligonucleotide represented by the base sequence of SEQ ID NO: 5, oligonucleotide represented by the base sequence of SEQ ID NO: 6 A primer (B.lag1R primer) consisting of: an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 7 Design primer (B.zol1F primer) and oligonucleotide primer (B.zol1R primer) represented by the nucleotide sequence of SEQ ID NO: 8, and request synthesis to Sigma-Aldrich Japan (Demineralized product) , 0.02 μmol scale).
Each of the above primers is designed based on the base sequence of each specific region of the β-tubulin gene of bisoclamis falba, bisoclamis nivea, bisoclamis laganclarie and bisoclamis zolernia as shown in FIGS.

(3) Preparation of specimens The fungi used for evaluation of the effectiveness of the designed primer, that is, the strains listed in Tables 1 to 3 were used as the genus Bisocramis and other heat-resistant molds and general molds. Regarding these fungi, strains stored by the National Institute of Technology and Evaluation managed by the National Institute of Technology and Evaluation, NBRC numbers, strains stored by the Center for Fungal Medicine, Chiba University School of Medicine and controlled by IFM numbers, The Centraalbureau voor Schimmelcultures Stocks stored and managed by CBS number, shares managed by RIKEN BioResource Center and managed by IAM number, shares managed by RIKEN BioResource Center and managed by JCM number, and The strains managed by the Fermentation Research Institute and managed by IFO numbers were obtained and used.
Each cell was cultured under optimal conditions. As for the culture conditions, potato dextrose medium (trade name: Pearl Core Potato Dextrose Agar Medium, manufactured by Eiken Chemical Co., Ltd.) was used at the optimum temperature, that is, general mold was cultured at 25 ° C. and heat-resistant mold was cultured at 30 ° C. for 7 days.

(4) Preparation of genomic DNA Each bacterial cell was collected from an agar medium using a platinum loop and placed on a potato dextrose agar medium (composition: potato extract 200 g, glucose 20 g and agar 15 g, Eiken Chemical Co., Ltd. per 1000 L of medium). Sowing. The cells were cultured for 2 to 5 days under the culture conditions of 25 to 30 ° C., and 1 platinum loop amount of hyphae was collected.
A genomic DNA solution was prepared from the collected cells using a genomic DNA preparation kit (PrepMan ultra (trade name) manufactured by Applied Biosystems). The concentration of the DNA solution was adjusted to 5 ng / μL.

(5) Detection of bisoclamis falba As a DNA template, 1 μL of the genomic DNA solution prepared in (4) above, 13 μL of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μL of sterile distilled water are mixed and described in SEQ ID NO: 1. 0.5 μL of a primer (B. fulva1F primer, 20 pmol / μL) consisting of an oligonucleotide represented by the nucleotide sequence of (B. fulva1R primer, 20 pmol) / ΜL) 0.5 μL was added to prepare a 25 μL PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 59 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute as one cycle. 35 cycles were performed.

  After the PCR reaction, 5 μL was taken from the PCR reaction solution, mixed well with the loading buffer, and subjected to electrophoresis (100 V for 45 minutes or 135 V for 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. 3 shows an electrophoretic diagram (100 V for 45 minutes) for the fungal sample shown in Table 1, and FIG. 4 shows an electrophoretic diagram (135 V for 25 minutes) for the fungal sample shown in Table 2. The numbers in the figure indicate that the samples were reacted using DNA extracted from the samples with corresponding sample numbers in the table. As a molecular weight marker, EZ Load ™ 100 bp (trade name, BIO RAD) was used.

  As a result, amplification of a gene fragment of about 250 bp was confirmed in a sample containing genomic DNA of B. clamis falba. On the other hand, amplification of gene fragments was not confirmed in samples containing genomic DNA of fungi widely distributed in the production environment of foods and other foods of the genus Bisoclamis other than bisoclamis farba. Therefore, by using a specific oligonucleotide, B. clamis falba can be specifically detected and identified at the species level.

(6) Detection of bisoclamis nivea As a DNA template, 1 μL of the genomic DNA solution prepared in the above (4), 13 μL of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μL of sterile distilled water are mixed and described in SEQ ID NO: 3 0.5 μL of a primer (B. nivea1F primer, 20 pmol / μL) composed of an oligonucleotide represented by the nucleotide sequence of (B. nivea1R primer, 20 pmol) / ΜL) 0.5 μL was added to prepare a 25 μL PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 55 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute in one cycle. 35 cycles were performed.

  After the PCR reaction, 5 μL was taken from the PCR reaction solution, mixed well with the loading buffer, and subjected to electrophoresis (100 V for 45 minutes or 135 V for 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. 5 shows an electrophoretic diagram (100 V for 45 minutes) for the fungal sample shown in Table 1, and FIG. 6 shows an electrophoretic diagram (135 V for 25 minutes) for the fungal sample shown in Table 3. The numbers in the figure indicate that the samples were reacted using DNA extracted from the samples with corresponding sample numbers in the table. As a molecular weight marker, EZ Load ™ 100 bp (trade name, BIO RAD) was used.

  As a result, amplification of a gene fragment of about 320 bp was confirmed in the sample containing genomic DNA of B. clamis nivea. On the other hand, amplification of the gene fragment was not confirmed in samples containing genomic DNA of fungus widely distributed in the production environment of foods and other foods of the genus Bisocramis other than Bisocramis nivea. Therefore, by using specific oligonucleotides, B. clamis nivea can be specifically detected and identified at the species level.

(7) Detection of Bisoclamis Lagankurarie As a DNA template, 1 μL of the genomic DNA solution prepared in (4) above, 13 μL of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μL of sterile distilled water are mixed and described in SEQ ID NO: 5 0.5 μL of a primer (B.lag1F primer, 20 pmol / μL) composed of an oligonucleotide represented by the nucleotide sequence of (B.lag1R primer, 20 pmol) / ΜL) 0.5 μL was added to prepare a 25 μL PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 59 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute as one cycle. 35 cycles were performed.

  After the PCR reaction, 5 μL was taken from the PCR reaction solution, thoroughly mixed with the loading buffer, and subjected to electrophoresis (100 V for 45 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. FIG. 7 shows an electrophoretic diagram of the fungal samples shown in Table 1. 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 (trade name, BIO RAD) was used.

  As a result, amplification of a gene fragment of about 390 bp was confirmed in a sample containing genomic DNA of B. clamis laganclarie. On the other hand, amplification of the gene fragment was not confirmed in samples containing genomic DNA of fungus widely distributed in the production environment of foods and other foods of the genus Bisoclamis other than Bisocramis laganclarie. Therefore, by using a specific oligonucleotide, B. clamis laganclare can be specifically detected and identified at the species level.

(8) Detection of bisoclamis zolernia As a DNA template, 1 μL of the genomic DNA solution prepared in (4) above, 13 μL of Pre Mix Taq (trade name, manufactured by Takara Bio Inc.), and 10 μL of sterile distilled water are mixed, and described in SEQ ID NO: 7 0.5 μL of a primer (B.zol1F primer, 20 pmol / μL) consisting of an oligonucleotide represented by the nucleotide sequence of (B.zol1R primer, 20 pmol) / ΜL) 0.5 μL was added to prepare a 25 μL PCR reaction solution.
The PCR reaction solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were (i) heat denaturation reaction at 95 ° C. for 1 minute, (ii) annealing reaction at 59 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute as one cycle. 35 cycles were performed.

  After the PCR reaction, 5 μL was taken from the PCR reaction solution, thoroughly mixed with the loading buffer, and subjected to electrophoresis (100 V for 45 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. FIG. 8 shows an electrophoretic diagram of the fungal samples shown in Table 1. 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 (trade name, BIO RAD) was used.

  As a result, amplification of a gene fragment of about 300 bp was confirmed in the sample containing genomic DNA of B. clamis zolernia. On the other hand, amplification of the gene fragment was not confirmed in samples containing the genome DNA of fungi widely distributed in the production environment of foods, and the genus Bisoclamis other than Bisochramis zolernia. Therefore, by using a specific oligonucleotide, B. clamis zolernia can be specifically detected and identified at the species level.

  From the above results, by using the oligonucleotide of the present invention, the presence of the base sequence of the β-tubulin gene of fungi belonging to the genus Bisoclamis can be confirmed, and the fungi belonging to the genus Bisoclamis can be specifically identified at the species level. It can be detected. Therefore, according to the method of the present invention, it is possible to detect fungi belonging to the genus Bisoclamis simply, rapidly and specifically.

Claims (19)

The following oligonucleotide pairs (a) and (b), the following oligonucleotide pairs (c) and (d), the following oligonucleotide pairs (e) and (f), and the following (g) and (h) oligos at least one fungi belonging to Byssochlamys (Byssochlamys) genus, characterized by using a pair of oligonucleotides containing Byssochlamys (Byssochlamys) conducting the identification at the species level of fungi belonging to the genus selected from the group consisting of nucleotide pairs Detection method.
(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 belongs to the genus Byssochlamys An oligonucleotide that can be used for detection at the species level of fungi belonging to (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, Oligonucleotide which is substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (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 the Byssochlamys (Byssochlamys) genus One base deletion 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 of fungi, substituted An oligonucleotide that is inserted or added and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (e) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5, or An oligonucleotide (f) described in SEQ ID NO: 6 in which one base is deleted, substituted, inserted or added in the described base sequence and can be used for detection at the species level of fungi belonging to the genus Byssochlamys one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 6, represented by the nucleotide sequence, substitution, interpolation Or and is appended Byssochlamys (Byssochlamys) oligonucleotides can be used to detect at the species level belonging fungi to the genus (g) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7, or SEQ ID NO: 7 Oligonucleotide (h) that has one base deleted, substituted, inserted or added in the base sequence and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (h) SEQ ID NO: 8 in one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 8 is represented, substituted, used in the detection of an insertion or are added and Byssochlamys (Byssochlamys) species level of fungi belonging to the genus Oligonucleotide
  In order to perform the identification, the oligonucleotide pairs (a) and (b), the oligonucleotide pairs (c) and (d), the oligonucleotide pairs (e) and (f), and the (g) And (h) using at least one oligonucleotide pair selected from the group consisting of oligonucleotide pairs, amplifying the partial base sequence of the β-tubulin gene of fungi belonging to the genus Bisoclamis and confirming the presence or absence of the amplified product The detection method according to claim 1, wherein:   The detection method according to claim 2, wherein the amplification reaction is performed by a polymerase chain reaction (PCR) method. The polymerase chain reaction method is performed using the oligonucleotide pair of (a) and (b) as a nucleic acid primer, and a partial base sequence of the β-tubulin gene of Byssochlamys fulva is amplified. Detection method. The polymerase chain reaction method is performed using the oligonucleotide pair of (c) and (d) as a nucleic acid primer, and a partial base sequence of the β-tubulin gene of Byssochlamys nivea is amplified. Detection method. The partial chain sequence of the β-tubulin gene of Byssochlamys lagunculariae is amplified by performing the polymerase chain reaction using the oligonucleotide pair of (e) and (f) as a nucleic acid primer. Detection method. The partial chain sequence of the β-tubulin gene of Byssochlamys zollernia is amplified by performing the polymerase chain reaction using the oligonucleotide pair (g) and (h) as a nucleic acid primer. Detection method. The following oligonucleotide pairs (a) and (b), the following oligonucleotide pairs (c) and (d), the following oligonucleotide pairs (e) and (f), and the following (g) and (h) oligos The presence of a nucleic acid represented by any one of the following base sequences (i) to (p) using at least one oligonucleotide pair selected from the group consisting of nucleotide pairs: Byssochlamys ) Method for detecting fungi belonging to the genus.
(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 belongs to the genus Byssochlamys An oligonucleotide that can be used for detection at the species level of fungi belonging to (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, Oligonucleotide which is substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (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 the Byssochlamys (Byssochlamys) genus One base deletion 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 of fungi, substituted An oligonucleotide that is inserted or added and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (e) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5, or An oligonucleotide (f) described in SEQ ID NO: 6 in which one base is deleted, substituted, inserted or added in the described base sequence and can be used for detection at the species level of fungi belonging to the genus Byssochlamys one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 6, represented by the nucleotide sequence, substitution, interpolation Or and is appended Byssochlamys (Byssochlamys) oligonucleotides can be used to detect at the species level belonging fungi to the genus (g) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7, or SEQ ID NO: 7 Oligonucleotide (h) that has one base deleted, substituted, inserted or added in the base sequence and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (h) SEQ ID NO: 8 in one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 8 is represented, substituted, used in the detection of an insertion or are added and Byssochlamys (Byssochlamys) species level of fungi belonging to the genus Oligonucleotide (i) partial base sequence of β-tubulin gene represented by SEQ ID NO: 9 or a phase thereof Complementary sequence (j) A nucleotide sequence in which one or several bases are deleted, substituted, inserted or added in the nucleotide sequence of SEQ ID NO: 9 and can be used for detection of Byssochlamys fulva , or its complement Sequence (k) a partial base sequence of the β-tubulin gene described in SEQ ID NO: 10 or its complementary sequence (l) In the base sequence described in SEQ ID NO: 10, one or several bases are deleted, substituted, inserted or added And a base sequence that can be used for detecting Byssochlamys nivea , or a complementary sequence (m) thereof, a partial base sequence of the β-tubulin gene described in SEQ ID NO: 11 or a complementary sequence thereof (n) SEQ ID NO: 11 1 or more nucleotides in the nucleotide sequence are deleted, substituted, inserted or added in and and Byssochlamys Ragankurarie of (Byssochlamys lagunculariae) according to Or a complementary sequence (o) thereof, a partial base sequence of the β-tubulin gene described in SEQ ID NO: 12 or a complementary sequence thereof (p) in the base sequence described in SEQ ID NO: 12 A nucleotide sequence that has been deleted, substituted, inserted or added and can be used to detect Byssochlamys zollernia or its complementary sequence
  The oligonucleotide pair (a) and (b), the oligonucleotide pair (c) and (d), the oligonucleotide pair (e) and (f), and the oligonucleotide (g) and (h) Using at least one oligonucleotide pair selected from the group consisting of nucleotide pairs, a part of the base sequence of any of (i) to (p) above is amplified, the presence or absence of an amplification product is confirmed, The detection method according to claim 8, wherein the presence of the nucleic acid represented by any one of the base sequences i) to (p) is confirmed.   The detection method according to claim 9, wherein the amplification reaction is performed by a polymerase chain reaction (PCR) method.   The detection according to claim 10, wherein the polymerase chain reaction method is performed using the oligonucleotide pair of (a) and (b) as a nucleic acid primer, and a part of the base sequence of (i) or (j) is amplified. Method.   The detection according to claim 10, wherein the polymerase chain reaction method is performed using the oligonucleotide pair of (c) and (d) as a nucleic acid primer to amplify a part of the base sequence of (k) or (l). Method.   The detection according to claim 10, wherein the polymerase chain reaction method is performed using the oligonucleotide pair of (e) and (f) as a nucleic acid primer, and a part of the base sequence of (m) or (n) is amplified. Method.   The detection according to claim 10, wherein the polymerase chain reaction method is performed using the oligonucleotide pair of (g) and (h) as a nucleic acid primer, and a part of the base sequence of (o) or (p) is amplified. Method. Be any oligonucleotide of the following (a) ~ (h), Byssochlamys (Byssochlamys) can be used as a detector oligonucleotide at the species level of fungi belonging to the genus oligo for fungi detected belonging to Byssochlamys (Byssochlamys) genus nucleotide.
(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 belongs to the genus Byssochlamys An oligonucleotide that can be used for detection at the species level of fungi belonging to (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, Oligonucleotide which is substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (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 the Byssochlamys (Byssochlamys) genus One base deletion 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 of fungi, substituted An oligonucleotide that is inserted or added and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (e) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5, or An oligonucleotide (f) described in SEQ ID NO: 6 in which one base is deleted, substituted, inserted or added in the described base sequence and can be used for detection at the species level of fungi belonging to the genus Byssochlamys one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 6, represented by the nucleotide sequence, substitution, interpolation Or and is appended Byssochlamys (Byssochlamys) oligonucleotides can be used to detect at the species level belonging fungi to the genus (g) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7, or SEQ ID NO: 7 Oligonucleotide (h) that has one base deleted, substituted, inserted or added in the base sequence and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (h) SEQ ID NO: 8 in one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 8 is represented, substituted, used in the detection of an insertion or are added and Byssochlamys (Byssochlamys) species level of fungi belonging to the genus Oligonucleotide
A nucleic acid probe or nucleic acid capable of hybridizing to a nucleic acid represented by any one of the following base sequences (i) to (p) and specifically detecting a fungus belonging to the genus Byssochlamys at the species level The oligonucleotide for detection according to claim 15, which can function as a primer.
(I) a partial base sequence of the β-tubulin gene shown in SEQ ID NO: 9 or its complementary sequence (j) one or several bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 9 And a base sequence that can be used for detecting Byssochlamys fulva , or a complementary sequence (k) thereof, a partial base sequence of the β-tubulin gene described in SEQ ID NO: 10 or its complementary sequence (l) in SEQ ID NO: 10 A nucleotide sequence in which one or several bases are deleted, substituted, inserted or added in the described nucleotide sequence and can be used for detection of Byssochlamys nivea , or its complementary sequence (m) described in SEQ ID NO: 11 A partial base sequence of the β-tubulin gene or its complementary sequence (n) in the base sequence set forth in SEQ ID NO: 11, wherein one or several bases are deleted, substituted, inserted or Nucleotide sequences can be used to detect pressure has been provided and Byssochlamys Ragankurarie (Byssochlamys lagunculariae), or its complementary sequence (o) SEQ ID NO: 12 beta-tubulin gene partial nucleotide sequence or its complementary sequence according to (p) SEQ ID NO: 12. A nucleotide sequence in which one or several bases are deleted, substituted, inserted or added in the nucleotide sequence described in 12, and can be used for detecting Byssochlamys zollernia , or a complementary sequence thereof   The oligonucleotide for detection according to claim 15 or 16, wherein the oligonucleotide is a nucleic acid primer. The following (a) and (b) oligonucleotide pairs, the following (c) and (d) oligonucleotide pairs, the following (e) and (f) oligonucleotide pairs, or the following (g) and (h) oligos Byssochlamys represented by nucleotide pairs (Byssochlamys) fungi detector oligonucleotide pairs belonging to the genus.
(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 belongs to the genus Byssochlamys An oligonucleotide that can be used for detection at the species level of fungi belonging to (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, Oligonucleotide which is substituted, inserted or added and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (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 the Byssochlamys (Byssochlamys) genus One base deletion 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 of fungi, substituted An oligonucleotide that is inserted or added and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (e) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5, or An oligonucleotide (f) described in SEQ ID NO: 6 in which one base is deleted, substituted, inserted or added in the described base sequence and can be used for detection at the species level of fungi belonging to the genus Byssochlamys one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 6, represented by the nucleotide sequence, substitution, interpolation Or and is appended Byssochlamys (Byssochlamys) oligonucleotides can be used to detect at the species level belonging fungi to the genus (g) an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7, or SEQ ID NO: 7 Oligonucleotide (h) that has one base deleted, substituted, inserted or added in the base sequence and can be used for detection at the species level of fungi belonging to the genus Byssochlamys (h) SEQ ID NO: 8 in one base deletion in the nucleotide sequence described in the oligonucleotide or SEQ ID NO: 8 is represented, substituted, used in the detection of an insertion or are added and Byssochlamys (Byssochlamys) species level of fungi belonging to the genus Oligonucleotide 18. oligonucleotide pair Byssochlamys (Byssochlamys) fungi detection kit belonging to the genus including description.