JP5956389B2 - Method for detecting thermostable Penicillium fungi - Google Patents

Description

  The present invention relates to a method for detecting a thermostable Penicillium fungus.

  Generally, fungi that can survive even after 30 minutes of heat treatment at 75 ° C. are called heat-resistant fungi, and many of these are considered to be harmful bacteria that cause food and beverage accidents. Thermostable fungi are widely inhabited in soil and the like, and are mixed in food and drink by contact with food ingredients. The heat-resistant fungus mixed in cannot be completely killed by ordinary heat sterilization treatment, but grows in the product and causes the product to decay or deteriorate. Known examples of detection of heat-resistant fungi from foods and drinks include grains, spices, fruits, fruit juices, tea leaves, boiled vegetables, fruit drinks, tea-based drinks, sports drinks, tomato juice, jams, marmalades, and the like.

In order to ensure the safety of foods and drinks, not only take preventive measures to prevent contamination by harmful bacteria, but also when the fungus is detected in raw materials and product inspection, etc. It is important to identify
Until now, the thermostable fungus detection method has been carried out by culturing the fungus in a medium and then observing and identifying its form with a microscope. However, this method requires a period of about two weeks until the characteristic morphogenesis of each bacterial species that serves as an index for determination, and skill is required for accurate determination. Therefore, it has been difficult to perform quick and accurate detection.
As a conventional method for morphological observation, a method for detecting and identifying a target fungus by a nucleic acid amplification reaction using a detection primer has been proposed. For example, a Paecilomyces (Paecilomyces) genus fungus is Byssochlamys (Byssochlamys) genus fungi and their anamorph is a heat-resistant fungus, specifically a method of detecting a nucleic acid amplification method has been proposed (e.g., see Patent Document 1).

A heat-resistant Penicillium fungus has been reported as one of the heat-resistant fungi. Among them, Penicillium Rapidosamu (Penicillium lapidosu m) and Penicillium brefeldianum Diana beam (Penicillium brefeldianum) 2 species of, there are a plurality of detection instances from processed foods, has been reported as a cause of the accident bacteria. Penicillium rapidum is separated from canned blueberries and frozen blueberry cake, and Penicillium brefeldinum is separated from marmalade, cream cheese and apple juice.
For this reason, detection and identification of Penicillium rapidum and Penicillium brefeldinum are particularly important among heat-resistant Penicillium fungi, but a rapid and accurate detection method for these bacteria has been established so far. It has not been.

JP 2010-004880 A

  One of the reasons why rapid and accurate detection of Penicillium rapidum and Penicillium brefeldinum is difficult is that genetic information is fragile and it is difficult to design highly specific primers. In addition, after designing a universal primer based on a gene region that is widely conserved in fungi, performing a nucleic acid amplification reaction, analyzing the base sequence of the obtained amplification product and comparing it with known sequence information There is also a method to determine the bacterial species using However, this method requires a certain amount of time for base sequence analysis and comparison, and is not sufficient from the viewpoint of rapidity.

  An object of the present invention is to provide a method for rapidly and accurately detecting and identifying Penicillium rapidum and Penicillium brefeldinum, which are heat-resistant Penicillium species, at the species level. Another object of the present invention is to provide an oligonucleotide, an oligonucleotide pair and a detection kit applicable to the method.

In view of the above problems, the present inventors have intensively studied a method for quickly and accurately detecting Penicillium rapidum and Penicillium brefeldinum at the species level.
On the gene sequences of Penicillium rapidum and Penicillium brefeldinum, DNA regions useful for designing primers capable of specifically detecting them were searched. As a result of molecular phylogenetic analysis based on the base sequence information of RPB2 (RNA polymerase II) gene of Penicillium rapidum and Penicillium brefeldinum and the base sequence information of RPB2 gene such as known Penicillium fungi, In the RPB2 gene region of Penicillium rapidum and Penicillium brefeldinum, a specific base sequence that can be distinguished from other bacterial species was found. The inventors have found that Penicillium rapidum and Penicillium brefeldinum can be detected quickly and accurately by using the base sequence as a detection target.
The present invention has been completed based on these findings.

That is, the present invention includes a step of detecting Penicillium lapidosum using an oligonucleotide pair consisting of the following oligonucleotides (a) and (b), and the oligonucleotides (c) and (d) below: A method for detecting a thermostable Penicillium fungus, comprising at least one step selected from the step of detecting Penicillium brefeldianum using an oligonucleotide pair comprising:
(A) an oligonucleotide having the base sequence shown in SEQ ID NO: 1 or a base sequence in which one or several bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 1 and penicillium rapidum Oligonucleotide capable of hybridizing to RPB2 gene region (b) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2, or one or several bases deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 2 Oligonucleotide consisting of a base sequence and capable of hybridizing to the RPB2 gene region of Penicillium rapidum (c) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3, or one or several bases missing in the base sequence shown in SEQ ID NO: 3 Consisting of base sequences deleted, substituted, inserted or added, and Oligonucleotide that can hybridize to the RPB2 gene region of Penicillium brefeldinum (d) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4, or one or several bases deleted or substituted in the base sequence shown in SEQ ID NO: 4 Oligonucleotides consisting of inserted or added nucleotide sequences and capable of hybridizing to the RPB2 gene region of Penicillium brefeldinum

The present invention also provides at least one oligonucleotide selected from the group consisting of the oligonucleotides (a) to (d), an oligonucleotide pair consisting of the oligonucleotides (a) and (b), or ( It relates to an oligonucleotide pair consisting of the oligonucleotides c) and (d).
The present invention also relates to a thermostable Penicillium fungus detection kit comprising at least one selected from the above-mentioned oligonucleotides and oligonucleotide pairs.
The “detection” in the present invention is a concept including “identification” used taxonomically.

The detection method of the present invention can quickly and accurately detect and identify the heat-resistant fungi Penicillium rapidum and Penicillium brefeldinum that are causative bacteria of food and beverage contamination accidents. According to this method, the time required to detect and identify the fungus can be significantly shortened as compared with conventional morphological observation methods.
Moreover, the oligonucleotide, oligonucleotide pair, and detection kit of the present invention can be suitably used in the method.

It is a figure which shows the molecular phylogenetic tree of Penicillium fungi created based on the base sequence of the RPB2 gene region of Penicillium fungi. FIG. 2 is an electrophoretogram of PCR products amplified using the oligonucleotide pairs (a) and (b) in the examples. 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 (c) and (d) in the examples. FIG. 2 is an electrophoretogram of PCR products amplified using the oligonucleotide pairs (a) and (b) in the examples. 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 (c) and (d) in the examples.

The method of the present invention is a method for detecting heat-resistant Penicillium fungi, and the heat-resistant Penicillium fungi to be detected are Penicillium lapidosum and Penicillium brefeldianum . Among these fungi belonging to the genus Penicillium, the heat resistance of ascospores is high, and there are reports of food and beverage accidents. These fungi have a sexual generation in the life cycle, and the sexual generation forms offspring spores with heat resistance. In the method of the present invention, both the sexual generation and the asexual generation of the fungus can be detected.
It should be noted that, in April 2011, in the International Commission of Penicillium and Aspergillus (ICPA ), it has been proposed to unify the genus Penicillium fungi associated with Yupenishiriumu (Eupenicillium) spp fungi Penicillium name (http: // see www.aspergilluspenicillium.org/index.php/penicillium-names). In the present invention, penicillium is used as the name according to this.

In the method of the present invention, using a base sequence specific for Penicillium rapidum or Penicillium brefeldinum in the RPB2 gene region, Penicillium rapidum or Penicillium brefeldinum can be detected in a species-specific manner. it can.
The present inventors performed alignment analysis between the base sequence of RPB2 gene of Penicillium rapidum and Penicillium brefeldinum and the base sequence of RPB2 gene of other fungi. As a result, in the base sequence of the RPB2 gene of Penicillium rapidum, a region with low conservation was found in other species belonging to the genus Penicillium and highly conserved within the same species, and this region was species-specific for Penicillium rapidum. It was useful as an index (target) for detecting and identifying automatically. Furthermore, in the base sequence of the RPB2 gene of Penicillium brefeldinum, a region having low conservability with other fungi belonging to the genus Penicillium and having high conservability within the same species was found. It was useful as an index (target) for detecting and identifying Ferdinham species-specifically. Hereinafter, these areas are also referred to as “detection target areas”.
The detection of Penicillium rapidum and the detection of Penicillium brefeldinum using this detection target region will be described in the following order. In the detection method of the present invention, the detection of penicillium / rapidosum and the detection of penicillium / brefeldinum may be performed independently or in combination.

1. Detection of Penicillium Rapidum In the method of the present invention, a nucleic acid represented by the following base sequence (A) or (B) is used as the detection target region for the detection of Penicillium rapidum. Preferably, a nucleic acid represented by the following base sequence (A) is used. The base sequence shown in SEQ ID NO: 5 is a partial base sequence of the RPB2 gene of Penicillium rapidum.
(A) Base sequence shown in SEQ ID NO: 5 or its complementary sequence (B) Base sequence in which one or several bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 5, or its complementary sequence

  In the above (B), the number of one or several bases is preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, and even more preferably 1.

Moreover, as the base sequence of (B), the base sequence of (B1) below is also preferable.
(B1) A base sequence having 85% or more identity (homology) with the base sequence shown in SEQ ID NO: 5 or a complementary sequence thereof In (B1), the identity of the base sequence may be 90% or more Preferably, it is 95% or more, more preferably 97% or more, and even more preferably 99% or more.
In the present invention, the identity of base sequences is calculated by the Lipman-Pearson method (Science, 1985, 227, p. 1435) or the like. Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the parameter Unit size to compare (ktup) is set to 2 and the calculation is performed. Can do.

Specifically, as a method for detecting Penicillium rapidosum using the base sequence of (A) or (B), an oligonucleotide capable of hybridizing to the nucleic acid represented by the base sequence of (A) or (B) is used. A method of detecting by a nucleic acid amplification method using a nucleic acid primer (hereinafter referred to as “nucleic acid amplification method”), an oligonucleotide capable of hybridizing to the nucleic acid represented by the base sequence of (A) or (B) above was used as a nucleic acid probe A method of detection by hybridization (hereinafter referred to as “hybridization method”), determining the base sequence of the RPB2 gene of the subject, and confirming that the base sequence of (A) or (B) is included in the sequence Method (hereinafter, “sequencing method”). Among these, from the viewpoint of rapidity, a nucleic acid amplification method or a hybridization method is preferable, and a nucleic acid amplification method is more preferable.
Hereinafter, each method will be described.

[Nucleic acid amplification method]
Detection by the nucleic acid amplification method is carried out by a nucleic acid amplification reaction using a nucleic acid prepared from a specimen sample as a template and using an oligonucleotide that can hybridize to the nucleic acid represented by the base sequence (A) or (B) as a nucleic acid primer. It is carried out by amplifying a nucleic acid consisting of a part or all of the base sequence (A) or (B) and confirming the presence or absence of the amplification product. If the amplification product is confirmed, it is identified as Penicillium rapidum.

  The oligonucleotide that can be hybridized to the nucleic acid represented by the base sequence (A) or (B) used as a nucleic acid primer (hereinafter, “detection oligonucleotide”) has the base sequence (A) or (B). Oligonucleotides that are capable of hybridizing under stringent conditions to the nucleic acid represented are preferred. Examples of “stringent conditions” include 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. for 8-16 hours Examples of the conditions include constant temperature and hybridization.

The oligonucleotide for detection is more preferably an oligonucleotide consisting of a partial sequence of the base sequence (A) or (B) and satisfying the following conditions (1) to (4).
(1) The 3 ′ end is a sequence specific to penicillium rapidum (2) The GC content is approximately 30 to 80% (3) The possibility of self-annealing is low (4) Tm value (melting temperature: melting) temperature) is about 55 to 65 ° C. In the condition (3), “the possibility of self-annealing is low” means that the oligonucleotides are not expected to bind to each other from the base sequence of the oligonucleotide. .

  The number of bases of the detection oligonucleotide is not particularly limited, but is preferably 13 to 30 bases, and more preferably 18 to 26 bases. The Tm value of the oligonucleotide at the time of hybridization is preferably in the range of 55 ° C to 65 ° C, and more preferably in the range of 59 ° C to 62 ° C. The GC content of the oligonucleotide is preferably 30% to 80%, more preferably 45% to 65%, and even more preferably around 55%.

More preferably, the oligonucleotide for detection (a) or (b) below is used.
(A) an oligonucleotide having the base sequence shown in SEQ ID NO: 1 or a base sequence in which one or several bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 1 and penicillium rapidum Oligonucleotide capable of hybridizing to RPB2 gene region (b) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2, or one or several bases deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 2 Oligonucleotide consisting of a base sequence and capable of hybridizing to the RPB2 gene region of Penicillium rapidum

The oligonucleotides (a) and (b) correspond to the sequences of positions 358 to 379 and positions 619 to 642 of the base sequence shown in SEQ ID NO: 5, respectively.
In (a) and (b), the base sequence of the RPB2 gene region of Penicillium rapidum is the base sequence of (A) or (B).
In the above (a) and (b), the number of one or several bases is preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, and even more preferably 1.

As the oligonucleotide (a) or (b), the following oligonucleotide (a1) or (b1) is also preferable.
(A1) An oligonucleotide having the base sequence shown in SEQ ID NO: 1 or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 having a identity of 85% or more and capable of hybridizing to the RPB2 gene region of Penicillium rapidum (B1) an oligonucleotide having the base sequence shown in SEQ ID NO: 2 or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 having a identity of 85% or more and capable of hybridizing to the RPB2 gene region of Penicillium rapidum In the above (a1) or (b1), the identity of the base sequence is preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and 99% or more. Even more preferably. The method described above can be used as a method for calculating the identity of base sequences.

As the oligonucleotide (a), the following oligonucleotide (a2) or (b2) is more preferable.
(A2) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 (b2) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2

The binding mode of the detection oligonucleotide may be not only a phosphodiester bond existing in a natural nucleic acid but also a phosphoramidate bond, a phosphorothioate bond, or the like.
The detection oligonucleotide can be prepared by a usual synthesis method. For example, it can be prepared by chemical synthesis using an automatic nucleic acid synthesizer or the like based on the sequence of the detection oligonucleotide. It is also possible to excise directly from the genome of Penicillium rapidosum using a restriction enzyme or the like, or to clone and isolate and purify the RPB2 gene and then excise 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.

  For detection by the nucleic acid amplification method, it is preferable to use a nucleic acid primer pair comprising the detection oligonucleotide. As the nucleic acid primer pair, an oligonucleotide pair consisting of the oligonucleotides (a) and (b) and an oligonucleotide pair consisting of the oligonucleotides (a1) and (b1) are preferable, and the above-mentioned (a2) and (b2) More preferred are oligonucleotide pairs consisting of oligonucleotides.

The method for amplifying a nucleic acid is not particularly limited, and includes polymerase chain reaction (PCR) method, real-time PCR method, LCR (Ligase Chain Reaction) method, SDA (Strand Displacement Amplification) method, NASBA (Nucleic Acid Sequence-based Amplification) method, Conventional nucleic acid amplification methods such as RCA (Rolling-circle amplification) and LAMP (Loop mediated isothermal amplification) can be used. In the present invention, the PCR method is preferably used from the viewpoint of rapidity.
Hereinafter, the case where an amplification reaction is performed by the PCR method will be described in detail. However, the present invention is not limited to these.

The PCR conditions are not particularly limited as long as the target DNA fragment can be amplified to a detectable level.
For example, when PCR is performed using an oligonucleotide pair consisting of the oligonucleotides of (a) and (b) as a nucleic acid primer, a preferable PCR condition is a heat denaturation reaction in which a double-stranded DNA is made into a single strand. An annealing reaction for hybridizing the primer pair to single-stranded DNA at 50 ° C. or higher and 98 ° C. or lower for 10 seconds or longer and 60 seconds or shorter (preferably 52 ° C. or higher, more preferably 55 ° C. or higher) 70 ° C. or lower (preferably Is 64 ° C. or lower, more preferably 60 ° C. or lower) for 5 seconds or longer (preferably 10 seconds or longer) and 120 seconds or shorter (preferably 60 seconds or shorter). This is performed for 60 seconds or less, and the cycle is performed for 30 cycles or more and 35 cycles or less.

In the present invention, the nucleic acid amplification product can be confirmed by an ordinary method. For example, electrophoresis of reaction products, confirmation of the presence or absence of a band corresponding to the size of the target amplification product, method of measuring the amount of amplification product over time, method of decoding the base sequence of the amplification product, etc. Can be mentioned. Of these, a method is preferred in which the reaction product is electrophoresed after the nucleic acid amplification reaction and the presence or absence of a band corresponding to the size of the target amplification product is confirmed.
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 amplification reaction, a method of using a primer labeled with a fluorescent substance, or the like, and fluorescence intensity by binding to DNA such as ethidium bromide between amplified DNA double strands. For example, a method of inserting a fluorescent material that increases the intensity of the light is used. Of these, a method is preferred in which a fluorescent substance whose fluorescence intensity is increased by binding to DNA between the amplified DNA double strands is inserted.
When the sample contains a fungus to be detected, PCR is performed using the oligonucleotide pair of the present invention as a primer set, and the obtained PCR product is electrophoresed to confirm a DNA fragment of a specific size. . Specifically, when the sample contains penicillium rapidosam, electrophoresis of the PCR product obtained using the oligonucleotide pair consisting of the oligonucleotides (a) and (b) as a nucleic acid primer yields about A 280 bp DNA fragment is confirmed. When the DNA fragment is confirmed, it can be identified that the sample contains penicillium rapidum.

[Hybridization method]
In the detection by the hybridization method, the oligonucleotide that can hybridize to the nucleic acid represented by the base sequence (A) or (B) is labeled to form a nucleic acid probe, and the probe is hybridized with the nucleic acid prepared from the specimen sample. , By detecting the label of the hybridized probe. When the probe hybridizes, it is identified as penicillium rapidum.

  As the nucleic acid probe, the detection oligonucleotide and the oligonucleotide pair described in the above-mentioned “Nucleic acid amplification method” can be used, and the preferable range is also the same. Among these, at least one oligonucleotide selected from the oligonucleotides (a), (b), (a1) and (b1) is preferable, and is selected from the oligonucleotides (a2) and (b2). More preferred is at least one oligonucleotide.

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. The oligonucleotide thus labeled is hybridized with a nucleic acid extracted from a specimen sample by a conventional method under stringent conditions, and then the label of the hybridized detection oligonucleotide is measured. Penicillium rapidum can be detected at the species level. Examples of stringent conditions include the conditions described above. As a method for measuring the label of the nucleic acid probe hybridized with the nucleic acid, a usual method (FISH method, dot blot method, Southern blot method, Northern blot method, etc.) can be used.
The detection 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 a target nucleic acid can be captured using the label as an indication.

[Sequencing method]
Detection by the sequencing method is performed by analyzing and determining the base sequence of the RPB2 gene in the nucleic acid prepared from the specimen sample, comparing the sequence with the base sequence of (A) or (B), and determining whether or not they match. Based on the above, penicillium rapidum is identified.
The method for analyzing the base sequence is not particularly limited, and a commonly used nucleic acid sequencing method 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.

2. Detection of Penicillium brefeldinum In addition, in the method of the present invention, for detection of Penicillium brefeldinum, a nucleic acid represented by the base sequence of (C) or (D) below is used as the detection target region. Use. Preferably, a nucleic acid represented by the following base sequence (C) is used. The base sequence shown in SEQ ID NO: 6 is a partial base sequence in the RPB2 gene of Penicillium brefeldinum.
(C) the base sequence shown in SEQ ID NO: 6 or its complementary sequence (D) the base sequence shown in SEQ ID NO: 6 in which one or several bases are deleted, substituted, inserted or added, or its complementary sequence

  In the above (D), the number of one or several bases is preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, and even more preferably 1.

In addition, the base sequence (D1) below is also preferable as the base sequence (D).
(D1) A nucleotide sequence having 85% or more identity with the nucleotide sequence shown in SEQ ID NO: 6, or a complementary sequence thereof In (D1), the identity of the nucleotide sequence is preferably 90% or more, and 95% More preferably, it is more preferably 97% or more, still more preferably 99% or more. The method for calculating the identity of base sequences has been described above.

Specifically, as a method for detecting Penicillium brefeldinum using the base sequence (C) or (D), an oligo that can hybridize to the nucleic acid represented by the base sequence (C) or (D) A method of detecting by a nucleic acid amplification method using nucleotides as nucleic acid primers (hereinafter referred to as “nucleic acid amplification method”), and an oligonucleotide capable of hybridizing to the nucleic acid represented by the base sequence of (C) or (D) as a nucleic acid probe A method of detecting by hybridization (hereinafter referred to as “hybridization method”), determining the base sequence of the RPB2 gene of the subject, and including the base sequence (C) or (D) in the sequence And a confirmation method (hereinafter, “sequencing method”). Among these, from the viewpoint of rapidity, a nucleic acid amplification method or a hybridization method is preferable, and a nucleic acid amplification method is more preferable.
Hereinafter, each method will be described.

[Nucleic acid amplification method]
The detection by the nucleic acid amplification method is performed by a nucleic acid amplification reaction using a nucleic acid prepared from a specimen sample as a template and using an oligonucleotide that can hybridize to the nucleic acid represented by the base sequence (C) or (D) as a nucleic acid primer. It is carried out by amplifying a nucleic acid consisting of a part or all of the base sequence (C) or (D) and confirming the presence or absence of the amplification product. If the amplification product is confirmed, it is identified as Penicillium brefeldinum.

  The oligonucleotide (hereinafter referred to as “detection oligonucleotide”) that can hybridize to the nucleic acid represented by the base sequence (C) or (D) used as a nucleic acid primer has the base sequence (C) or (D). Oligonucleotides that are capable of hybridizing under stringent conditions to the nucleic acid represented are preferred. Examples of “stringent conditions” include 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. for 8-16 hours Examples of the conditions include constant temperature and hybridization.

The oligonucleotide for detection is more preferably an oligonucleotide consisting of a partial sequence of the base sequence (C) or (D) and satisfying the following conditions (1) to (4).
(1) The 3 ′ end is a sequence specific to Penicillium brefeldinum (2) The GC content is approximately 30 to 80% (3) The possibility of self-annealing is low (4) Tm value (melting temperature: melting temperature) is about 55 to 65 ° C. In the condition (3), “the possibility of self-annealing is low” means that the oligonucleotides are not expected to bind to each other from the base sequence of the oligonucleotide. say.

  The number of bases of the detection oligonucleotide is not particularly limited, but is preferably 13 to 30 bases, and more preferably 18 to 26 bases. The Tm value of the oligonucleotide at the time of hybridization is preferably in the range of 55 ° C to 65 ° C, and more preferably in the range of 59 ° C to 62 ° C. The GC content of the oligonucleotide is preferably 30% to 80%, more preferably 45% to 65%, and even more preferably around 55%.

More preferred as the oligonucleotide for detection is the oligonucleotide (c) or (d) below.
(C) an oligonucleotide having the base sequence shown in SEQ ID NO: 3 or a base sequence in which one or several bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 3 and Penicillium brefeldi Oligonucleotide that can hybridize to RPB2 gene region of Anam (d) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4, or one or several bases deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 4 Oligonucleotides consisting of a defined nucleotide sequence and capable of hybridizing to the RPB2 gene region of Penicillium brefeldinum

The oligonucleotides (c) and (d) correspond to the sequences from position 507 to position 531 and position 687 to position 710 of the base sequence shown in SEQ ID NO: 6, respectively.
The base sequence of the Penicillium brefeldinum RPB2 gene region in (c) and (d) is the base sequence of (C) or (D).
In the above (c) and (d), the number of one or several bases is preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, and even more preferably 1.

As the oligonucleotide (c) or (d), the following oligonucleotide (c1) or (d1) is also preferable.
(C1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 or a base sequence having 85% or more identity with the base sequence shown in SEQ ID NO: 3 and capable of hybridizing to the RPB2 gene region of Penicillium brefeldinum Oligonucleotide (d1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 or a base sequence having 85% or more identity with the base sequence shown in SEQ ID NO: 4 and hybridizing to the RPB2 gene region of Penicillium brefeldinum Oligonucleotide capable of soybean In the above (c1) or (d1), the identity of the base sequence is preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, More preferably, it is 99% or more. The method described above can be used as a method for calculating the identity of base sequences.

As the oligonucleotide (c) or (d), the following oligonucleotide (c2) or (d2) is more preferable.
(C2) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 (d2) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4

The binding mode of the detection oligonucleotide may be not only a phosphodiester bond existing in a natural nucleic acid but also a phosphoramidate bond, a phosphorothioate bond, or the like.
The detection oligonucleotide can be prepared by a usual synthesis method. For example, it can be prepared by chemical synthesis using an automatic nucleic acid synthesizer or the like based on the sequence of the detection oligonucleotide. It is also possible to excise directly from the genome of Penicillium brefeldinum using a restriction enzyme or the like, or to clone and isolate and purify the RPB2 gene and then excise 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.

  For detection by the nucleic acid amplification method, it is preferable to use a nucleic acid primer pair comprising the detection oligonucleotide. As the nucleic acid primer pair, an oligonucleotide pair consisting of the oligonucleotides (c) and (d) and an oligonucleotide pair consisting of the oligonucleotides (c1) and (d1) are preferred, and the above-mentioned (c2) and (d2) More preferred are oligonucleotide pairs consisting of oligonucleotides.

The method for amplifying a nucleic acid is not particularly limited, and includes polymerase chain reaction (PCR) method, real-time PCR method, LCR (Ligase Chain Reaction) method, SDA (Strand Displacement Amplification) method, NASBA (Nucleic Acid Sequence-based Amplification) method, Conventional nucleic acid amplification methods such as RCA (Rolling-circle amplification) and LAMP (Loop mediated isothermal amplification) can be used. In the present invention, the PCR method is preferably used from the viewpoint of rapidity.
Hereinafter, the case where an amplification reaction is performed by the PCR method will be described in detail. However, the present invention is not limited to these.

The PCR conditions are not particularly limited as long as the target DNA fragment can be amplified to a detectable level.
For example, when PCR is performed using an oligonucleotide pair consisting of the oligonucleotides (c) and (d) as a nucleic acid primer, a preferable PCR condition is a heat denaturation reaction in which a double-stranded DNA is converted into a single strand. An annealing reaction for hybridizing the primer pair to the single-stranded DNA at 50 ° C. or higher and 98 ° C. or lower for 10 seconds or longer and 60 seconds or shorter (preferably 52 ° C. or higher, more preferably 55 ° C. or higher) 65 ° C. or lower (preferably Is 63 ° C. or lower, more preferably 60 ° C. or lower) for 5 seconds or longer (preferably 10 seconds or longer) and 120 seconds or shorter (preferably 60 seconds or shorter). This is performed for 60 seconds or less, and the cycle is performed for 30 cycles or more and 35 cycles or less.

In the present invention, the nucleic acid amplification product can be confirmed by an ordinary method. For example, electrophoresis of reaction products, confirmation of the presence or absence of a band corresponding to the size of the target amplification product, method of measuring the amount of amplification product over time, method of decoding the base sequence of the amplification product, etc. Can be mentioned. Of these, a method is preferred in which the reaction product is electrophoresed after the nucleic acid amplification reaction and the presence or absence of a band corresponding to the size of the target amplification product is confirmed.
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 amplification reaction, a method of using a primer labeled with a fluorescent substance, or the like, and fluorescence intensity by binding to DNA such as ethidium bromide between amplified DNA double strands. For example, a method of inserting a fluorescent material that increases the intensity of the light is used. Of these, a method is preferred in which a fluorescent substance whose fluorescence intensity is increased by binding to DNA between the amplified DNA double strands is inserted.
When the sample contains a fungus to be detected, PCR is performed using the oligonucleotide pair of the present invention as a primer set, and the obtained PCR product is electrophoresed to confirm a DNA fragment of a specific size. . Specifically, when Penicillium brefeldinum is included in the specimen, electrophoresis of the PCR product obtained using the oligonucleotide pair consisting of the oligonucleotides (c) and (d) as a nucleic acid primer is performed. A DNA fragment of about 200 bp is confirmed. When the DNA fragment is confirmed, it can be identified that Penicillium brefeldinum is contained in the specimen.

[Hybridization method]
Detection by the hybridization method is carried out by labeling an oligonucleotide that can hybridize with the nucleic acid represented by the base sequence (C) or (D) to form a nucleic acid probe, and then hybridizing the probe with a nucleic acid prepared from a specimen sample. , By detecting the label of the hybridized probe. When the probe hybridizes, it is identified as Penicillium brefeldinum.

  As the nucleic acid probe, the detection oligonucleotide and the oligonucleotide pair described in the above-mentioned “Nucleic acid amplification method” can be used, and the preferable range is also the same. Among these, at least one kind of oligonucleotide selected from the oligonucleotides (c), (d), (c1) and (d1) is preferable, and is selected from the oligonucleotides (c2) and (d2). More preferred is at least one oligonucleotide.

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. The oligonucleotide thus labeled is hybridized with a nucleic acid extracted from a specimen sample by a conventional method under stringent conditions, and then the label of the hybridized detection oligonucleotide is measured. Penicillium brefeldinum can be detected at the species level. Examples of stringent conditions include the conditions described above. As a method for measuring the label of the nucleic acid probe hybridized with the nucleic acid, a usual method (FISH method, dot blot method, Southern blot method, Northern blot method, etc.) can be used.
The detection 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 a target nucleic acid can be captured using the label as an indication.

[Sequencing method]
Detection by the sequencing method is performed by analyzing and determining the base sequence of the RPB2 gene in the nucleic acid prepared from the specimen sample, comparing the sequence with the base sequence of (C) or (D), and determining whether there is a match or difference. To identify Penicillium brefeldinum.
The method for analyzing the base sequence is not particularly limited, and a commonly used nucleic acid sequencing method 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.

  There is no restriction | limiting in particular in the sample to which the detection method of this invention is applied, The food / beverage products, the raw material of food / beverage products, an isolated microbial cell, a cultured cell, food / beverage products, or the packaging container of the raw material can be used.

  The method for preparing a nucleic acid from a sample is not particularly limited as long as it is a method capable of obtaining a nucleic acid having an amount sufficient for detection and a degree of purification, and a normal nucleic acid extraction method or a commercially available extraction kit can be used. As the nucleic acid, DNA obtained by reverse transcription of RNA in a specimen may be used. The extracted nucleic acid may be further subjected to treatments such as separation, extraction, concentration, and purification.

  The detection kit of the present invention contains at least one selected from the oligonucleotide for detection and the oligonucleotide pair as a nucleic acid probe or a nucleic acid primer. In addition to the nucleic acid probe and the nucleic acid primer, the kit of the present invention includes a label detection substance, a buffer solution, a nucleic acid synthase (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), an enzyme substrate (dNTP, rNTP, etc.) depending on the purpose. ) And the like, which are usually used for detecting fungi. The kit of the present invention may contain a positive control (positive control) for confirming that detection is possible with the oligonucleotide or oligonucleotide pair of the present invention. Examples of the positive control include genomic DNA containing a region amplified by the method of the present invention.

  Regarding the above-described embodiments, the present invention further discloses the following detection methods, oligonucleotides or oligonucleotide pairs, detection kits, uses, and methods.

<1> A step of detecting Penicillium lapidosum using an oligonucleotide pair comprising the following oligonucleotides (a) and (b), and an oligonucleotide comprising the following oligonucleotides (c) and (d) A method for detecting a thermostable Penicillium fungus, comprising at least one step selected from the step of detecting Penicillium brefeldianum using a nucleotide pair.
(A) One or several oligonucleotides (preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, even more preferably) in the oligonucleotide having the base sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 1. (1) an oligonucleotide having a base sequence in which a base is deleted, substituted, inserted or added and capable of hybridizing to the RPB2 gene region of Penicillium rapidosum (b) an oligonucleotide having a base sequence shown in SEQ ID NO: 2, Alternatively, one or several (preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, more preferably 1) bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 2. Oligonucleotides consisting of a defined nucleotide sequence and capable of hybridizing to the RPB2 gene region of Penicillium rapidum c) Oligonucleotide having the base sequence shown in SEQ ID NO: 3 or 1 or several in the base sequence shown in SEQ ID NO: 3 (preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, more preferably 1) an oligonucleotide having a base sequence deleted, substituted, inserted or added and capable of hybridizing to the RPB2 gene region of Penicillium brefeldinum (d) an oligonucleotide having the base sequence shown in SEQ ID NO: 4 Or 1 or several (preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, more preferably 1) of the base sequence shown in SEQ ID NO: 4 is deleted, substituted, inserted or It consists of an added base sequence and can hybridize to the RPB2 gene region of Penicillium brefeldinum Oligo nucleotide

<2> The detection method according to <1>, wherein the oligonucleotides (a) to (d) are the oligonucleotides (a1) to (d1) below.
(A1) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 or 85% or more identity with the base sequence shown in SEQ ID NO: 1 (preferably 90% or more, more preferably 95% or more, more preferably 97% or more More preferably 99% or more) and an oligonucleotide that can hybridize to the RPB2 gene region of Penicillium rapidum (b1) RPB2 of Penicillium Rapidosum comprising a base sequence having 85% or more identity with the base sequence (preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and still more preferably 99% or more) Oligonucleotide (c1) SEQ ID NO: capable of hybridizing to gene region 85% or more of the nucleotide sequence shown in SEQ ID NO: 3 or preferably the nucleotide sequence shown in SEQ ID NO: 3 (preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, still more preferably 99 % Or more) and can hybridize to the RPB2 gene region of Penicillium brefeldinum (d1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4, or a base sequence shown in SEQ ID NO: 4 and 85 % Of the RPB2 gene region of Penicillium brefeldinum comprising a base sequence having at least% identity (preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 99% or more) That can hybridize to

<3> The detection method according to <1> or <2>, wherein the detection step includes a step of performing nucleic acid amplification using the oligonucleotide pair as a primer pair, and a step of detecting an amplification product.
<4> The detection method according to <3>, wherein the nucleic acid amplification is performed by a polymerase chain reaction (PCR) method.
<5> In the step of detecting the amplification product, when the amplification product is confirmed using the oligonucleotide pair consisting of the oligonucleotides of (a) and (b), it is identified as Penicillium rapidum, (c) And when an amplification product is confirmed using the oligonucleotide pair consisting of the oligonucleotide of (d), it is identified as Penicillium brefeldinum, the oligonucleotide pair consisting of the oligonucleotides of (a) and (b) and The detection according to <2> or <3>, wherein when an amplification product is confirmed using an oligonucleotide pair consisting of the oligonucleotides of (c) and (d), it is identified as Penicillium rapidum or Penicillium brefeldinum Method.
<6> The detection method according to any one of <3> to <5>, wherein the region amplified by the nucleic acid amplification is a part of the RPB2 gene region.

<7> The detection method according to any one of <1> to <6>, wherein the base sequence of the RPB2 gene region of the Penicillium rapidum is the base sequence of (A) or (B) below.
(A) 1 or several (preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, in the base sequence shown in SEQ ID NO: 5 or its complementary sequence (B) SEQ ID NO: 5 <7> The base sequence in which (preferably 1) base is deleted, substituted, inserted or added, or its complementary sequence <8> The base sequence of (B) is the base sequence of (B1) below <7> The detection method according to item.
(B1) a base sequence having 85% or more identity (preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, even more preferably 99% or more) with the base sequence shown in SEQ ID NO: 5 Or its complementary sequence

<9> The detection method according to any one of <1> to <6>, wherein the base sequence of the RPB2 gene region of Penicillium brefeldinum is the base sequence of (C) or (D) below.
(C) 1 or several (preferably 1 or more, 3 or less, more preferably 1 or more and 2 or less, in the base sequence shown in SEQ ID NO: 6 or its complementary sequence (D) SEQ ID NO: 6; <9> The base sequence in which (preferably 1) base is deleted, substituted, inserted or added, or its complementary sequence <10> The base sequence of (D) is <9> The detection method according to item.
(D1) A base sequence having 85% or more identity (preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, even more preferably 99% or more) with the base sequence shown in SEQ ID NO: 6. Or its complementary sequence

<11> At least one kind of oligonucleotide selected from the group consisting of the oligonucleotides (a) to (d), an oligonucleotide pair consisting of the oligonucleotides (a) and (b), or (c) and An oligonucleotide pair consisting of the oligonucleotide of (d).
<12> The oligonucleotide or the oligonucleotide pair according to <11>, wherein the oligonucleotides (a) to (d) are the oligonucleotides (a1) to (d1).
<13> The oligonucleotide or oligonucleotide pair according to <11> or <12>, wherein the oligonucleotides (a) to (d) are nucleic acid probes or nucleic acid primers.

<14> A thermostable Penicillium fungus detection kit comprising at least one selected from the oligonucleotide and the oligonucleotide pair according to any one of <11> to <13>.

<15> A method for detecting Penicillium rapidum, wherein the nucleic acid represented by the base sequence (A) or (B) is used to identify penicillium rapidum.
<16> An oligo that can hybridize to a region in the nucleic acid represented by the base sequence (A) or (B), which satisfies the following four conditions (1) to (4): The detection method according to <15>, wherein nucleotides are used.
(1) The 3 ′ end is a sequence specific to penicillium rapidum (2) The GC content is about 30 to 80% (3) The possibility of self-annealing is low (4) The Tm value is about 55 to 65 Around ℃

<17> A method for detecting Penicillium brefeldinum, wherein the nucleic acid represented by the base sequence (C) or (D) is used to identify Penicillium brefeldinum.
<18> An oligo that can hybridize to a region in the nucleic acid represented by the base sequence of (C) or (D), which satisfies the following four conditions (1) to (4): The detection method according to <17>, wherein nucleotides are used.
(1) The 3 ′ end is a sequence specific to Penicillium brefeldinum (2) The GC content is about 30 to 80% (3) The possibility of self-annealing is low (4) The Tm value is about 55 ~ 65 ℃

<19> Use of the oligonucleotides (a) and (b) as a nucleic acid primer for detection of penicillium / rapidosum.
<20> Use of the oligonucleotides (a) and (b) above for the production of a nucleic acid primer for detection of Penicillium rapidum.
<21> A method of using the oligonucleotides (a) and (b) as a nucleic acid primer for detecting Penicillium rapidum.

<22> Use of the oligonucleotides (c) and (d) as a nucleic acid primer for detection of Penicillium brefeldinum.
<23> Use of the oligonucleotides (c) and (d) above for the production of a nucleic acid primer for detection of Penicillium brefeldinum.
<24> A method of using the oligonucleotides (c) and (d) as a nucleic acid primer for detecting Penicillium brefeldinum.

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

Example 1. RPB2 gene analysis of Penicillium fungus The base sequence of the RPB2 gene was determined for Penicillium fungi including Penicillium rapidum and Penicillium brefeldinum and related fungi by the following method.
Gentolic DNA was extracted from the fungal cells cultured in a dark place on a potato dextrose agar slant at 25 ° C. for 7 days using Gen Toru-kun (trade name, manufactured by Takara Bio Inc.). PCR amplification of the target site was performed using PuReTaq ™ Ready-To-Go PCR Beads (manufactured by GE Healthcare) as a DNA polymerase, RPB2 5F (5′-gaygaymgwgatcayttygg-3 ′: SEQ ID NO: 7), RPB2 7CR (primary as a primer) 5′-cccatrgcttgyttrcccat-3 ′: SEQ ID NO: 8) was used. In the sequences of SEQ ID NOs: 7 and 8, “y” is “t or c”, “m” is “a or c”, “w” is “a or t”, “r” is “g” Or a ”respectively. Amplification was performed by heat denaturation: 94 ° C. for 1 minute, annealing: 51 ° C. for 1 minute, extension reaction; 72 ° C. for 1 minute, 35 cycles, followed by extension reaction at 72 ° C. for 10 minutes. PCR products were labeled using a BigDye terminator Cycle sequencing kit (Applied Biosystems) and electrophoresed with an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Software ATGC Ver.4 (manufactured by Genetyx) was used to determine the base sequence from the fluorescence signal during electrophoresis, and software ATSQ (manufactured by Genetyx) was used to bind the forward and reverse base sequences.

2. Preparation of molecular phylogenetic tree Clustal X (http: /) based on the RPB2 gene sequence of each fungus determined by the sequencing analysis of and the RPB2 gene sequences of Penicillium and related fungi obtained from the DNA data bank of Japan (DDBJ) Phylogenetic analysis was performed using /www.clustal.org/). Based on the phylogenetic analysis results, a molecular phylogenetic tree was created using NJ-plot.
The created molecular phylogenetic tree is shown in FIG.

  As shown in FIG. 1, it has been clarified that Penicillium rapidum and Penicillium brefeldinum are each divided into independent branched chains.

3. Detection of Penicillium rapidum (1) Primer design 1) The nucleotide sequence region of the RPB2 gene of Penicillium rapidum obtained in 1), 3) the 3 ′ end is a sequence specific to Penicillium rapidum, 2) the GC content is approximately 30-80%, 3) self A partial region satisfying the four conditions of 4) where the possibility of annealing is low and 4) the Tm value is approximately 55 to 65 ° C. was examined.
Based on the partial base sequence information corresponding to the above conditions, one primer pair consisting of a forward primer (SEQ ID NO: 1) and a reverse primer (SEQ ID NO: 2) was designed. The primer was purchased from Hokkaido System Science Co., Ltd. (demineralized product, 0.2 μmol scale) and purchased.
Subsequently, the effectiveness of the designed primer for penicillium rapidum detection was verified.

(2) Preparation of specimens For primer evaluation, Penicillium fungi and related fungi including Penicillium rapidosum described in Tables 1-1 and 1-2, Penicillium rapidosum described in Table 2, thermostable fungi, other Common fungi were used. These fungi were obtained and used by the Chiba University Research Center for Fungal Medicine and managed by IFM numbers.
Each strain was cultured under optimal growth conditions. Culture, potato dextrose medium (trade name: Pearlcore potato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.) using, Byssochlamys spp., Talaromyces sp., In Hamigera genera, 25 ℃ except Neosartorya the genus is the optimal growth temperature, Byssochlamys genus, Talaromyces genus, Hamigera genus and Neosartorya genus were cultured at 30 ° C. which is the optimum temperature for growth for 7 days.

(3) Preparation of genomic DNA Each cultured microbial cell is recovered from the agar medium using a platinum loop, and is collected from the microbial cell using a genomic DNA preparation kit (trade name: Gen Toru-kun, manufactured by Takara Bio Inc.). A genomic DNA solution was prepared. The concentration of the DNA solution was adjusted to 50 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, and one reagent of a PCR reaction kit containing DNA polymerase (trade name: illustra PuRe Taq PCR kit, manufactured by GE Healthcare), sterile distilled water 23 μl was mixed, a primer (5′-gggtcgttgtcaatagtgaaaaga-3 ′) consisting of 0.5 μl of a primer consisting of the base sequence shown in SEQ ID NO: 1 (5′-gcctagcgagcctatcattgac-3 ′, 20 pmol / μl) and a base sequence shown in SEQ ID NO: 2 20 pmol / μl) 0.5 μl was added to prepare a 25 μl PCR solution.
This PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) a heat denaturation reaction at 97 ° C. for 1 minute, (ii) an annealing reaction at 61 ° C. for 1 minute, and (iii) an extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified DNA product After the PCR reaction, 5 μl was taken from the PCR reaction solution and electrophoresed on a 2% agarose gel. Moreover, EZ Load 100 bp Molecular Ruler (made by BIO RAD) was used as a molecular weight marker. After electrophoresis, the DNA was stained with SYBR Safe DNA gel stain in 1 × TAE (manufactured by Invitrogen), and the presence or absence of the amplified DNA fragment (RPB2 gene fragment) was confirmed.
The electropherograms of the agarose gel are shown in FIGS. FIG. 2 shows the results of electrophoresis of PCR reaction products obtained using the genomic DNA extracted from each fungus in Tables 1-1 and 1-2 as a template. FIG. 3 shows the genomic DNA extracted from each fungus in Table 2 as a template. The electrophoresis results of the obtained PCR reaction products are shown respectively. In addition, the lane number in each figure respond | corresponds to the sample number described in each table | surface.

As a result, amplification of a DNA fragment of about 280 bp was confirmed from a sample using the genomic DNA of Penicillium rapidosum as a template (lanes 1 to 3 in FIG. 2, 1 and 2 lanes in FIG. 3). On the other hand, amplification of DNA fragments was not confirmed in samples using genomic DNA of Penicillium genus other than Penicillium / rapidosum and other fungi as a template.
From the above results, it was found that only the penicillium rapidum can be specifically detected by the primer pair consisting of the oligonucleotides of SEQ ID NOs: 1 and 2.

4). Detection of Penicillium brefeldinum (1) Primer design 1) The base sequence region of the RPB2 gene of Penicillium brefeldinum obtained in 1), the 3 ′ end is a sequence specific to Penicillium brefeldinum, and 2) the GC content is approximately 30 to 80%. A partial region satisfying the four conditions of 3) low possibility of self-annealing and 4) a Tm value of approximately 55 to 65 ° C. was examined.
Based on the partial base sequence information corresponding to the above conditions, one primer pair consisting of a forward primer (SEQ ID NO: 3) and a reverse primer (SEQ ID NO: 4) was designed. The primer was purchased from Hokkaido System Science Co., Ltd. (demineralized product, 0.2 μmol scale) and purchased.
Subsequently, the effectiveness of the designed primer for detecting Penicillium brefeldinum was verified.

(2) Preparation of specimen For primer evaluation, Penicillium genus fungi including Penicillium brefeldinum described in Tables 3-1 and 3-2 and related fungi, Penicillium brefeldinum described in Table 2 above, heat resistance Sex fungi and other common fungi were used. These fungi were obtained and used by the Chiba University Research Center for Fungal Medicine and managed by IFM numbers.
Each strain was cultured under optimal growth conditions. Cultivation was performed using a potato dextrose medium (trade name: Pearl Core potato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.) at 25 ° C., which is the optimum temperature for growth, for 7 days.

(3) Preparation of genomic DNA Each cultured cell was recovered from the agar medium using a platinum loop.
A genomic DNA solution was prepared from the cells using a genomic DNA preparation kit (trade name: Gen Toru-kun, manufactured by Takara Bio Inc.). The concentration of the DNA solution was adjusted to 2 ng / μl.

(4) PCR reaction As a DNA template, 1 μl of the genomic DNA solution prepared above, and one reagent of a PCR reaction kit containing DNA polymerase (trade name: illustra PuRe Taq PCR kit, manufactured by GE Healthcare), sterile distilled water 23 μl was mixed, primer consisting of the base sequence shown in SEQ ID NO: 3: Ebre_F3 (5′-tgaccccacccatctagtgaacaca-3 ′, 20 pmol / μl) 0.5 μl and primer consisting of the base sequence shown in SEQ ID NO: 4: Ebre_R3 (5′- (atgtgctccttgttgaggacgaga-3 ′, 20 pmol / μl) 0.5 μl was added to prepare a 25 μl PCR solution.
This PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions include 35 cycles of (i) heat denaturation reaction at 97 ° C. for 1 minute, (ii) annealing reaction at 61 ° C. for 1 minute, and (iii) extension reaction at 72 ° C. for 1 minute. Cycled.

(5) Confirmation of amplified DNA product After the PCR reaction, 5 μl was taken from the PCR reaction solution and electrophoresed on a 2% agarose gel. Moreover, EZ Load 100 bp Molecular Ruler (made by BIO RAD) was used as a molecular weight marker. After electrophoresis, the DNA was stained with SYBR Safe DNA gel stain in 1 × TAE (manufactured by Invitrogen), and the presence or absence of the amplified DNA fragment (RPB2 gene fragment) was confirmed.
The electropherograms of the agarose gel are shown in FIGS. FIG. 4 shows the electrophoresis results of PCR reaction products obtained using the genomic DNA extracted from each fungus in Tables 3-1 and 3-2 as a template, and FIG. 5 shows the genomic DNA extracted from each fungus in Table 2 as a template. The electrophoresis results of the obtained PCR reaction products are shown respectively. In addition, the lane number in each figure respond | corresponds to the sample number described in each table | surface.

As a result, amplification of a DNA fragment of about 200 bp was confirmed in samples using the genomic DNA of Penicillium brefeldinum (1 lane and 2 lanes in FIG. 4, 3 lanes and 4 lanes in FIG. 5). On the other hand, amplification of DNA fragments was not confirmed in samples using the genomic DNAs of Penicillium and other fungi other than Penicillium brefeldinum as templates.
From the above results, it was found that only Penicillium brefeldinum can be specifically detected by the primer pair consisting of the oligonucleotides of SEQ ID NOs: 3 and 4.

5. Verification of annealing temperature conditions Next, PCR was performed using a primer pair for detecting Penicillium rapidum consisting of oligonucleotides of SEQ ID NOs: 1 and 2, or a primer pair for detecting Penicillium brefeldinum consisting of oligonucleotides of SEQ ID NOs: 3 and 4. The reaction was performed and the detection specificity when the annealing temperature condition was changed was verified.

(1) Preparation of genomic DNA from bacterial cells The fungi shown in Table 4 were used. In addition, the fungi in lane numbers 1 to 3 in Table 4 were used for the primer pair for Penicillium / rapidosum detection, and the fungus in lane numbers 5 to 7 in Table 4 was used for the primer pair for Penicillium / brefeldinum detection.
Each bacterial cell is as described in 1. above. After culturing in the same manner as above, the cells were collected using platinum ears, and a genomic DNA solution was prepared from the cells using Gen Toru-kun (trade name, manufactured by Takara Bio Inc.).

(2) PCR reaction As a DNA template, for the fungi of lane numbers 1 to 3 in Table 4, 1 μl of the genomic DNA solution prepared above and a PCR reaction kit containing DNA polymerase (trade name: SapphireAmp ™ Fast PCR Master Mix, Takara Bio 25 μl and sterile distilled water 23 μl are mixed, 0.5 μl of a primer consisting of the base sequence shown in SEQ ID NO: 1 (20 pmol / μl) and a primer consisting of the base sequence shown in SEQ ID NO: 2 (20 pmol / μl) 0.5 μl was added to prepare 50 μl of PCR solution. For fungi in lane numbers 5 to 7 in Table 4, 1 μl of the genomic DNA solution prepared above and a PCR reaction kit (trade name: illustra PuRe Taq PCR kit, manufactured by GE Healthcare) containing sterile DNA 23 μl of water was added, 0.5 μl of a primer consisting of the base sequence shown in SEQ ID NO: 3 (20 pmol / μl) and 0.5 μl of a primer consisting of the base sequence shown in SEQ ID NO: 4 (20 pmol / μl) were added, and 25 μl of PCR was added. A solution was prepared.
This PCR solution was subjected to gene amplification using an automatic gene amplification apparatus thermal cycler DICE (Takara Bio). PCR reaction conditions were (i) a heat denaturation reaction at 97 ° C. for 5 to 60 seconds, (ii) an annealing reaction at 55 to 64 ° C. for 5 to 60 seconds, and (iii) extension at 72 ° C. for 5 to 60 seconds. After 35 cycles of one reaction, 72 ° C. extension reaction was carried out for 5 minutes.

(3) Confirmation of amplified DNA product After the PCR reaction, 5 μl was fractioned from the PCR reaction solution and electrophoresed on a 2% agarose gel. Moreover, EZ Load 100 bp Molecular Ruler (made by BIO RAD) was used as a molecular weight marker. After electrophoresis, the DNA was stained with SYBR Safe DNA gel stain in 1 × TAE (manufactured by Invitrogen), and the presence or absence of the amplified DNA fragment (RPB2 gene fragment) was confirmed.
Electropherograms of agarose gels of PCR products using the primers shown in SEQ ID NOs: 1 and 2 are shown in FIGS. 6 and 7, and electrophoretic diagrams of agarose gels of PCR products using the primers shown in SEQ ID NOs: 3 and 4 are shown. 8 and FIG. FIG. 6 shows annealing temperatures 55 ° C., 56 ° C., 57 ° C., 58 ° C. and 59 ° C. from the left. FIG. 7 shows the electrophoresis results of the PCR reaction at annealing temperatures of 60 ° C., 61 ° C., 62 ° C., 63 ° C. and 64 ° C. from the left. FIG. 8 shows annealing temperatures 55 ° C., 56 ° C., 57 ° C., 58 ° C., 59 ° C. from the left. FIG. 9 shows the electrophoresis results of PCR reaction at annealing temperatures of 60 ° C., 61 ° C., 62 ° C., 63 ° C. and 64 ° C. from the left. The lane numbers in each figure correspond to the lane numbers in Table 4.

As a result of PCR using a primer pair for detecting penicillium rapidosam consisting of the oligonucleotides of SEQ ID NOS: 1 and 2, specific for only samples using penicillium rapidosam genomic DNA at all annealing temperatures from 55 ° C to 64 ° C A typical band was confirmed (lane 1 in FIGS. 6 and 7).
Moreover, as a result of PCR using a primer pair for detection of Penicillium brefeldinum consisting of the oligonucleotides of SEQ ID NOs: 3 and 4, the genomic DNA of Penicillium brefeldinum was templated at all annealing temperatures from 55 ° C to 64 ° C. A specific band was confirmed only for the sample (lane 5 in FIGS. 8 and 9).

Claims (10)

A step of detecting Penicillium lapidosum using an oligonucleotide pair comprising the following oligonucleotides (a) and (b), and an oligonucleotide pair comprising the following oligonucleotides (c) and (d): A method for detecting a heat-resistant Penicillium fungus, comprising at least one step selected from the steps of using Penicillium brefeldianum .
(A) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 or an RPB2 gene of Penicillium rapidum comprising a base sequence in which one base is deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 1 Oligonucleotide that can hybridize to the region (b) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 or a base sequence in which one base is deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 2 And an oligonucleotide that can hybridize to the RPB2 gene region of Penicillium rapidosum (c) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3, or one base in the base sequence shown in SEQ ID NO: 3 is deleted, substituted, inserted, or It consists of an added base sequence and Penicillium brefeldi Oligonucleotide that can hybridize to the RPB2 gene region of Nam (d) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4, or one base is deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 4 Oligonucleotide consisting of a base sequence and capable of hybridizing to the RPB2 gene region of Penicillium brefeldinum   The detection method according to claim 1, wherein the detection step includes a step of performing nucleic acid amplification using the oligonucleotide pair as a primer pair, and a step of detecting an amplification product.   The detection method according to claim 2, wherein the nucleic acid amplification is performed by a polymerase chain reaction (PCR) method.   In the step of detecting the amplification product, when the amplification product is confirmed using the oligonucleotide pair consisting of the oligonucleotides of (a) and (b), it is identified as Penicillium rapidum, and (c) and (d) When the amplification product is confirmed using the oligonucleotide pair consisting of the oligonucleotide of (a), it is identified as Penicillium brefeldinum, the oligonucleotide pair consisting of the oligonucleotide of (a) and (b) and (c) The detection method according to claim 2 or 3, wherein when an amplification product is confirmed using an oligonucleotide pair comprising the oligonucleotide of (d), it is identified as Penicillium rapidum or Penicillium brefeldinum.   The detection method according to any one of claims 2 to 4, wherein the region amplified by the nucleic acid amplification is a part of the RPB2 gene region. The detection method according to any one of Claims 1 to 5, wherein the base sequence of the RPB2 gene region of Penicillium rapidum is the base sequence of (A ) below.
(A) nucleotide sequence or its complementary sequence shown in SEQ ID NO: 5 The detection method according to any one of claims 1 to 5, wherein the base sequence of the RPB2 gene region of Penicillium brefeldinum is the following base sequence (C ) .
(C) a nucleotide sequence or its complementary sequence shown in SEQ ID NO: 6 Under Symbol (a) and (b) the oligonucleotide pair consisting of an oligonucleotide, or (c) below and oligonucleotide pair consisting of an oligonucleotide of (d).
(A) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 or an RPB2 gene of Penicillium rapidum comprising a base sequence in which one base is deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 1 Oligonucleotide that can hybridize to the region (b) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 or a base sequence in which one base is deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 2 And an oligonucleotide that can hybridize to the RPB2 gene region of Penicillium rapidosum (c) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3, or one base in the base sequence shown in SEQ ID NO: 3 is deleted, substituted, inserted, or It consists of an added base sequence and Penicillium brefeldi Oligonucleotide that can hybridize to the RPB2 gene region of Nam (d) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4, or one base is deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 4 Oligonucleotide consisting of a base sequence and capable of hybridizing to the RPB2 gene region of Penicillium brefeldinum Wherein (a) ~ oligonucleotide (d) is a nucleic acid primer, oligonucleotide pair of claim 8. Heat resistance Penicillium fungi detection kit comprising at least one selected from claims 8 or 9, wherein the oligonucleotide pairs.