JP6400981B2 - Oligonucleotide and primer pair used for detection of species belonging to the genus Talalomyces, and detection kit and detection method for species belonging to the genus Talalomyces - Google Patents

Description

The present invention relates to an oligonucleotide and a primer pair used for detecting a species belonging to the genus Talaromyces, which is a kind of heat-resistant mold, and a detection kit and a detection method for the species belonging to the genus Talaromyces.

  Fungi containing fungi are widely distributed in nature, such as heat-resistant strains that cause problems in production of foods and soft drinks, and strains that produce harmful mycotoxins that have carcinogenic properties. Including. In particular, killing heat-resistant fungi is difficult in canned fruits, juices, jams and the like that are pasteurized at 100 ° C. or lower. Therefore, it is extremely important to manage and control these fungi by preventing the introduction of heat-resistant fungi from the raw materials and the production environment and objectively evaluating the possibility of the contamination.

Heat fungi are generally Byssochlamys (Byssochlamys) genus Hamigera (Hamigera) genus Neosartorya (Neosartorya) genus, such as Talaromyces genus, those belonging to ascomycetes fungi. Among the species belonging to the genus Talalomyces, Talalomyces macrosporus and Talalomyces trachyspermus can actually be detected from acidic foods such as canned fruits, juices, and jams. (See Non-Patent Document 1 Table 1).

  Thus, Patent Document 1, Patent Document 2, and Non-Patent Document 2 describe methods for detecting heat-resistant fungi such as Talamomyces using nucleic acids.

JP 2010-4876 A JP 2010-4878 A

Shunichi Udagawa, “Problems of Food Mycology-Hazard from Heat-resistant Molds”, Mycotoxins, Japan Mycotoxin Society, 2000, vol. 50 No. 1, p. 3-p. 11 S. Nakayama, “Rapid identification and identification method of heat-resistant mold using PCR”, soft drink technical data, general soft drink industry association, April 2012, No. 1, p. 43-60

  However, with the detection methods described in Patent Document 1 and Patent Document 2, it has been difficult to specifically detect Talaromyces macrosporus and Tallalomyces traxpermus, which are species belonging to the genus Talaromyces. For this reason, it has been difficult to take effective measures against these bacteria, which are considered to be particularly problematic.

  In view of the circumstances as described above, an object of the present invention is to provide an oligonucleotide, a primer pair, a detection kit, and a detection method capable of specifically detecting a species belonging to the genus Talalomyces.

  The inventors of the present invention have intensively studied to achieve the above object. As a result, the present inventors have found an oligonucleotide, a primer pair, a detection kit, and a detection method that can detect species-specific and sensitively Talaromyces macrosporus and Tallalomyces traxpermus, which are species belonging to the genus Talalomyces, and complete the present invention. It came to.

That is, the present invention
(1) An oligonucleotide used for detection of a species belonging to the genus Tallaromyces,
The following (A) and the following (C) and (the following (C) used when detecting Talaromyces traxpermus, or the oligonucleotide represented by any one of the following base sequences (A) and (B) used to detect Talaromyces macrosporus An oligonucleotide represented by any one of the base sequences of D).
(A) The base sequence described in SEQ ID NO: 1 or its complementary sequence, or one or several bases in the base sequence or its complementary sequence is deleted, substituted or added, and Talaromyces macrosporus can be detected Base sequence (B) The base sequence shown in SEQ ID NO: 2 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof are deleted, substituted or added, and Talaromyces macrosporus can be detected. Possible base sequence (C) Detection of Tallalomyces traxpermus with one or several bases deleted, substituted or added in the base sequence described in SEQ ID NO: 3 or its complementary sequence, or in the base sequence or its complementary sequence (D) the nucleotide sequence set forth in SEQ ID NO: 4 or a nucleotide sequence thereof A complementary sequence, or a nucleotide sequence in which one or several bases have been deleted, substituted or added in the nucleotide sequence or its complementary sequence and can detect Talamomyces traxpermus (2) Used for detection of species belonging to the genus Talalomyces An oligonucleotide comprising:
An oligonucleotide capable of species-specific hybridization to a gene region encoding the isocitrate lyase of Tallalomyces macrosporus or Tallalomyces traxpermus.
(3) An oligonucleotide used for detection of a species belonging to the genus Talamomyces according to (2),
The following (K) and the following (K) used for detecting the oligonucleotide represented by any one of the following base sequences (I) and (J), or Tallaromyces traxpermus, which is used when detecting Tallaromyces macrosporus ( L) an oligonucleotide represented by any one of the nucleotide sequences.
(I) One or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 9 or a complementary sequence thereof, or the base sequence or the complementary sequence thereof, and Talaromyces macrosporus can be detected. Base sequence (J) The base sequence described in SEQ ID NO: 10 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof are deleted, substituted, or added, and Thallomyces macrosporus can be detected. Possible base sequence (K) Detection of Talalomyces traxpermus, wherein one or several bases are deleted, substituted or added in the base sequence described in SEQ ID NO: 11 or a complementary sequence thereof, or in the base sequence or the complementary sequence thereof The base sequence described in SEQ ID NO: 12 The complementary sequence, or the nucleotide sequence in which one or several bases have been deleted, substituted or added in the nucleotide sequence or the complementary sequence and can detect Talalomyces traxpermus (4) For detection of species belonging to the genus Talalomyces A primer pair used,
A primer pair consisting of an oligonucleotide represented by the base sequence of (E) below and an oligonucleotide represented by the base sequence of (F) below or a primer pair (M) below used when detecting Tallaromyces macrosporus by the PCR method A primer pair consisting of an oligonucleotide represented by the base sequence and an oligonucleotide represented by the base sequence (N) below, or the base sequence of the following (G) used when detecting Tallalomyces traxpermus by the PCR method. A primer pair comprising an oligonucleotide and an oligonucleotide represented by the base sequence of (H) below, or an oligonucleotide represented by the base sequence of (O) below and an oligonucleotide represented by the base sequence of (P) below A primer pair consisting of
(E) the base sequence described in SEQ ID NO: 1, or the base sequence in which one or several bases are deleted, substituted or added in the base sequence and Tallomyces macrosporus can be detected (F) SEQ ID NO: 2 Or a base sequence in which one or several bases are deleted, substituted or added in the base sequence, and Talaromyces macrosporus can be detected (G), the base sequence described in SEQ ID NO: 3, Or a base sequence described in SEQ ID NO: 4 in which one or several bases are deleted, substituted or added in the base sequence and Talaromyces traxpermus can be detected, or in the base sequence 1 or A base that has several bases deleted, substituted, or added and that can detect Talalomyces traxpermus Sequence (M) SEQ ID NO: 9, or a base sequence (N) SEQ ID NO in which one or several bases are deleted, substituted or added in the base sequence, and Thallomyces macrosporus can be detected Or a base sequence described in SEQ ID NO: 11, wherein one or several bases are deleted, substituted or added in the base sequence, and Talaromyces macrosporus can be detected. Or a base sequence described in SEQ ID NO: 12 in which one or several bases are deleted, substituted or added in the base sequence and Talamomyces traxpermus can be detected, or 1 in the base sequence Or several bases have been deleted, substituted or added and Talaromyces traxpermus is detected. A primer pair used for detection of a species belonging to the genus Talamomyces described in the possible base sequences (5) and (4),
A primer pair consisting of an oligonucleotide represented by the base sequence of (e) below and an oligonucleotide represented by the base sequence of (f) below or a primer pair (m) below used when detecting Tallaromyces macrosporus by the PCR method A primer pair consisting of an oligonucleotide represented by the base sequence and an oligonucleotide represented by the base sequence (n) below, or the base sequence of the following (g) used when detecting Tallalomyces traxpermus by the PCR method. A primer pair consisting of the oligonucleotide represented by the nucleotide sequence shown in the following (h) or the oligonucleotide represented by the nucleotide sequence shown in (p) below and the oligonucleotide represented by the nucleotide sequence shown in (p) below A primer pair consisting of
(E) Base sequence described in SEQ ID NO: 1 (f) Base sequence described in SEQ ID NO: 2 (g) Base sequence described in SEQ ID NO: 3 (h) Base sequence described in SEQ ID NO: 4 (m) SEQ ID NO: 9 (N) Base sequence described in SEQ ID NO: 10 (o) Base sequence described in SEQ ID NO: 11 (p) Base sequence described in SEQ ID NO: 12 (6) Kit for detection of species belonging to the genus Talaromyces Because
The oligonucleotide represented by the base sequence of (A) used when detecting Talaromyces macrosporus, the oligonucleotide represented by the base sequence of (B) used when detecting Talaromyces macrosporus, and Thallomyces macross Oligonucleotide represented by the base sequence (I) used when detecting a porus and Talaromyces macrosporus used for detecting the oligonucleotide and Talaromyces traxperumus used when detecting a macrosporus The oligonucleotide represented by the base sequence of (C) and Talaromyces traxpermus used in the case of detecting the oligonucleotide and the Talaromyces traxpermus used in the case of detecting the base sequence of (D) used in the case of Oligonucleotide represented by the base sequence of (K) used in the case of releasing, and at least one oligo of the oligonucleotide represented by the base sequence of (L) used when detecting Tallalomyces traxpermus A kit for detecting a species belonging to the genus Talalomyces comprising a nucleotide.
(7) A kit for detecting a species belonging to the genus Talamomyces according to (6),
A primer pair consisting of an oligonucleotide represented by the base sequence of (E) and an oligonucleotide represented by the base sequence of (F), which is used when detecting Tallalomyces macrosporus by PCR, PCR of Talaromyces macrosporus A primer pair comprising the oligonucleotide represented by the base sequence of (M) and the oligonucleotide represented by the base sequence of (N), and Tallalomyces traxpermus used in the case of detecting by the PCR method A primer pair consisting of the oligonucleotide represented by the base sequence of (G) and the oligonucleotide each represented by the base sequence of (H), and the Talromyces traxpermus used in the case of detecting by PCR method ( O A kit for detecting a species belonging to the genus Talaromyces comprising at least one primer pair of an oligonucleotide represented by the nucleotide sequence of () and an oligonucleotide pair represented by the nucleotide sequence of (P).
(8) A method for detecting a species belonging to the genus Talamomyces according to (7),
When detecting Talaromyces macrosporus, PCR is performed using the oligonucleotide represented by the base sequence of (E) and the oligonucleotide represented by the base sequence of (F) as a primer pair, and Talaromyces traxpermus is detected. A method for detecting a species belonging to the genus Tallaromyces, comprising performing PCR using the oligonucleotide represented by the base sequence (G) and the oligonucleotide represented by the base sequence (H) as a primer pair.
(9) A method for detecting a species belonging to the genus Tallaromyces,
When detecting Tallaromyces macrosporus, the oligonucleotide represented by the base sequence of (A), the oligonucleotide represented by the base sequence of (B), the oligonucleotide represented by the base sequence of (I), and When detecting at least one of the oligonucleotides represented by the base sequence of (J) and detecting Tallalomyces traxpermus, the oligonucleotide represented by the base sequence of (C), the base sequence of (D) At least one selected from the group consisting of the oligonucleotide represented by the base sequence (K) and the oligonucleotide represented by the base sequence (L). Detecting species to which it belongs
(10) A method for detecting a species belonging to the genus Talamomyces according to (9),
When detecting Tallaromyces macrosporus, PCR is performed using the oligonucleotide represented by the base sequence of (E) and the oligonucleotide represented by the base sequence of (F) as a primer pair, and the step (M) Including at least one step of performing PCR using the oligonucleotide represented by the base sequence and the oligonucleotide represented by the base sequence of (N) as a primer pair, and detecting (G) Talaromyces traxpermus PCR using the oligonucleotide represented by the nucleotide sequence of (H) and the oligonucleotide represented by the nucleotide sequence of (H) as a primer pair, and the oligonucleotide represented by the nucleotide sequence of (O) and (P ) Oligonucleotide represented by the base sequence Species detection methods belonging to Talaromyces genus, characterized in that it comprises at least one of the steps of the steps of performing PCR as a primer pair.
(11) A method for detecting a species belonging to the genus Talaromyces according to (10),
When detecting Talaromyces macrosporus, PCR is performed using the oligonucleotide represented by the base sequence of (e) and the oligonucleotide represented by the base sequence of (f) as a primer pair, and the step (m) Including at least one step of performing PCR using the oligonucleotide represented by the base sequence and the oligonucleotide represented by the base sequence of (n) as a primer pair, and detecting the Tallaromyces traxpermus (g) PCR using the oligonucleotide represented by the nucleotide sequence of (a) and the oligonucleotide represented by the nucleotide sequence of (h) as a primer pair, and the oligonucleotide represented by the nucleotide sequence of (o) and (p ) Oligonucleotide represented by the base sequence Species detection methods belonging to Talaromyces genus, characterized in that it comprises at least one of the steps of the steps of performing PCR as a primer pair,
It is.

  According to the present invention, it is possible to provide an oligonucleotide, a primer pair, a detection kit, and a detection method capable of specifically detecting a species belonging to the genus Tallaromyces.

It is a figure which shows reaction of a glyoxylic acid cycle. It is an electrophoresis image of the amplification product by PCR method of Test Example 1 using Example 1 according to the embodiment of the present invention. It is an electrophoresis image of the amplification product by PCR method of Test Example 1 using Example 2 according to the embodiment of the present invention. It is an electrophoresis image of the amplification product by PCR method of Test Example 2 according to the embodiment of the present invention, A shows the results using Example 1, and B shows the results using Example 2. It is an electrophoresis image of the amplification product by PCR method of Test Example 3 according to the embodiment of the present invention, A shows the result using Example 1, and B shows the result using Example 2. It is an electrophoresis image of the amplification product by PCR method of Test Example 4 according to the embodiment of the present invention, A shows the results of the strains shown in Table 8, and B shows the results of the strains shown in Table 9. It is an electrophoresis image of the amplification product by PCR method of Test Example 5 according to the embodiment of the present invention, A shows the results of the strains shown in Table 10, and B shows the results of the strains shown in Table 11. It is an electrophoresis image of the amplification product by PCR method of Test Example 6 using Example 3 according to the embodiment of the present invention. It is an electrophoresis image of the amplification product by PCR method of Test Example 6 using Example 4 according to the embodiment of the present invention. It is an electrophoretic image of the amplification product by PCR method of Test Example 7 according to the embodiment of the present invention, A shows the result using Example 3 and B shows the result using Example 4.

  Hereinafter, the present invention will be described in detail.

<Features of the present invention>
The present invention provides an oligonucleotide and a primer pair used for specifically detecting a species belonging to the genus Talaromyces, and a detection kit and a detection method capable of specifically detecting a species belonging to the genus Talaromyces. It is. Conventionally, it has been difficult to specifically detect species belonging to the genus Tallaromyces (see Patent Document 1 and Patent Document 2). Therefore, the inventors of the present invention focused on a hydrophobin gene specific to molds and mushrooms, a part of the microorganism and an isocitrate lyase gene (described later) held by the plant, and thus a species belonging to the genus Talalomyces. We have found a technology that specifically detects.

<Genus Talaromyces>
The genus Taralomyces is a kind of heat-resistant mold and belongs to the Ascomycota fungus. Ascospores are fungi that are widely present in soil in a solid state where hard seeds are wrapped in a hard shell. Some molds of the genus Talalomyces have heat resistance even at 90 ° C or higher, so it is generally sterilized at a low temperature of 100 ° C or lower, such as fruit processed foods such as canned fruits and jams, juices and green tea beverages It becomes a problem in the production of soft drinks.

<Species belonging to the genus Talaromyces>
Species belonging to Talaromyces genus, a fungus belonging to the Talaromyces genus, specifically, Talaromyces Macross Porras, Talaromyces Torakisuperumusu, Talaromyces flavus (Talaromyces flavus), Talaromyces luteus (Talaromyces luteus), Talaromyces Walt Mann knee (Talaromyces wortmannii) And Talaromyces bacilis porus .

<Talalomyces macrosporus and Talalomyces trakispermus>
In the present invention, there is provided a technique for specifically detecting Talaromyces macrosporus and Tallalomyces traxpermus from species belonging to the genus Talaromyces. Talaromyces macrosporus and Talalomyces traxpermus are more likely to be detected from fruit processed foods such as canned fruits and jams, or soft drinks such as juices and green tea beverages, compared to other bacteria belonging to the genus Talaromyces. In addition to this, heat resistance and D value are high even when compared with other bacteria belonging to the genus Taralomyces, and killing by low-temperature heating at 100 ° C. or lower is difficult (see Non-Patent Document 1 Table 1). For this reason, it is extremely important to detect the above-mentioned two bacteria in a species-specific manner in order to ensure food safety.

<Hydrophobin gene>
The hydrophobin gene is one of genes targeted for detection using the oligonucleotide or primer pair according to the present invention. The hydrophobin gene is a gene that is considered to be specific for fungi and mushrooms, and encodes a low molecular weight protein called hydrophobin. Hydrophobin is universally present on the surface of cells such as molds and mushrooms that form structures such as mycelia and fruit bodies in the air, and has a function of imparting water repellency to the surface.

<Isocitrate lyase>
Isocitrate lyase is one of the enzymes that catalyzes the reaction of the glyoxylate cycle, and is unique to the glyoxylate cycle. The glyoxylic acid cycle is a metabolic circuit found in some microorganisms and plants, and can produce malic acid and succinic acid from isocitrate, and can further produce oxaloacetic acid. (See "Life and death in a macrophage: role of the glyoxylate cycle in virulence" Eukaryotic Cell, 2002, vol.1 (5), pp.657-662)

  FIG. 1 is a diagram showing the reaction of the glyoxylic acid cycle. As shown in the figure, isocitrate lyase catalyzes a reaction for producing glyoxylate from isocitrate.

<Gene region encoding isocitrate lyase>
The gene region encoding isocitrate lyase is one of the gene regions to which the oligonucleotide and primer pair according to the present invention can hybridize. In the following description, the “gene encoding isocitrate lyase” is also simply referred to as “isocitrate lyase gene”.

  “Hybridize” means that the oligonucleotide of the present invention binds complementarily to a specific sequence in the target gene region.

<Oligonucleotide>
The oligonucleotide according to the present invention refers to DNA in which 10 to 30 bases, 15 to 25 bases, and further 18 to 20 bases are combined. The oligonucleotide according to the present invention is an oligonucleotide used for detection of a species belonging to the genus Talalomyces, and more specifically, is used for species-specific detection of Talalomyces macrosporus or Talalomyces traxpermus.

  The oligonucleotide of the present invention is an oligonucleotide that can hybridize in a species-specific manner using the hydrophobin gene of Talalomyces macrosporulus or Talalomyces traxpermus as a target. Alternatively, the oligonucleotide of the present invention is an oligonucleotide that can hybridize species-specifically to a gene region encoding the isocitrate lyase of Talaromyces macrosporus or Talaromyces traxpermus. The oligonucleotide of the present invention can be used in various molecular biological detection methods such as PCR (polymerase chain reaction) primer, hybridization nucleic acid probe, and complementary probe bound to a solid phase carrier. In addition, when using the oligonucleotide of this invention for PCR method, the oligonucleotide which concerns on this invention should just be used for at least one primer, and it is also possible to make another primer into another oligonucleotide.

  The oligonucleotide according to the present invention may be used without being labeled, or may be used as a nucleic acid probe or primer after being labeled with a radioactive substance, a fluorescent / luminescent substance, a label such as an enzyme or an enzyme substrate.

<Oligonucleotide: Oligonucleotide used when detecting Tallaromyces macrosporus>
The oligonucleotide used in detecting Talaromyces macrosporus is an oligonucleotide represented by one of the following base sequences (A) and (B), or the following (I) and (J) It is an oligonucleotide represented by any one of the base sequences. These oligonucleotides enable species-specific detection of Talaromyces macrosporus.
(A) The base sequence described in SEQ ID NO: 1 or its complementary sequence, or one or several bases in the base sequence or its complementary sequence is deleted, substituted or added, and Talaromyces macrosporus can be detected Base sequence (B) The base sequence shown in SEQ ID NO: 2 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof are deleted, substituted or added, and Talaromyces macrosporus can be detected. Possible base sequence (I) The base sequence described in SEQ ID NO: 9 or its complementary sequence, or one or several bases in the base sequence or its complementary sequence has been deleted, substituted or added, and Talaromyces macrosporus Detectable nucleotide sequence (J) SEQ ID NO: 10 nucleotide sequence or its Complement sequence, or the nucleotide sequence or one or several bases are deleted in their complementary sequence, substitution or addition by which and Talaromyces Macross Porras detection of possible base sequences

<Oligonucleotide: Oligonucleotide used for detecting Talalomyces traxpermus>
The oligonucleotide used for detecting Talalomyces traxpermus is an oligonucleotide represented by one of the following base sequences (C) and (D), or the bases (K) and (L) below. It is an oligonucleotide represented by any one of the base sequences. The oligonucleotide makes it possible to detect Talamomyces traxpermus in a species-specific manner.
(C) a base sequence of SEQ ID NO: 3 or a complementary sequence thereof, or a base in which one or several bases have been deleted, substituted or added in the base sequence or the complementary sequence thereof, and Thallomyces traxpermus can be detected Sequence (D) The base sequence described in SEQ ID NO: 4 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof is deleted, substituted or added, and Talaromyces traxpermus can be detected Base sequence (K) The base sequence described in SEQ ID NO: 11 or a complementary sequence thereof, or one or several bases in the base sequence or the complementary sequence thereof are deleted, substituted, or added, and Talaromyces traxpermus can be detected Nucleotide sequence (L) The nucleotide sequence set forth in SEQ ID NO: 12 Its complementary sequence, or the nucleotide sequence or one or several bases are deleted in their complementary sequence, substitution or addition by which and detection of Talaromyces Torakisuperumusu capable nucleotide sequence

<Base sequence described in SEQ ID NO: 1-4>
The base sequences described in SEQ ID NOs: 1 and 2 are base sequences complementary to a predetermined base sequence in the hydrophobin gene of Talaromyces macrosporus. Similarly, the base sequences described in SEQ ID NOs: 3 and 4 are base sequences that are complementary to a predetermined base sequence in the hydrophobin gene of Talalomyces traxpermus.

The base sequences described in SEQ ID NOs: 1 to 4 were searched as follows. First, the DNA region predicted to be the hydrophobin gene region of Tallalomyces macrosporulus and Tallalomyces traxpermus was amplified by the PCR method, and the base sequence of the amplified product was analyzed by sequence analysis or the like. Subsequently, it was confirmed that the analyzed nucleotide sequence showed a relatively high homology with hydrophobin genes such as Aspergillus , and the amplification products were the Talaromyces macrosporus and Talaromyces traxpermus hydrophobin genes, respectively. I inferred. Subsequently, a plurality of primer pairs including primers having the nucleotide sequences of SEQ ID NOs: 1 to 4 are designed based on the analyzed nucleotide sequences, and these primer pairs specifically identify Talaromyces macrosporus and Tallalomyces traxpermus by PCR. It was examined whether it could be detected. As a result, as described later, Talaromyces macrosporus is specifically detected by the primer pair having the nucleotide sequences of SEQ ID NOs: 1 and 2, and Talaromyces traxpermus is specifically detected by the primer pair having the nucleotide sequences of SEQ ID NOs: 3 and 4. It was confirmed that the base sequences described in SEQ ID NOs: 1 to 4 were found.

<Base sequence described in SEQ ID NO: 9-12>
The base sequences described in SEQ ID NOs: 9 and 10 are base sequences complementary to a predetermined base sequence in the isocitrate lyase gene of Talalomyces macrosporus. Similarly, the base sequences described in SEQ ID NOs: 11 and 12 are base sequences complementary to a predetermined base sequence in the isocitrate lyase gene of Talalomyces traxpermus.

The base sequences described in SEQ ID NOs: 9 to 12 were searched as follows. First, DNA regions predicted to be isocitrate lyase gene regions of Talaromyces macrosporus and Talaromyces traxpermus were each amplified by PCR, and the base sequences of the amplified products were analyzed by sequence analysis and the like. Subsequently, the analyzed nucleotide sequences Aspergillus, Fusarium (Fusarium) genus, check the Penicillium (Penicillium) to exhibit a relatively high homology to the isocitrate lyase gene of the genus such as, amplification products respectively Talaromyces MACROSS Porras and It was inferred to be the isocitrate lyase gene of Talalomyces traxpermus. Subsequently, a plurality of primer pairs including primers having the nucleotide sequences of SEQ ID NOs: 9 to 12 are designed based on the analyzed nucleotide sequences, and these primer pairs are used for species-specification of Tallalomyces macrosporus and Talalomyces traxpermus by PCR. It was examined whether it could be detected. As a result, as described later, Talaromyces macrosporus is specifically detected by the primer pair having the nucleotide sequences of SEQ ID NOs: 9 and 10, and Talaromyces traxpermus is specifically detected by the primer pair having the nucleotide sequences of SEQ ID NOs: 11 and 12. It was confirmed that the base sequences described in SEQ ID NOs: 9 to 12 were found.

<Complementary sequence>
The complementary sequence according to the present invention refers to a base sequence that can be a complementary strand of a certain base sequence. The complementary sequence of the base sequences described in SEQ ID NOs: 1 to 4 is hybridized in a species-specific manner to the hydrophobin gene region of Talaromyces macrosporus or Talaromyces traxpermus, as in the base sequences described in SEQ ID NOs: 1 to 4. Is possible.
Similarly, the complementary sequence of the base sequences described in SEQ ID NOs: 9 to 12 can hybridize species-specifically to the isocitrate lyase gene region of Talaromyces macrosporulus or Talaromyces traxpermus.
Therefore, the complementary sequence according to the present invention can be used as the oligonucleotide and primer pair according to the present invention.

<A base sequence in which one or several bases are deleted, substituted or added in the base sequence or its complementary sequence, and Talaromyces macrosporus or Talaromyces traxpermus can be detected>
In the present invention, “a base sequence in which one or several bases are deleted, substituted or added in the base sequence or a complementary sequence thereof and which can detect Talaromyces macrosporus or Talaromyces traxpermus” refers to the base sequence or a sequence thereof. A base sequence that is substantially homologous to a complementary sequence. More specifically, it is a base sequence in which 1 to 3 bases are deleted, substituted or added, and refers to a base sequence capable of species-specific detection of Talaromyces macrosporus or Talaromyces traxpermus And

<Primer pair>
The primer pair according to the present invention is a combination of two oligonucleotides and is used as a primer pair in the PCR method. The primer pair according to the present invention is a primer pair used for detection of a species belonging to the genus Talalomyces, and more specifically, used for species-specific detection of Talalomyces macrosporus or Talalomyces traxpermus.

  The primer pair according to the present invention is capable of amplifying a predetermined region in the hydrophobin gene region of Talaromyces macrosporus or Talaromyces traxpermus by PCR. Alternatively, the primer pair according to the present invention is capable of amplifying a predetermined region in the isocitrate lyase gene region of Talaromyces macrosporulus or Talaromyces traxpermus by PCR. That is, each primer of the primer pair according to the present invention can hybridize to the 3 ′ side of both strands of double-stranded DNA in the region to be amplified in the hydrophobin gene region or the isocitrate lyase gene region. Configured.

  The primer pair according to the present invention is typically used as it is as a primer pair, but can also be used after being labeled with a label. In addition, the primer pair of the present invention can be prepared, for example, at a concentration of 5 to 20 μM, further 8 to 12 μM, and used for PCR. The PCR method will be described later.

<Primer pair: Oligonucleotide used to detect Talaromyces macrosporus>
The primer pair used when detecting Tallaromyces macrosporus by PCR is a primer pair consisting of an oligonucleotide represented by the base sequence of (E) below and an oligonucleotide represented by the base sequence of (F) below, or It can be a primer pair consisting of an oligonucleotide represented by the base sequence of (M) below and an oligonucleotide represented by the base sequence of (N) below. The primer pair enables rapid and species-specific detection of Talaromyces macrosporus.
(E) the base sequence described in SEQ ID NO: 1, or the base sequence in which one or several bases are deleted, substituted or added in the base sequence and Tallomyces macrosporus can be detected (F) SEQ ID NO: 2 Or a base sequence in which one or several bases are deleted, substituted or added in the base sequence, and Talaromyces macrosporus can be detected, the base sequence described in SEQ ID NO: 9, Alternatively, in the base sequence, one or several bases are deleted, substituted or added, and the base sequence capable of detecting Talaromyces macrosporus (N) is the base sequence set forth in SEQ ID NO: 10, or 1 in the base sequence Alternatively, a base sequence in which several bases have been deleted, substituted or added, and Talaromyces macrosporus can be detected. Column

Furthermore, the primer pair which consists of the oligonucleotide represented with the base sequence of the following (e) and the oligonucleotide represented with the base sequence of the following (f) among the said primer pairs may be sufficient. Alternatively, a primer pair consisting of an oligonucleotide represented by the base sequence (m) below and an oligonucleotide represented by the base sequence (n) below may be used. By using these primer pairs, it is possible to detect Talaromyces macrosporus quickly and more accurately, for example, even when the concentration of template DNA is 10 to 100 pg / μl.
(E) Base sequence described in SEQ ID NO: 1 (f) Base sequence described in SEQ ID NO: 2 (m) Base sequence described in SEQ ID NO: 9 (n) Base sequence described in SEQ ID NO: 10

<Primer pair: Talaromyces Oligonucleotide used for detecting Trachyspermus>
The primer pair used when detecting Tallalomyces traxpermus by the PCR method is a primer pair consisting of an oligonucleotide represented by the base sequence of the following (G) and an oligonucleotide represented by the base sequence of the following (H), or It can be a primer pair consisting of an oligonucleotide represented by the base sequence (O) below and an oligonucleotide represented by the base sequence (P) below. The primer pair enables rapid and species-specific detection of Talamomyces traxpermus.
(G) the nucleotide sequence of SEQ ID NO: 3, or the nucleotide sequence (H) of SEQ ID NO: 4 in which one or several bases have been deleted, substituted or added and capable of detecting Tallalomyces traxpermus The nucleotide sequence described in the above, or the nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence and capable of detecting Tallalomyces traxpermus, or the nucleotide sequence described in SEQ ID NO: 11, or 1 or several bases are deleted, substituted or added in the base sequence, and Talaromyces traxpermus can be detected (P) The base sequence set forth in SEQ ID NO: 12, or one or several bases in the base sequence Can detect Talaromyces traxpermus Nucleotide sequence

Furthermore, the primer pair which consists of the oligonucleotide represented with the base sequence of the following (g) and the oligonucleotide represented with the base sequence of the following (h) among the said primer pairs may be sufficient. Alternatively, a primer pair consisting of an oligonucleotide represented by the base sequence (o) below and an oligonucleotide represented by the base sequence (p) below may be used. By using these primer pairs, it is possible to detect Talamomyces traxpermus rapidly and more accurately. For example, even when the concentration of the template DNA is 10 to 100 pg / μl, it is possible to detect it.
(G) Base sequence described in SEQ ID NO: 3 (h) Base sequence described in SEQ ID NO: 4 (o) Base sequence described in SEQ ID NO: 11 (p) Base sequence described in SEQ ID NO: 12

<Primer pair: forward primer / reverse primer>
When detecting Tallaromyces macrosporus by PCR, for example, among the primer pair consisting of the oligonucleotide represented by the base sequence of (E) above and the oligonucleotide represented by the base sequence of (F) above, The oligonucleotide represented by the base sequence (E) can be used as a forward primer, and the oligonucleotide represented by the base sequence (F) can be used as a reverse primer. Further, for example, among the primer pair consisting of the oligonucleotide represented by the base sequence of (e) above and the oligonucleotide represented by the base sequence of (f) above, the oligonucleotide represented by the base sequence of (e) above A nucleotide can be used as a forward primer, and an oligonucleotide represented by the base sequence (f) can be used as a reverse primer.
Similarly, of the primer pair consisting of the oligonucleotide represented by the base sequence of (M) and the oligonucleotide represented by the base sequence of (N), the oligonucleotide represented by the base sequence of (M) Can be used as a forward primer, and the oligonucleotide represented by the base sequence (N) can be used as a reverse primer. Further, for example, of the primer pair consisting of the oligonucleotide represented by the base sequence of (m) and the oligonucleotide represented by the base sequence of (n) above, the oligonucleotide represented by the base sequence of (m) above A nucleotide can be used as a forward primer, and an oligonucleotide represented by the base sequence (n) can be used as a reverse primer.

When detecting Tallalomyces traxpermus by the PCR method, for example, among the primer pair consisting of the oligonucleotide represented by the base sequence of (G) and the oligonucleotide represented by the base sequence of (H) above, The oligonucleotide represented by the base sequence (G) can be used as a forward primer, and the oligonucleotide represented by the base sequence (H) can be used as a reverse primer. Further, for example, among the primer pair consisting of the oligonucleotide represented by the base sequence of (g) and the oligonucleotide represented by the base sequence of (h), the oligonucleotide represented by the base sequence of (g) The nucleotide can be used as a forward primer, and the oligonucleotide represented by the base sequence (h) can be used as a reverse primer.
Similarly, of the primer pair consisting of the oligonucleotide represented by the base sequence of (O) and the oligonucleotide represented by the base sequence of (P), the oligonucleotide represented by the base sequence of (O) Can be used as a forward primer, and the oligonucleotide represented by the base sequence (P) can be used as a reverse primer. Further, for example, among the primer pair consisting of the oligonucleotide represented by the base sequence of (o) above and the oligonucleotide represented by the base sequence of (p) above, the oligonucleotide represented by the base sequence of (o) above The nucleotide can be used as a forward primer, and the oligonucleotide represented by the base sequence (p) can be used as a reverse primer.

<Detection kit>
The detection kit of a species belonging to the genus Tallaromyces according to the present invention includes the oligonucleotides represented by the base sequences (A) to (D), (I) to (L), and (E) to (H ), (M) to (P), (e) to (h), and (m) to (p) at least one primer pair represented by the base sequence, / Or Talaromyces traxpermus. The detection kit of the present invention includes these as primers and nucleic acid probes, and is used as a kit for various molecular biological detection methods such as a kit used for PCR (polymerase chain reaction) and a kit used for hybridization. Can be configured.

Specifically, the detection kit according to the present invention comprises an oligonucleotide represented by any one of the base sequences (A) and (B) used for detecting Talaromyces macrosporus, and Tallalomyces traxperumus. It comprises at least one oligonucleotide of the oligonucleotides represented by any one of the base sequences (C) and (D) used for detection.
Alternatively, the detection kit according to the present invention detects an oligonucleotide represented by any one of the base sequences (I) and (J), and Tallalomyces traxpermus, which is used when detecting Tallaromyces macrosporus. It may contain at least one oligonucleotide of oligonucleotides represented by any one of the base sequences (K) and (L) used in the case.

  In addition, the detection kit according to the present invention is an oligonucleotide represented by the base sequence of (A) used when detecting Talaromyces macrosporus, or the above-mentioned (B) used when detecting Talaromyces macrosporus. Oligonucleotide represented by the base sequence and the base sequence of the above (J) used when detecting the oligonucleotide represented by the base sequence of the above (I) and Talaromyces macrosporus used when detecting the Talaromyces macrosporus The oligonucleotide represented by the above, the oligonucleotide represented by the base sequence of (C) used when detecting Talaromyces traxpermus, and the base sequence of (D) used when detecting Talaromyces traxpermus Oligonucleotide Among the oligonucleotides represented by the base sequence of (K) used for detecting Talaromyces traxpermus and the oligonucleotides represented by the base sequence of (L) used when detecting Talaromyces traxpermus It can also comprise at least one oligonucleotide.

  In addition, the detection kit of the present invention includes an oligonucleotide represented by the base sequence of (E) and an oligonucleotide represented by the base sequence of (F), which are used when detecting Tallaromyces macrosporus by PCR. A primer pair consisting of an oligonucleotide represented by the base sequence of (M) and an oligonucleotide represented by the base sequence of (N) used when detecting Talaromyces macrosporus by PCR method, PCR used to detect Talalomyces traxpermus by the PCR method, a primer pair consisting of an oligonucleotide represented by the base sequence of (G) above and an oligonucleotide each represented by the base sequence of (H) above, and PCR By law It shall contain at least one primer pair of the primer pair consisting of the oligonucleotide represented by the base sequence of (O) and the oligonucleotide represented by the base sequence of (P) used for detection. You can also.

  Furthermore, the primer pair may be a primer pair consisting of an oligonucleotide represented by the base sequence of (e) above and an oligonucleotide represented by the base sequence of (f) above, and the base of (m) above It may be a primer pair consisting of an oligonucleotide represented by the sequence and an oligonucleotide represented by the base sequence (n) above. Alternatively, the primer pair may be a primer pair consisting of an oligonucleotide represented by the base sequence of (g) and an oligonucleotide represented by the base sequence of (h), and the base of (o) It may be a primer pair consisting of an oligonucleotide represented by the sequence and an oligonucleotide represented by the base sequence (p) above.

  The kit of the present invention may contain, in addition to the oligonucleotide and the primer pair, substances usually used for detecting bacteria such as a DNA extraction reagent for test bacteria, a PCR reagent, and a label detection substance. Such a kit makes it possible to more easily and species-specifically detect Talaromyces macrosporus and / or Talaromyces traxpermus.

<Method for detecting species belonging to the genus Talalomyces>
The method for detecting a species belonging to the genus Tallaromyces according to the present invention includes the oligonucleotides represented by the base sequences of (A) to (D), (I) to (L), and (E) to (H), Using at least one of the primer pairs represented by the base sequences of (M) to (P), (e) to (h), and (m) to (p), Talaromyces macrosporus and / or Or, it is a method for detecting Talaromyces trakispermus. The detection method according to the present invention can be a method for detecting Talaromyces macrosporus and / or Talalomyces traxpermus by molecular biology by bringing a nucleic acid extracted from a specimen into contact with the above-described oligonucleotide or the like. Specific examples of such a detection method include a PCR method, a hybridization method, and capture of a target nucleic acid using a capture probe.

  Specifically, in the detection method according to the present invention, when detecting Tallaromyces macrosporus, the oligonucleotide represented by the base sequence of (A) above, the oligonucleotide represented by the base sequence of (B) above, ( When at least one of the oligonucleotide represented by the base sequence of I) and the oligonucleotide represented by the base sequence of (J) above is used and Talamomyces traxpermus is detected, it is represented by the base sequence of (C) above. At least of the oligonucleotide represented by the base sequence of (D) above, the oligonucleotide represented by the base sequence of (K) above and the oligonucleotide represented by the base sequence of (L) above One is used.

  In addition, the detection method according to the present invention is a PCR method using the oligonucleotide represented by the base sequence (E) and the oligonucleotide represented by the base sequence (F) as a primer pair when detecting Tallaromyces macrosporus. When Talamomyces traxpermus is detected, PCR is performed using the oligonucleotide represented by the base sequence (G) and the oligonucleotide represented by the base sequence (H) as a primer pair.

  Similarly, the detection method according to the present invention uses the oligonucleotide represented by the base sequence of (I) above and the oligonucleotide represented by the base sequence of (J) above as a primer pair when detecting Tallaromyces macrosporus. When PCR is performed and Talamomyces traxpermus is detected, PCR may be performed using the oligonucleotide represented by the base sequence (K) and the oligonucleotide represented by the base sequence (L) as a primer pair.

  In addition, the detection method according to the present invention is a PCR method using the oligonucleotide represented by the base sequence (E) and the oligonucleotide represented by the base sequence (F) as a primer pair when detecting Tallaromyces macrosporus. And at least one step of performing PCR using the oligonucleotide represented by the base sequence of (M) and the oligonucleotide represented by the base sequence of (N) as a primer pair, When detecting Trachyspermus, PCR is performed using the oligonucleotide represented by the base sequence of (G) and the oligonucleotide represented by the base sequence of (H) as a primer pair, and the base sequence of (O) And the base sequence of (P) above It may include at least one step of performing PCR using the oligonucleotide represented by the column as a primer pair.

Furthermore, the primer pair may be a primer pair consisting of an oligonucleotide represented by the base sequence of (e) above and an oligonucleotide represented by the base sequence of (f) below, and the base of (g) above It may be a primer pair comprising an oligonucleotide represented by the sequence and an oligonucleotide represented by the base sequence (h) below.
Alternatively, the primer pair may be a primer pair consisting of an oligonucleotide represented by the base sequence of (m) above and an oligonucleotide represented by the base sequence of (n) below, and the base of (o) above A primer pair consisting of an oligonucleotide represented by the sequence and an oligonucleotide represented by the base sequence (p) below may be used.

<Method for detecting species belonging to the genus Talaromyces: PCR method>
As the PCR method according to the present invention, for example, a PCR method using DNA as a template (hereinafter referred to as a normal PCR method), an RT-PCR method, a real-time PCR method, etc. can be used. The method is preferred.

  In the PCR method, first, a PCR reaction solution is prepared by mixing a template nucleic acid (DNA or RNA), a primer pair, DNA polymerase, dNTP, a PCR reaction buffer, sterilized distilled water, and the like. Next, a thermal denaturation reaction, an annealing reaction, and an extension reaction are successively performed using a thermal cycler or the like, and a PCR reaction is performed by repeating this for a predetermined cycle. Then, the amplification product in the reaction solution after the PCR reaction is confirmed to confirm whether or not the desired DNA fragment is amplified. As the template nucleic acid, a solution obtained by directly extracting a nucleic acid from a specimen to be described later, or a solution obtained by extracting a nucleic acid from cultured bacteria can be used.

  For the primer pair in the PCR method according to the present invention, the oligonucleotide according to the present invention may be used for at least one primer, and the other primer may be another oligonucleotide. More preferably, the primer pair according to the present invention can be used as a primer pair. The concentration and amount of other elements contained in the PCR reaction solution can be determined as appropriate. PCR reaction conditions and the like can also be appropriately determined in consideration of the type of polymerase used and the like.

  The method for confirming the amplification product is not particularly limited, and for example, agarose gel electrophoresis, capillary electrophoresis, a method for determining a base sequence by sequence analysis, and the like can be applied. Among these, the agarose gel electrophoresis method and the capillary electrophoresis method are more preferable because the amplification product can be easily confirmed.

<Detection method of species belonging to the genus Talalomyces: detection method using nucleic acid extracted from specimen>
The detection method according to the present invention can detect a species belonging to the genus Tallaromyces using a nucleic acid extracted from a specimen. That is, when detecting Talaromyces macrosporus, the oligonucleotide represented by the base sequence of (A) above, the oligonucleotide represented by the base sequence of (B) above, on the nucleic acid extracted from the specimen, (I) And at least one of the oligonucleotide represented by the base sequence of (J) above is hybridized. Further, when detecting Tallalomyces traxpermus, the oligonucleotide represented by the base sequence of (C) above, the oligonucleotide represented by the base sequence of (D) above, the base of (K) above, in the extracted nucleic acid By hybridizing at least one of the oligonucleotide represented by the sequence and the oligonucleotide represented by the base sequence (L) above, a species belonging to the genus Talaromyces can be detected. For example, DNA can be used as the nucleic acid.

  In addition, the method for extracting nucleic acid from a specimen is not particularly limited, and for example, bacteria may be cultured from a specimen such as a production raw material, and nucleic acids such as DNA may be extracted from the cultured bacteria. Alternatively, the nucleic acid may be extracted directly from a sample such as a production raw material.

  In addition, when performing PCR, PCR can be performed using the extracted nucleic acid as a template. When the nucleic acid is DNA, the concentration of template DNA may be 10 pg / μl to 10 ng / μl, and preferably 100 pg / μl to 10 ng / μl.

<Method for detecting species belonging to the genus Talaromyces: specimen>
Examples of the specimen according to the present invention include a strain that may be a bacterium belonging to the genus Talalomyces, a production raw material that may contain a bacterium belonging to the genus Talalomyces, and the like. Examples of production raw materials include food production raw materials. The food may be, for example, processed fruit foods such as canned fruits and jams, soft drinks such as juices and green tea beverages, where contamination by the genus Tallaromyces is a problem.

<Method for identifying species belonging to the genus Talaromyces>
The present invention can further provide a method for identifying a species belonging to the genus Tallaromyces included in a specimen. The identification method according to the present invention includes at least one of the following steps (i) and (ii) in the nucleic acid extracted from the specimen. Step (i) includes the oligonucleotide represented by the base sequence of (A), the oligonucleotide represented by the base sequence of (B), the oligonucleotide represented by the base sequence of (I), and the above Talaromyces macrosporus is detected by hybridizing at least one of the oligonucleotides represented by the base sequence of (J). Step (ii) comprises the oligonucleotide represented by the base sequence of (C), the oligonucleotide represented by the base sequence of (D), the oligonucleotide represented by the base sequence of (K) and the above ( L. Talaromyces traxpermus is detected by hybridizing at least one of the oligonucleotides represented by the base sequence of L).

  When Talaromyces macrosporus is detected by performing step (i) above, the species contained in the specimen can be identified as Talaromyces macrosporus. On the other hand, when the above step (ii) is performed and Talaromyces traxpermus is detected, the bacteria contained in the specimen can be identified as Talalomyces traxpermus.

  Only one of step (i) and step (ii) may be performed. Or you may perform both the said step (i) and step (ii). When both are performed, there is no particular limitation on the time before and after step (i) and step (ii).

<Operational effect of the present invention>
Each of the oligonucleotide and the primer pair according to the present invention has the above-described base sequence, whereby each of Talaromyces macrosporus and Talaromyces traxpermus can be detected in a species-specific manner. As described above, Talaromyces macrosporus and Tallalomyces traxpermus are more likely to be detected from processed fruit foods and soft drinks than other bacteria belonging to the genus Talalomyces, as well as heat resistance and D value. High and difficult to kill by low temperature heating. Therefore, when these bacteria are detected, it is necessary to quickly take measures such as removal of raw materials for production and review of the production environment instead of heat sterilization. According to the present invention, Talaromyces macrosporus and Talaromyces traxpermus can be detected in a species-specific manner, so that the execution of the above treatment can be determined quickly and appropriately. Therefore, the spread of contamination can be prevented.

  Furthermore, according to the present invention, it is possible to easily detect Talaromyces macrosporus and / or Talaromyces traxpermus using a PCR method, a hybridization method, or the like. In particular, when the PCR method is used, rapid detection is possible by performing PCR using a nucleic acid extracted from a specimen and the primer pair according to the present invention. Thereby, complicated work like what is called nested PCR (refer nonpatent literature 2) becomes unnecessary.

  In addition, the detection method of the present invention is excellent in detection limit. For example, in the PCR method, as shown in Test Example 3 and Test Example 7 described later, each of the above bacteria can be detected even when the concentration of the template DNA is 10 to 100 pg / μl. Therefore, even if only a small amount of bacteria can be collected from the production raw material, it can be detected, and it is possible to contribute to thorough hygiene management.

  Hereinafter, the present invention will be further described based on examples and the like.

[Example 1: Primer for detecting Talaromyces macrosporus]
A primer pair for PCR having the nucleotide sequences of SEQ ID NOs: 1 and 2 was prepared for detecting Talaromyces macrosporus. These primers were prepared using the hydrophobin gene as a target. Each primer was commissioned to Operon Biotechnology Co., Ltd. Table 1 shows the base sequences of the created primer pairs.

[Example 2: Talaromyces traxpermus detection primer]
A primer pair for PCR having the nucleotide sequences of SEQ ID NOs: 3 and 4 was prepared as a primer for detecting Talalomyces traxpermus. These primers were prepared using the hydrophobin gene as a target. Each primer was commissioned to Operon Biotechnology Co., Ltd. in the same manner as in Example 1. Table 1 shows the base sequences of the created primer pairs.

[Test Example 1]
As Test Example 1, using the primer pair according to Example 1 and Example 2, it was confirmed whether or not Talaromyces macrosporus and Talalomyces traxpermus can be detected in a species-specific manner from the specimen bacteria.

(Sample preparation)
First, DNA was extracted from the specimen to prepare a DNA solution. As specimens, the strains shown in Tables 2 and 3 were used. Among them, Nos. 1 and 2 in Table 2 and Nos. 1, 2, 5, and 6 in Table 3 are strains belonging to Talaromyces macrosporus, Nos. 3 and 4 in Table 2, and 3, 4, 7, and 8 in Table 3. No. is a strain belonging to Talalomyces traxpermus. These strains were obtained from RIKEN BioResource Center, Microbial Materials Development Office (JCM) and National Institute of Technology and Evaluation Biotechnology Center (NBRC).

  Each strain was cultured in a PDA (Potato Dextrose Agar) medium or the like at about 25 to 30 ° C. for 7 to 10 days. From the recovered strain, DNA was extracted using the ZR Fungal / Bacterial DNA Kit (manufactured by ZYMO RESEARCH) according to the attached protocol. The extracted DNA was prepared as a DNA solution having a concentration of 10 to 50 ng / μl.

(Gene amplification by PCR method)
Subsequently, PCR for detection of Talaromyces macrosporus was performed using each of the prepared 45 types of DNA solutions and the primer pair according to Example 1. Similarly, PCR for Talaromyces traxpermus detection was performed using 45 prepared DNA solutions and the primer pair according to Example 2.

  First, each PCR reaction solution was prepared. In each PCR, only the primer and the template DNA were different, and the composition of the PCR reaction solution was the same. Table 4 shows the composition of the PCR reaction solution. The template DNA was each DNA solution prepared in “Preparation of specimen”. Quick Taq (registered trademark) HS DyeMix (manufactured by Toyobo Co., Ltd.) is a PCR reagent in which DNA polymerase (Taq DNA Polymerase), a PCR reaction buffer, and dNTP are mixed in advance. In the primer pair according to Example 1, the forward primer was a primer having the base sequence of SEQ ID NO: 1, and the reverse primer was a primer having the base sequence of SEQ ID NO: 2. In the primer pair according to Example 2, the forward primer was a primer having the base sequence of SEQ ID NO: 3, and the reverse primer was a primer having the base sequence of SEQ ID NO: 4.

  Next, the prepared PCR reaction solution was subjected to gene amplification using a thermal cycler. The conditions of the treatment in the detection of Talalomyces macrospora are (1) heat denaturation reaction at 94 ° C. for 2 minutes, (2) heat denaturation reaction at 94 ° C. for 30 seconds, (3) annealing reaction at 55 ° C. for 30 seconds, ( 4) An extension reaction at 68 ° C. for 20 seconds and (5) an extension reaction at 68 ° C. for 5 minutes, and (2) to (4) were performed for 30 cycles. The conditions for the treatment in the detection of Talalomyces traxpermus are (1) heat denaturation reaction at 94 ° C. for 2 minutes, (2) heat denaturation reaction at 94 ° C. for 30 seconds, (3) annealing reaction at 57 ° C. for 30 seconds, (4 (3) An extension reaction at 68 ° C. for 20 seconds and (5) an extension reaction at 68 ° C. for 5 minutes, and (2) to (4) were performed for 30 cycles.

(Confirmation of PCR amplification products)
Subsequently, 5 to 10 μl was taken from the PCR reaction solution and electrophoresed on an agarose gel having a concentration of 1.3% (1 to 1.5%). As the buffer, 1 × TAE buffer was used. After electrophoresis, the agarose gel was stained according to a conventional method, and the agarose gel was imaged on an ultraviolet (UV) light box. PCR amplification products were confirmed based on the captured images.

(result)
FIG. 2 is an electrophoretic image of Example 1, and FIG. 3 is an electrophoretic image of Example 2. Moreover, in each figure, A shows the result of each specimen described in Table 2, and B shows the result of each specimen described in Table 3. The numbers of the strains given in Tables 2 and 3 correspond to the numbers of the lanes given to the electrophoresis images of FIGS. M lanes indicate size markers.

  First, as shown in FIG. 2, bands of about 300 to 400 bp were confirmed only in the lanes 1 and 2 of A and the lanes 1, 2, 5, and 6 of B. The strains corresponding to these lanes are strains belonging to Talaromyces macrosporus with reference to Table 2. From this, it was confirmed that the primer pair according to Example 1 can detect Talaromyces macrosporus in a species-specific manner.

  As shown in FIG. 3, bands of about 300 to 400 bp were confirmed only in the 3rd and 4th lanes of A and the 3rd, 4th, 7th and 8th lanes of B. The strains corresponding to these lanes are strains belonging to Talaromyces trakispermus, referring to Table 3. From this, it was confirmed that the primer pair according to Example 2 can detect Talaromyces traxpermus in a species-specific manner.

[Test Example 2]
In Test Example 2, a strain isolated from the production raw material was used as a specimen, and it was confirmed whether Talaromyces macrosporus and Talalomyces traxpermus could be detected in a species-specific manner as in Test Example 1.

(Sample preparation)
First, a strain was isolated from a production raw material as a specimen, DNA was extracted, and a DNA solution was prepared. Production raw materials were pineapple, raspberry, blueberry, strawberry, and the like, and heating was performed at 80 ° C. for 30 minutes, and the surviving strain was isolated. The isolated strain was identified by morphological observation and nucleotide sequence analysis. The identified strains are shown in Table 5. Among the strains described in Table 5, Nos. 1 and 13 are strains belonging to Talalomyces macrosporus, and Nos. 2, 3, and 10 are strains belonging to Talalomyces traxpermus.

  Each strain was cultured in PDA (Potato Dextrose Agar) medium at about 25 ° C. for 7 days. One platinum loop of the isolated strain was placed in 30 μl of sterilized water, heated in a microwave for 5 minutes, and stirred well with a vortex mixer. Centrifugation was performed at 1,200 rpm for 5 minutes, and the supernatant was used as a DNA extract.

(Gene amplification by PCR method)
Subsequently, PCR was carried out using the prepared DNA solution and the primer pair according to Example 1 and Example 2 to detect Talaromyces macrosporus and Talaromyces traxpermus. Since this process is the same as that of Test Example 1, description thereof is omitted.

(Confirmation of PCR amplification products)
Subsequently, the PCR reaction solution contained in the sample tube was set in a microchip electrophoresis apparatus MCE-202 MultiNA (Shimadzu Corporation) and subjected to electrophoresis. After electrophoresis, the PCR amplification product was confirmed by data analysis software (MultiNA Viewer).

(result)
4 is an electrophoretic image of Test Example 2. A shows the results of Example 1 and B shows the results of Example 2. FIG. The numbers of the lanes attached to the electrophoretic image of FIG. 4 correspond to the numbers of the strains attached to Table 5, respectively. M1 represents a high molecular marker, and M2 represents a low molecular marker. P represents the peak of the primer in the PCR reaction solution.

  First, in FIG. 4A, a band having the same size as that of Test Example 1 was confirmed only in lanes 1 and 13. The strains corresponding to these lanes are strains belonging to Talaromyces macrosporus with reference to Table 5. From this, it was confirmed that the primer pair according to Example 1 can detect Talaromyces macrosporus in a species-specific manner.

  Next, in FIG. 4B, bands of the same size as in Test Example 1 were confirmed only in the lanes 2, 3, and 10. The strains corresponding to these lanes are strains belonging to Tallalomyces traxpermus with reference to Table 5. From this, it was confirmed that the primer pair according to Example 2 can detect Talaromyces traxpermus in a species-specific manner.

[Test Example 3]
As Test Example 3, in order to confirm the detection limit of Talaromyces macrosporus and Talaromyces traxpermus in PCR using the primer pairs according to Example 1 and Example 2, DNA solutions having different concentrations were prepared from the specimens and tested. .

(Sample preparation)
First, DNA was extracted from the specimen to prepare DNA solutions having different concentrations. As samples, Talaromyces macrosporus JCM22818 strain and Talaromyces traxpermus NBRC31757 strain were used. The sources of these strains are the same as in Test Example 1.

  DNA was extracted from each strain in the same manner as in Test Example 1. The extracted DNAs were prepared as DNA solutions having the concentrations shown in Table 6, and used as samples 1 to 5, respectively.

(Gene amplification by PCR method)
Subsequently, PCR for Talaromyces macrosporus detection was performed using the DNA solutions of Samples 1 to 5 and the primer pair according to Example 1. In addition, PCR for detecting Talaromyces traxpermus was performed using the DNA solutions of Samples 1 to 5 and the primer pair according to Example 2. Since this process is the same as that of Test Example 1, description thereof is omitted.

(Confirmation of PCR amplification products)
Subsequently, in order to confirm the PCR amplification product, electrophoresis was performed on an agarose gel having a concentration of 1.3% (1 to 1.5%). Since this step is also the same as Test Example 1, the description thereof is omitted.

(result)
FIG. 5 is an electrophoretic image of Test Example 3. A shows the results of Example 1 and B shows the results of Example 2. FIG. The numbers of the lanes attached to the electrophoretic image in FIG. 5 correspond to the numbers of the samples shown in Table 6, respectively. M lanes indicate size markers.

  First, in FIG. 5A, it was observed that the fluorescence intensity of the band decreased as the DNA concentration of the sample decreased. Thereby, it was confirmed anew that the primer pair according to Example 1 can specifically detect Talaromyces macrosporus. In addition, the bands of Samples 1 to 3 with DNA concentrations of 100 pg / μl to 10 ng / μl can be confirmed, and the bands of Samples 4 and 5 with DNA concentrations of 10 pg / μl and 1 pg / μl, respectively, cannot be confirmed. It was. From this, it was suggested that the detection limit of the primer pair according to Example 1 is 10 to 100 pg / μl in terms of the DNA concentration prepared from the specimen.

  In FIG. 5B, the same result as in FIG. 5A was obtained. That is, it was observed that the fluorescence intensity of the band decreased as the sample DNA concentration decreased. Thereby, it was confirmed once again that the primer pair according to Example 2 can specifically detect Talaromyces traxpermus. In addition, the bands of Samples 1 to 3 having DNA concentrations of 100 pg / μl to 10 ng / μl were confirmed, and the bands of Samples 4 and 5 having DNA concentrations of 10 pg / μl and 1 pg / μl were not confirmed. From this, it was suggested that the detection limit of the primer pair according to Example 2 was 10 to 100 pg / μl as the DNA concentration prepared from the specimen.

  Subsequently, a primer having a sequence that does not correspond to any of the sequences (A) to (H) and (e) to (h) is prepared, and whether or not Talaromyces macrosporus and Talaromyces traxpermus can be detected in a species-specific manner. confirmed.

[Comparative Example 1]
In order to detect at least one of Talaromyces macrosporus and Talalomyces traxpermus, a primer pair for PCR having the nucleotide sequences of SEQ ID NOs: 5 and 6 was prepared. Each primer was commissioned to Operon Biotechnology Co., Ltd. Table 7 shows the base sequences of the created primer pairs.

[Comparative Example 2]
As a primer for detecting Talaromyces macrosporus, a primer pair for PCR having the nucleotide sequences of SEQ ID NOs: 7 and 8 was prepared. Primers were synthesized from Operon Biotechnology Co., Ltd. in the same manner as in Example 1. Table 7 shows the base sequences of the prepared primers.

[Test Example 4]
As Test Example 4, using the primer pair according to Comparative Example 1, it was confirmed whether one of Talaromyces macrosporus and Talalomyces traxpermus was able to be detected in a species-specific manner from the specimen bacteria. In addition, since this test example was performed with the flow similar to Test example 1, the description is abbreviate | omitted about the overlapping part.

  In this test example, the strains shown in Table 8 and Table 9 were used as specimens. No. 14 in Table 8 is a strain belonging to Taralomyces traxpermus, and Nos. 12 and 13 in Table 9 are strains belonging to Taralomyces macrosporus. In addition, the strains shown in Table 8 were obtained from the National Institute of Technology and Evaluation Biotechnology Center (NBRC) similar to Test Example 1, and the strains shown in Table 9 were the same administrative agency RIKEN as Test Example 1. It was obtained from the Research Institute, BioResource Center, Microbial Materials Development Office (JCM). The composition of the PCR reaction solution was the composition described in Table 4 of Test Example 1. In the primer pair according to Comparative Example 1, the forward primer was a primer having the base sequence of SEQ ID NO: 5, and the reverse primer was a primer having the base sequence of SEQ ID NO: 6. PCR conditions were as follows: (1) heat denaturation reaction at 94 ° C for 2 minutes, (2) heat denaturation reaction at 94 ° C for 30 seconds, (3) annealing reaction at 53 ° C for 30 seconds, and (4) 20 ° C at 68 ° C. (5) An extension reaction at 68 ° C. for 5 minutes was performed, and (2) to (4) were performed 30 cycles. Subsequently, in the same manner as in Test Example 1, electrophoresis was performed on an agarose gel having a concentration of 1.3% (1 to 1.5%), and PCR amplification products were confirmed.

(result)
6 is an electrophoretic image of Test Example 4, wherein A shows the results of the strains shown in Table 8, and B shows the results of the strains shown in Table 9. FIG. In addition, the number of each strain attached | subjected to Table 8 and Table 9 and the number of each lane attached | subjected to the electrophoresis image of FIG. 6A and B respond | correspond, respectively. M lanes indicate size markers.

As shown in FIG. 6A, a band was confirmed in the 14th lane relating to the strain belonging to Talaromyces traxpermus. In addition, although it is difficult to confirm from the figure, the inventors also confirmed a band with the naked eye in the 15th lane related to the strain belonging to Thermoscus crustaceus . Thereby, it was suggested that the primer pair which concerns on the comparative example 1 detects also about strains other than the genus Talaromyces.

  On the other hand, as shown in FIG. 6B, a band was confirmed only in the 12th lane related to the strain belonging to Talaromyces macrosporus. However, no band was confirmed from the thirteenth lane related to the strain belonging to the same Tallalomyces macrosporus. From this, it was confirmed that the primer pair according to Comparative Example 1 is difficult to detect species-specifically of either Talaromyces macrosporus or Talaromyces traxpermus.

[Test Example 5]
As Test Example 5, using the primer pair according to Comparative Example 2, it was confirmed whether or not Tallaromyces macrosporus can be detected in a species-specific manner from the specimen bacteria. In addition, since this test example was performed with the flow similar to Test example 1 and Test example 4, the description is abbreviate | omitted about the overlapping part.

  In this test example, the strains shown in Table 10 and Table 11 were used as specimens. Among them, Nos. 12 and 13 in Table 11 are strains belonging to Talaromyces macrosporus. In addition, the strains shown in Table 10 were obtained from the National Institute of Technology and Evaluation Biotechnology Center (NBRC), which is the same as that of Test Example 1, and the strains shown in Table 11 were the same administrative agency, Riken, as in Test Example 1. It was obtained from the Research Institute, BioResource Center, Microbial Materials Development Office (JCM). The composition of the PCR reaction solution was the composition described in Table 4 of Test Example 1. In the primer pair according to Comparative Example 2, the forward primer was a primer having the base sequence of SEQ ID NO: 7, and the reverse primer was a primer having the base sequence of SEQ ID NO: 8. PCR conditions were as follows: (1) heat denaturation reaction at 94 ° C for 2 minutes, (2) heat denaturation reaction at 94 ° C for 30 seconds, (3) annealing reaction at 53 ° C for 30 seconds, and (4) 20 ° C at 68 ° C. (5) An extension reaction at 68 ° C. for 5 minutes was performed, and (2) to (4) were performed 30 cycles. Subsequently, in the same manner as in Test Example 1, electrophoresis was performed on an agarose gel having a concentration of 1.3% (1 to 1.5%), and PCR amplification products were confirmed.

(result)
FIG. 7 is an electrophoresis image of Test Example 5, in which A indicates the results of the strains described in Table 10 and B indicates the results of the strains described in Table 11. The numbers of the strains given in Tables 10 and 11 correspond to the numbers of the lanes attached to the electrophoresis images of FIGS. 7A and 7B, respectively. M lanes indicate size markers.

  No band could be confirmed from the electrophoresis image shown in FIG. 7A. This is considered to be a reasonable result because the strains listed in Table 10 corresponding to FIG. 7A do not include strains belonging to Talaromyces macrosporus.

  On the other hand, as shown in FIG. 7B, a band was confirmed only in the twelfth lane related to the strain belonging to Talaromyces macrosporus. However, no band was confirmed from the 13th lane related to the strain belonging to Talaromyces macrosporus. From this, it was confirmed that the primer pair according to Comparative Example 2 is difficult to perform species-specific detection of Talaromyces macrosporus.

  From the results of Test Example 4 and Test Example 5, the primer pairs according to Comparative Example 1 and Comparative Example 2 that do not correspond to any of the sequences (A) to (H) and (e) to (h) are species-specific. It was confirmed that automatic detection was impossible. Therefore, it was confirmed that the sequence of primers and the like is very important for species-specific detection of Talaromyces macrosporus and Talaromyces traxpermus. The present invention has found the above-described sequences for species-specific detection of Talaromyces macrosporus and Talalomyces traxpermus, among countless possible sequences.

[Example 3: Primer for detecting Talaromyces macrosporus]
Furthermore, a primer pair for PCR having the nucleotide sequences of SEQ ID NOs: 9 and 10 was prepared for detecting Talaromyces macrosporus. These primers were prepared using the isocitrate lyase gene as a target. Each primer was commissioned to Operon Biotechnology Co., Ltd. in the same manner as in Examples 1 and 2. Table 12 shows the base sequences of the created primer pairs.

[Example 4: Talaromyces traxpermus detection primer]
A primer pair for PCR having the nucleotide sequences of SEQ ID NOs: 11 and 12 was prepared for detection of Talalomyces traxpermus. These primers were prepared using the isocitrate lyase gene as a target. Table 12 shows the base sequences of the created primer pairs.

[Test Example 6]
As Test Example 6, using the primer pair according to Example 3 and Example 4, it was confirmed whether or not Talaromyces macrosporus and Talalomyces traxperumus can be detected in a species-specific manner from the specimen bacteria. In addition, description is abbreviate | omitted about the part which overlaps with the test example 1. FIG.

(Sample adjustment)
First, DNA was extracted from the specimen, and a DNA solution was prepared in the same manner as in Test Example 1. As specimens, the strains shown in Tables 2 and 3 were used. Furthermore, each strain was cultured in a PDA (Potato Dextrose Agar) medium or the like at about 25 to 30 ° C. for 7 to 10 days. DNA was extracted from the recovered strain in the same manner as in Test Example 1. The extracted DNA was prepared as a DNA solution having a concentration of 10 to 50 ng / μl.

(Gene amplification by PCR method)
Subsequently, PCR for detection of Talaromyces macrosporus was performed using each of the 45 prepared DNA solutions and the primer pair according to Example 3. Similarly, PCR was carried out using the 45 prepared DNA solutions and the primer pair according to Example 4 to detect Talaromyces traxpermus.

  First, each PCR reaction solution was prepared. In each PCR, only the primer and the template DNA were different, and the composition of the PCR reaction solution was the same. Table 13 shows the composition of the PCR reaction solution. The template DNA was each DNA solution prepared in “Preparation of specimen”. In the primer pair according to Example 3, the forward primer was a primer having the base sequence of SEQ ID NO: 9, and the reverse primer was a primer having the base sequence of SEQ ID NO: 10. In the primer pair according to Example 4, the forward primer was a primer having the base sequence of SEQ ID NO: 11, and the reverse primer was a primer having the base sequence of SEQ ID NO: 12.

  Next, the prepared PCR reaction solution was subjected to gene amplification using a thermal cycler. The conditions of the treatment in the detection of Talaromyces macrosporus were (1) heat denaturation reaction at 94 ° C. for 2 minutes, (2) heat denaturation reaction at 94 ° C. for 30 seconds, (3) annealing reaction at 53 ° C. for 30 seconds, ( 4) An extension reaction at 68 ° C. for 20 seconds and (5) an extension reaction at 68 ° C. for 5 minutes, and (2) to (4) were performed for 30 cycles. The conditions of the treatment in the detection of Talalomyces traxpermus are (1) heat denaturation reaction at 94 ° C. for 2 minutes, (2) heat denaturation reaction at 94 ° C. for 30 seconds, (3) annealing reaction at 50 ° C. for 30 seconds, (4 (3) An extension reaction at 68 ° C. for 30 seconds and (5) an extension reaction at 68 ° C. for 5 minutes, and (2) to (4) were performed for 30 cycles.

(Confirmation of PCR amplification products)
Subsequently, 5 to 10 μl was taken from the PCR reaction solution and subjected to electrophoresis on an agarose gel having a concentration of 1.3% (1 to 1.5%) in the same manner as in Test Example 1. After electrophoresis, the agarose gel was imaged in the same manner as in Test Example 1. PCR amplification products were confirmed based on the captured images.

(result)
FIG. 8 is an electrophoretic image of Example 3, and FIG. 9 is an electrophoretic image of Example 4. Moreover, in each figure, A shows the result of each specimen described in Table 2, and B shows the result of each specimen described in Table 3. The numbers of the strains given in Tables 2 and 3 correspond to the numbers of the lanes given to the electrophoresis images of FIGS. M lanes indicate size markers.

  First, as shown in FIG. 8, a band of about 300 to 400 bp was confirmed only in the 1st and 2nd lanes of A and the 1st, 2nd, 5th and 6th lanes of B. The strains corresponding to these lanes are strains belonging to Talaromyces macrosporus with reference to Table 2. From this, it was confirmed that the primer pair according to Example 3 can detect Talaromyces macrosporus in a species-specific manner.

  As shown in FIG. 9, bands of about 300 to 400 bp were confirmed only in the 3rd and 4th lanes of A and the 3rd, 4th, 7th and 8th lanes of B. The strains corresponding to these lanes are strains belonging to Talaromyces trakispermus, referring to Table 3. From this, it was confirmed that the primer pair according to Example 4 can detect Talamomyces traxpermus in a species-specific manner.

[Test Example 7]
As in Test Example 7, as in Test Example 3, in order to confirm the detection limit of Talaromyces macrosporus and Talaromyces traxpermus in PCR using the primer pairs according to Example 3 and Example 4, DNA solutions having different concentrations from the specimen were used. Created and tested. Note that the sample and the method for preparing the sample are the same as in Test Example 3, and the method for gene amplification and confirmation is the same as in Test Example 6, and thus the description thereof is omitted.

(result)
FIG. 10 is an electrophoretic image of Test Example 7. A shows the results of Example 3 and B shows the results of Example 4. Note that the numbers of the lanes attached to the electrophoretic image of FIG. 10 correspond to the numbers of the samples shown in Table 6, respectively. M lanes indicate size markers.

  First, in FIG. 10A, it was observed that the fluorescence intensity of the band decreased as the DNA concentration of the sample decreased. Thereby, it was confirmed once again that the primer pair according to Example 3 can specifically detect Talaromyces macrosporus. In addition, the bands of Samples 1 to 3 with DNA concentrations of 100 pg / μl to 10 ng / μl can be confirmed, and the bands of Samples 4 and 5 with DNA concentrations of 10 pg / μl and 1 pg / μl, respectively, cannot be confirmed. It was. From this, it was suggested that the detection limit of the primer pair according to Example 3 is 10 to 100 pg / μl in terms of the DNA concentration prepared from the specimen.

  In FIG. 10B, the same result as in FIG. 10A was obtained. That is, it was observed that the fluorescence intensity of the band decreased as the sample DNA concentration decreased. Thereby, it was confirmed anew that the primer pair according to Example 4 can specifically detect Talaromyces traxpermus. In addition, the bands of Samples 1 to 3 having DNA concentrations of 100 pg / μl to 10 ng / μl were confirmed, and the bands of Samples 4 and 5 having DNA concentrations of 10 pg / μl and 1 pg / μl were not confirmed. From this, it was suggested that the detection limit of the primer pair according to Example 4 was 10 to 100 pg / μl as the DNA concentration prepared from the specimen.

  From the results of Test Example 6 and Test Example 7, species-specific detection of Talaromyces macrosporus and Talalomyces traxpermus is also possible with the primer pairs of Examples 3 and 4 that hybridize to the isocitrate lyase gene region. Was confirmed. This is because the inventors searched for the gene region from among a number of gene regions and found the primer pair that hybridizes in a species-specific manner.

Claims (11)

A primer pair used for detecting a species belonging to the genus Talaromyces,
A primer pair consisting of an oligonucleotide represented by the following base sequence (E) and an oligonucleotide represented by the following base sequence (F), which is used when detecting Tallaromyces macrosporus by the PCR method.
(E) the base sequence set forth in SEQ ID NO: 1, or a base sequence in which one or several bases have been deleted, substituted or added in the base sequence and which can detect Talaromyces macrosporus
(F) the base sequence set forth in SEQ ID NO: 2, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence, and Talaromyces macrosporus can be detected A primer pair used for detecting a species belonging to the genus Talaromyces,
A primer pair consisting of an oligonucleotide represented by the base sequence of (G) below and an oligonucleotide represented by the base sequence of (H) below, which are used when detecting Tallalomyces traxpermus by the PCR method.
(G) the base sequence set forth in SEQ ID NO: 3, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence and which can detect Talalomyces traxpermus
(H) the nucleotide sequence set forth in SEQ ID NO: 4, or a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence and capable of detecting Tallalomyces traxpermus A primer pair used for detecting a species belonging to the genus Talaromyces,
A primer pair comprising an oligonucleotide represented by the following base sequence (M) and an oligonucleotide represented by the following base sequence (N), which is used when detecting Tallaromyces macrosporus by PCR.
(M) The base sequence set forth in SEQ ID NO: 9, or a base sequence in which one or several bases have been deleted, substituted or added in the base sequence and capable of detecting Talaromyces macrosporus
(N) the base sequence set forth in SEQ ID NO: 10, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence, and Talaromyces macrosporus can be detected A primer pair used for detecting a species belonging to the genus Talaromyces ,
Below (O) the primer pair consisting respectively represented an oligonucleotide with a base sequence of the oligonucleotide and the following represented by the nucleotide sequence (P) which is used in detecting Talaromyces Torakisuperumusu by PCR.
(O) the base sequence described in SEQ ID NO: 11, or the base sequence (P) SEQ ID NO: 12 in which one or several bases are deleted, substituted or added in the base sequence and Tallomyces traxpermus can be detected The base sequence described above, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence and which can detect Talaromyces traxpermus Following (e) of the primer consisting of the represented oligonucleotide base sequence pair or following oligonucleotides and the following represented by the nucleotide sequence (f) which is used in detecting the data Raromaisesu MACROSS Porras by PCR (m) A primer pair consisting of an oligonucleotide represented by the nucleotide sequence of (1) and an oligonucleotide represented by the nucleotide sequence of (n) below, or a base sequence of (g) below used when detecting Tallomyces traxpermus by the PCR method. A primer pair consisting of an oligonucleotide represented by the nucleotide sequence shown in the following (h) or an oligonucleotide represented by the nucleotide sequence shown in (o) below and an oligonucleotide represented by the nucleotide sequence shown in (p) below Primer pair consisting of nucleotides.
(E) Base sequence described in SEQ ID NO: 1 (f) Base sequence described in SEQ ID NO: 2 (g) Base sequence described in SEQ ID NO: 3 (h) Base sequence described in SEQ ID NO: 4 (m) SEQ ID NO: 9 The base sequence described in (n) The base sequence described in SEQ ID NO: 10 (o) The base sequence described in SEQ ID NO: 11 (p) The base sequence described in SEQ ID NO: 12 Talaromyces belongs to the genus Talalomyces comprising a primer pair consisting of an oligonucleotide represented by the base sequence of (E) below and an oligonucleotide represented by the base sequence of (F) below used when detecting Macrosporus by the PCR method Species detection kit.
(E) the base sequence set forth in SEQ ID NO: 1, or a base sequence in which one or several bases have been deleted, substituted or added in the base sequence and which can detect Talaromyces macrosporus
(F) the base sequence set forth in SEQ ID NO: 2, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence, and Talaromyces macrosporus can be detected Talalomyces belongs to the genus Talalomyces comprising a primer pair consisting of an oligonucleotide represented by the base sequence of (G) below and an oligonucleotide represented by the base sequence of (H) below, which is used when detecting Trachyspermus by PCR. Species detection kit.
(G) the base sequence set forth in SEQ ID NO: 3, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence and which can detect Talalomyces traxpermus
(H) the nucleotide sequence set forth in SEQ ID NO: 4, or a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence and capable of detecting Tallalomyces traxpermus A primer pair consisting of an oligonucleotide represented by the base sequence of (M) below and an oligonucleotide represented by the base sequence of (N) below used when detecting Tallaromyces macrosporus by the PCR method
Kit for detection of species belonging to the genus Talalomyces.
(M) The base sequence set forth in SEQ ID NO: 9, or a base sequence in which one or several bases have been deleted, substituted or added in the base sequence and capable of detecting Talaromyces macrosporus
(N) the base sequence set forth in SEQ ID NO: 10, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence, and Talaromyces macrosporus can be detected A primer pair consisting of an oligonucleotide represented by the following base sequence (O) and an oligonucleotide represented by the following base sequence (P) used when detecting Tallalomyces traxpermus by the PCR method
Kit for detection of species belonging to the genus Talalomyces.
(O) the nucleotide sequence set forth in SEQ ID NO: 11, or a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence and capable of detecting Tallalomyces traxpermus
(P) the base sequence set forth in SEQ ID NO: 12, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence and which can detect Tallalomyces traxpermus Under Symbol (e) a primer pair consisting of the represented oligonucleotide with a base sequence of the base sequence represented oligonucleotides and the (f) which is used in detecting the data Raromaisesu MACROSS Porras by PCR, Talaromyces MACROSS Porras the lower is used in detecting by PCR SL (m) of the oligonucleotides and under Symbol represented by the nucleotide sequence (n) a primer pair consisting of the represented oligonucleotide with a base sequence of the PCR method Talaromyces Torakisuperumusu under Symbol (g) a primer pair consisting respectively represented an oligonucleotide with a base sequence of the oligonucleotide and the lower Symbol represented by the nucleotide sequence (h) which is used in detecting, as well as Talaromyces Torakisuperumusu detected by PCR If the bottom is used to Symbol (o Species detection kit belonging to Talaromyces genus containing at least one primer pair of the primer pair consisting respectively represented an oligonucleotide with a base sequence of the oligonucleotide and the lower Symbol represented by the nucleotide sequence (p)) of.
(E) the nucleotide sequence set forth in SEQ ID NO: 1
(F) the nucleotide sequence set forth in SEQ ID NO: 2
(G) the nucleotide sequence set forth in SEQ ID NO: 3
(H) the nucleotide sequence set forth in SEQ ID NO: 4
(M) the nucleotide sequence set forth in SEQ ID NO: 9
(N) the nucleotide sequence set forth in SEQ ID NO: 10
(O) the nucleotide sequence set forth in SEQ ID NO: 11
(P) the nucleotide sequence set forth in SEQ ID NO: 12 Performing a PCR the oligonucleotide represented by the nucleotide sequence under Symbol oligonucleotides and lower Symbol represented by the nucleotide sequence of (e) (f) as a primer pair in the case of detecting the data Raromaisesu MACROSS Porras, and lower Stories wherein at least one of the steps of the steps of performing PCR oligonucleotide as a primer pair represented by the nucleotide sequence of the nucleotide sequence in the oligonucleotide and the lower Symbol represented (n) of the (m), when detecting Talaromyces Torakisuperumusu Table with a base sequence of steps of performing PCR using an oligonucleotide represented by the nucleotide sequence under Symbol oligonucleotides and lower Symbol represented by the nucleotide sequence of (g) (h) as a primer pair, and under SL (o) is oligonucleotide represented by the nucleotide sequence of the oligonucleotide and the lower SL (p) is Species detection methods belonging to Talaromyces genus, characterized in that it comprises at least one of the steps of the steps of performing PCR as a primer pair.
(E) the nucleotide sequence set forth in SEQ ID NO: 1
(F) the nucleotide sequence set forth in SEQ ID NO: 2
(G) the nucleotide sequence set forth in SEQ ID NO: 3
(H) the nucleotide sequence set forth in SEQ ID NO: 4
(M) the nucleotide sequence set forth in SEQ ID NO: 9
(N) the nucleotide sequence set forth in SEQ ID NO: 10
(O) the nucleotide sequence set forth in SEQ ID NO: 11
(P) the nucleotide sequence set forth in SEQ ID NO: 12