JP2018143147A - Mold detection carrier, mold detection method, and mold detection kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a mold detection carrier, a mold detection method, and mold detection kit that can specifically detect with high speed and precision heat resistant mold such as Talaromyces macrosporus, Talaromyces trachyspermus, Eupenicillium crustaceum, Eupenicillium javanicum, Eupenicillium brefeldianum, Eupenicillium lapidosum, Byssochlamys fulva, Byssochlamys nivea, Talaromyces wortmannii, Hamigera avellanea, and Hamigera striata.SOLUTION: The mold detection carrier has immobilized thereon at least two probes selected from the probe group consisting of a probe (a) having a specific base sequence, a probe (b) that can hybridize under stringent conditions to a nucleic acid fragment made of a base sequence complementary to that of the probe (a), and a probe (c) having a base sequence complementary to the probe (a) or probe (b).SELECTED DRAWING: None

Description

  The present invention relates to a mold detection carrier for detecting heat-resistant mold, a mold detection method, and a mold detection kit.

In recent years, in food production sites, clinical sites, agricultural sites, cultural property protection environments, etc., it has been checked whether microorganisms such as mold exist to confirm safety and soundness, and prevent their reproduction It is important.
In such mold inspection, generally, mold is collected from the environment, pre-cultured, and then cultured for about 20 days in an optimal medium for each bacterial species, followed by observation of morphological characteristics. Thus, a morphological observation method (culturing method) for identifying mold is performed (see Patent Document 1).

  However, this method has a problem that the inspection process becomes complicated because it is necessary to separate and culture for each mold species. In addition, since culture takes a long time, there is a problem that it is inappropriate for tests that require quickness, such as indoor inspections and food inspections where humans live, and plant disease diagnosis in the agricultural field. . In addition, there is a problem that the identification cannot be performed unless the spore representing the morphological feature is formed, and the labor may be wasted.

  Recently, identification methods using genes have been carried out for mold inspection. For example, after culturing mold collected from the environment, DNA is extracted from the cultured mold, the target region is amplified by PCR (polymerase chain reaction) method, etc., and the amplified product is analyzed. The mold has been identified. As a method for analyzing an amplification product, a method for analyzing the size of the amplification product by electrophoresis, a method using a DNA chip on which a probe that binds complementarily to the amplification product is immobilized, and the like have been proposed (Patent Document 2). 3).

JP 2007-195454 A Japanese Patent No. 5670623 Japanese Patent No. 5196848 Japanese Patent No. 5548345 Japanese Patent No. 5548385

In the case of using a DNA chip, a probe that specifically binds to a target region in the mold DNA to be detected is first prepared and immobilized on the DNA chip. Then, mold collected from the environment or product or plant specimen is cultured to extract mold DNA, and a target region in the mold DNA is amplified by PCR or the like. Further, the amplification product is dropped on the DNA chip, the amplification product is hybridized to the probe, and mold is detected by measuring the fluorescence intensity of the label contained in the amplification product.
By the way, since there are various types of molds to be detected in the environment, it is desirable that a plurality of molds can be detected simultaneously by one DNA chip. For this purpose, it is necessary to immobilize a probe selected from a plurality of molds on a DNA chip.

On the other hand, the amplification product of the target region to be hybridized to the probe can be obtained by PCR using a predetermined primer set, but depending on the type of mold, a false target reaction is likely to occur only with a single target region. Thus, it may be desirable to simultaneously amplify multiple target regions with multiple primer sets. Also, rather than amplifying the cultured molds individually, it is possible to rapidly test by proliferating multiple types of molds at the same time, and detecting molds using a DNA chip with a probe selected from these multiple types of molds immobilized. Improves.
However, when performing multiplex PCR that simultaneously amplifies a plurality of target regions, probes that function with higher accuracy are also required. Further, if the number of molds to be detected that are amplified simultaneously increases, the accuracy required of the probe becomes higher.

The present inventors are heat-resistant molds such as Talaromyces macrosporus, Talaromyces trachyspermus, Eupenicillium crustaceum, Eupenicillium javanicum, Penicillium brefeldianum, Eupenicillium lapidosum, Byssochlamys fulva, Byssochlamys nivea, Talaromyces waltmann, or Teraromies ) And 11 types of Hamigera striata (Hamigera striata) were used as molds to be detected, and extensive research was conducted to create a mold detection carrier capable of detecting them.
Among the specific target regions in the DNA of each of these types of molds, a probe is selected by selecting a base sequence that specifically binds only to each mold and does not bind to other types of molds. Theoretically, it is theoretically possible to specifically detect these multiple types of molds with each probe.

  However, actually, even a probe obtained in this manner does not always function effectively. Therefore, in order to create each probe, it was necessary to find a probe that functions effectively by repeating trial and error while confirming the effect of the probe through experiments.

Here, Patent Document 4 describes a primer set for amplifying and detecting a target region in DNA of Talamomyces spp., Bisoclamis spp., And Hamigella spp. By the LAMP method. In addition, a primer set for amplifying and detecting a target region in the DNA of these bacteria by PCR is described in Patent Document 5.
However, Talalomyces macrosporus, Talalomyces trakispermus, Eupenicilium clusteraceum, Eupenicilium jabanicum, Eupenicilium breferdianum, Eupenicilium rapidsum, Bisoclamis fulba, Bisoclamis nivea, Talalomyces waltmany, Hamigueri There is no known mold detection carrier on which a probe capable of specifically detecting 11 types of molds of Abernea and Hamigella striata with high accuracy and specificity is immobilized.

  The present invention has been made in view of the above circumstances, and is a heat-resistant mold, Tallaromyces macrosporus, Tallaromyces trakispermus, Eupenicilium clusterceum, Eupenicilium jabanicam, Eupenicilium brefeldinum, Eupenicilium A mold detection carrier capable of quickly and accurately detecting 11 types of molds such as rapidsum, bisoclamis fulva, bisoclamis nivea, taralomyces waltmany, hamiguera abernea, and hamiguera striata An object is to provide a detection method and a mold detection kit.

In order to achieve the above object, the mold detection carrier of the present invention immobilizes two or more probes selected from the probe group having the base sequence described in the following (a), (b), or (c) This is a mold detecting carrier characterized by the above.
(A) A probe having the base sequence shown in SEQ ID NOs: 1 to 47.
(B) A probe capable of hybridizing under stringent conditions to a nucleic acid fragment consisting of a base sequence complementary to the base sequences shown in SEQ ID NOs: 1 to 47.
(C) A probe having a base sequence complementary to the probe of (a) or (b).

  The fungus detection carrier of the present invention comprises a probe having a base sequence shown in SEQ ID NO: 1 selected from the ITS region for detecting Talaromyces macrosporus, and a β-tubulin gene. A base sequence shown in SEQ ID NO: 3 selected from the ITS region for detecting the first probe group consisting of the selected probe having the base sequence shown in SEQ ID NO: 2, Talaromyces trachyspermus And a second probe group consisting of probes having the nucleotide sequence shown in SEQ ID NO: 4 selected from the β-tubulin gene, selected from the ITS region for detecting Eupenicillium crustaceum A probe having the base sequence shown in SEQ ID NO: 5 and β-thio Selected from the ITS region for detecting the third group of probes, Eupenicillium javanicum, consisting of at least one of the probes having the nucleotide sequences shown in SEQ ID NOs: 6 to 7 selected from the burin gene A fourth probe group consisting of a probe having the base sequence shown in SEQ ID NO: 8 and a probe having the base sequence shown in SEQ ID NO: 9 selected from the β-tubulin gene, Eupenicillium brefeldianum At least one of a probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region and a probe having the base sequence shown in SEQ ID NOs: 10-15 selected from the β-tubulin gene A fifth group of probes, and Eupenicillium A probe having a base sequence shown in SEQ ID NO: 16 selected from the ITS region and a base sequence shown in SEQ ID NOs: 17 to 19 selected from the β-tubulin gene for detecting Pupsum (Eupenicillium lapidosum) It is preferable that the probe of the sixth probe group consisting of at least one of the probes is fixed.

  In addition, the mold detection carrier of the present invention is at least one of probes having a base sequence shown in SEQ ID NOs: 20 to 26 selected from a β-tubulin gene for detecting Byssochlamys fulva. A seventh probe group, and a probe comprising at least one of probes having a base sequence shown in SEQ ID NOs: 27 to 33 selected from the β-tubulin gene for detecting Byssochlamys nivea It is preferable that the probes of the eight probe groups are fixed.

  The fungus detection carrier of the present invention is a probe having a base sequence shown in SEQ ID NO: 34 selected from the ITS region for detecting Byssochlamys fulva or Byssochlamys nivea, and / Or selected from the ITS region for detecting a ninth probe group, Talaromyces wortmannii, consisting of probes having the base sequence shown in SEQ ID NO: 35 selected from the β-tubulin gene A tenth probe comprising at least one of the probes having the base sequences shown in SEQ ID NOs: 36 to 38 and / or at least one of the probes having the base sequences shown in SEQ ID NOs: 39 to 42 selected from the β-tubulin gene; Probe group for detecting Hamigera avellanea An eleventh probe group comprising a probe having the base sequence shown in SEQ ID NO: 43 selected from the ITS region and / or a probe having the base sequence shown in SEQ ID NO: 44 selected from the β-tubulin gene; In order to detect Hamigera striata, at least one of the probes having the nucleotide sequence shown in SEQ ID NOs: 45 to 46 selected from the ITS region and / or the β-tubulin gene was selected. It is preferable that the probe of the twelfth probe group consisting of the probe having the base sequence shown in SEQ ID NO: 47 is immobilized.

  The mold detection carrier of the present invention comprises the probes of the first to sixth probe groups, the probes of the seventh to eighth probe groups, and the probes of the ninth to twelfth probe groups. A fixed configuration is preferable.

  The mold detection method of the present invention is a mold detection method comprising a step of amplifying a target region in a mold DNA to be detected by PCR and confirming the presence or absence of an amplification product, wherein A primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a forward primer consisting of the base sequence shown in SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a reverse primer consisting of the base sequence shown in SEQ ID NO: 59 and a sample are contained, and the sample contains Talaromyces macrosporus, Talaromyces macrosporus, (Talaromyces trachyspermus), Eupenicilli When at least one of DNA of Eupenicillium crustaceum, Eupenicillium javanicum, Eupenicillium brefeldianum, and Eupenicillium lapidosum is contained, The mold detection carrier on which the target region in the DNA contained in the sample is amplified using the PCR reaction solution, and the obtained amplification product is immobilized with a probe for detecting these bacteria. This is a method of adding a probe having a complementary base sequence by dropping it onto the probe.

  The mold detection method of the present invention is a mold detection method comprising a step of amplifying a target region in a mold DNA to be detected by PCR and confirming the presence or absence of an amplification product, wherein A primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 58 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59, and a sample, When at least one DNA of Byssochlamys fulva and Byssochlamys nivea is contained, the PCR reaction solution is used to determine the target region in the DNA contained in the sample. The above-mentioned mold on which the probe for detecting these bacteria was immobilized was amplified. This is a method in which it is dropped onto a detection carrier and combined with a probe having a complementary base sequence.

  The mold detection method of the present invention is a mold detection method comprising a step of amplifying a target region in a mold DNA to be detected by PCR and confirming the presence or absence of an amplification product, wherein A primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a forward primer consisting of the base sequence shown in SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a reverse primer consisting of the base sequence shown in SEQ ID NO: 59 and a sample are contained, and the sample contains, for example, Byssochlamys fulva, Byssochlamys nivea (Byssochlamys) nivea, Talaromyces wortm When at least one DNA of annii), Hamigera avellanea, and Hamigera striata is contained, using the PCR reaction solution, the DNA in the sample contains As a method of amplifying the target region and dropping the obtained amplification product onto the mold detection carrier on which the probe for detecting these bacteria is immobilized, and binding the probe with a complementary base sequence is there.

  The mold detection method of the present invention is a mold detection method comprising a step of amplifying a target region in a mold DNA to be detected by PCR and confirming the presence or absence of an amplification product, wherein A primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a forward primer consisting of the base sequence shown in SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a reverse primer consisting of the base sequence shown in SEQ ID NO: 59 and a sample are contained, and the sample contains Talaromyces macrosporus, Talaromyces macrosporus, (Talaromyces trachyspermus), Eupenicilli Eupenicillium crustaceum, Eupenicillium javanicum, Eupenicillium brefeldianum, Eupenicillium lapidosum, Byssochlamys Byssochlamys Byssochlamys nivea), at least one of Talaromyces wortmannii, Hamigera avellanea, and Hamigera striata, the PCR reaction solution is used. The target region in the DNA contained in the sample is amplified, and the obtained amplification product is dropped on the mold detection carrier on which the probe for detecting these bacteria is immobilized, and a complementary base is added. Have array There a method for binding a probe that.

  Further, the mold detection kit of the present invention is a mold detection kit comprising a primer set for amplifying a target region in a mold DNA to be detected, and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed. The primer set is a primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of the base sequence shown and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59, wherein the carrier detects Talaromyces macrosporus To select from ITS area A first probe group consisting of a probe having the base sequence shown in SEQ ID NO: 1 and a probe having the base sequence shown in SEQ ID NO: 2 selected from the β-tubulin gene, Talaromyces trachyspermus A second probe comprising a probe having the base sequence shown in SEQ ID NO: 3 selected from the ITS region and a probe having the base sequence shown in SEQ ID NO: 4 selected from the β-tubulin gene A probe group, a probe having a base sequence shown in SEQ ID NO: 5 selected from the ITS region for detecting Eupenicillium crustaceum, and SEQ ID NOS: 6 to 7 selected from the β-tubulin gene A third probe group consisting of at least one of probes having the base sequence shown in A probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region for detecting Eupenicillium javanicum, and the base sequence shown in SEQ ID NO: 9 selected from the β-tubulin gene A fourth group of probes comprising the probes having, the probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region for detecting Eupenicillium brefeldianum, and the β-tubulin gene A fifth probe group consisting of at least one of the probes having the base sequences shown in SEQ ID NOs: 10 to 15 selected from the above, and the ITS region for detecting Eupenicillium lapidosum A probe having the base sequence shown in SEQ ID NO: 16, and The probe of the sixth probe group consisting of at least one of the probes having the base sequences shown in SEQ ID NOs: 17 to 19 selected from the β-tubulin gene is immobilized.

  Further, the mold detection kit of the present invention is a mold detection kit comprising a primer set for amplifying a target region in a mold DNA to be detected, and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed. The primer set includes a primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 58 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59, A seventh probe group consisting of at least one of probes having a base sequence represented by SEQ ID NOs: 20 to 26 selected from a β-tubulin gene for detecting Byssochlamys fulva, and , Β-chu to detect Byssochlamys nivea There as immobilized constituting the eighth probe group of probes comprising at least one probe having a nucleotide sequence shown in SEQ ID NO: 27 to 33 which is selected from phosphoric gene.

  Further, the mold detection kit of the present invention is a mold detection kit comprising a primer set for amplifying a target region in a mold DNA to be detected, and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed. The primer set is a primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of the base sequence shown and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59, wherein the carrier is Byssochlamys fulva or Bisoclamis fulva Detect Nivea (Byssochlamys nivea) A ninth probe group consisting of probes having the base sequence shown in SEQ ID NO: 34 selected from the ITS region and / or probes having the base sequence shown in SEQ ID NO: 35 selected from the β-tubulin gene In order to detect Talaromyces wortmannii, at least one of the probes having the nucleotide sequences shown in SEQ ID NOs: 36 to 38 selected from the ITS region and / or selected from the β-tubulin gene A tenth probe group consisting of at least one of the probes having the base sequences shown in SEQ ID NOs: 39 to 42, a base shown in SEQ ID NO: 43 selected from the ITS region for detecting Hamigera avellanea A probe having a sequence and / or a sequence selected from a β-tubulin gene The eleventh probe group consisting of probes having the base sequence shown in No. 44, and the base sequence shown in SEQ ID Nos. 45-46 selected from the ITS region for detecting Hamigera striata The probe of the twelfth probe group consisting of the probe having the base sequence shown in SEQ ID NO: 47 selected from at least one of the probes and / or the β-tubulin gene is immobilized.

  Further, the mold detection kit of the present invention is a mold detection kit comprising a primer set for amplifying a target region in a mold DNA to be detected, and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed. The primer set is a primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of the base sequence shown and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59, wherein the carrier detects Talaromyces macrosporus To select from ITS area A first probe group consisting of a probe having the base sequence shown in SEQ ID NO: 1 and a probe having the base sequence shown in SEQ ID NO: 2 selected from the β-tubulin gene, Talaromyces trachyspermus A second probe comprising a probe having the base sequence shown in SEQ ID NO: 3 selected from the ITS region and a probe having the base sequence shown in SEQ ID NO: 4 selected from the β-tubulin gene A probe group, a probe having a base sequence shown in SEQ ID NO: 5 selected from the ITS region for detecting Eupenicillium crustaceum, and SEQ ID NOS: 6 to 7 selected from the β-tubulin gene A third probe group consisting of at least one of probes having the base sequence shown in A probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region for detecting Eupenicillium javanicum, and the base sequence shown in SEQ ID NO: 9 selected from the β-tubulin gene A fourth group of probes comprising the probes having, the probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region for detecting Eupenicillium brefeldianum, and the β-tubulin gene SEQ ID NO: selected from the ITS region for detecting a fifth probe group consisting of at least one of the probes having the base sequences shown in SEQ ID NOs: 10 to 15 selected from the above, Eupenicillium lapidosum And a probe having the base sequence shown in FIG. Selected from β-tubulin gene for detecting a sixth probe group, Byssochlamys fulva, comprising at least one of the probes having the nucleotide sequences shown in SEQ ID NOs: 17 to 19 selected from the burin gene SEQ ID NO: selected from the β-tubulin gene for detecting a seventh probe group consisting of at least one of the probes having the base sequences shown in SEQ ID NOs: 20 to 26, Byssochlamys nivea A sequence selected from the ITS region for detecting an eighth probe group, Byssochlamys fulva or Byssochlamys nivea, comprising at least one of the probes having the base sequences shown in 27-33 A probe having the base sequence shown in No. 34, and / or SEQ ID NO: 36 selected from the ITS region for detecting a ninth group of probes consisting of probes having the base sequence shown in SEQ ID NO: 35 selected from the β-tubulin gene, Talaromyces wortmannii A tenth probe group comprising at least one of probes having the base sequence shown in -38 and / or at least one of probes having the base sequence shown in SEQ ID NOs: 39-42 selected from the β-tubulin gene , A probe having the base sequence shown in SEQ ID NO: 43 selected from the ITS region and / or the base shown in SEQ ID NO: 44 selected from the β-tubulin gene for detecting Hamigera avellanea An eleventh probe group comprising probes having a sequence, and Hamigera sp. At least one of the probes having the nucleotide sequence shown in SEQ ID NOs: 45 to 46 selected from the ITS region and / or the SEQ ID NO: 47 selected from the β-tubulin gene for detecting riata (Hamigera striata) The probe of the twelfth probe group consisting of probes having the base sequence shown in FIG.

  In the present specification and claims, the “probe group” means a set of probes and does not mean only a case of a plurality of probes, but includes a probe. It is called a probe group.

  According to the present invention, the heat-resistant molds Tallaromyces macrosporus, Tallaromyces trakispermus, Eupenicilium clusteraceum, Eupenicilium jabanicam, Eupenicilium brefeldinum, Eupenicilium rapidum, Bisoclamis fulva, Bisoclamis Nivea, Talalomyces wortmany, Hamigella abernea, and Hamigella striata can be detected quickly, accurately and specifically.

Probes immobilized on a mold detection carrier according to an embodiment of the present invention (molds to be detected: Talaromyces macrosporas (1), Talaromyces traxpermus (2), Eupenicillium clusterceum (3), Eupenicillium -It is a figure which shows the base sequence of Javanicum (4), Eupenicillium brefeldinum (5), Eupenicillium rapidum (6)). It is a figure which shows the base sequence of the probe (Mold of detection object: Bisoclamis fluva (7), Bisoclamis nivea (8)) fix | immobilized by the support | carrier for a mold detection which concerns on embodiment of this invention. Probes immobilized on the mold detection carrier according to the embodiment of the present invention (molds to be detected: Bisoclamis fulba (7), Bisoclamis nivea (8), Talaromyces wortmany (9), Hamigera abernea (10 ) And Hamiguera striata (11)). Probes that can be immobilized on a mold detection carrier according to an embodiment of the present invention (molds to be detected: Neosartria Fischeri (12), Eurotium Cevaeri (13), Pesilomyces barriotti (14), Pesilomyces relucinas (15 It is a figure which shows the base sequence of)). It is a figure which shows the base sequence of the primer for amplifying the target area | region of the mold of the object detected by the support | carrier for mold detection which concerns on embodiment of this invention. Probes immobilized on a mold detection carrier according to an embodiment of the present invention (molds to be detected: Tallalomyces macrosporus (1), Talalomyces traxpermus (2), Eupenicillium clusterceum (3), Eupenicilium It is a figure which shows the S / N ratio value of the fluorescence intensity detected about Javanicum (4), Eupenicillium brefeldinum (5), Eupenicillium rapidum (6)). The S / N ratio value of the fluorescence intensity detected about the probe fixed to the support | carrier for a mold detection which concerns on embodiment of this invention (mold to be detected: Bisoclamis fluva (7), bisoclamis nivea (8)) is shown. FIG. Probes immobilized on a mold detection carrier according to an embodiment of the present invention (molds to be detected: Bisoclamis fulva (7), Bisoclamis nivea (8), Talalomyces wortmany (9), Hamigera abernea (10) , Hamigera Striata (11)) is a diagram showing the S / N ratio value of the fluorescence intensity detected. Probes immobilized on a mold detection carrier according to an embodiment of the present invention (molds to be detected: Neosartoria Fischeri (12), Eurotium Cevaeri (13), Pesilomyces barriotti (14), Pesilomyces relasinas (15 It is a figure which shows the S / N ratio value of the fluorescence intensity detected about)). It is a figure which shows the arrangement | sequence number of the probe corresponding to the sample which contains 5 types of molds of the object detected by the support | carrier for mold detection which concerns on embodiment of this invention, and each detection object mold. It is a figure which shows the S / N ratio value of the fluorescence intensity for every probe detected about the sample containing 5 types of molds of the object detected by the support | carrier for mold detection which concerns on embodiment of this invention.

  Hereinafter, an embodiment of a mold detection carrier, a mold detection method, and a mold detection kit of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiment and examples described later.

[Mold detection carrier]
(Detectable mold)
Molds to be detected by the mold detection carrier of the present embodiment are heat-resistant molds, Talaromyces macrosporus, Talaromyces trachyspermus, Eupenicillium crustaceum, Eupenicillium javanicum, Eupenicillium brefeldianum, Eupenicillium lapidosum, Byssochlamys fulva, Byssochlamys fulva, Bysochis lamyta Talaromyces wortmannii), Hamigera avellanea, and Hamigera striata.

  These heat-resistant molds can survive even if heat treatment is performed at 75 ° C. for 30 minutes by holding the spores in a thick shell such as an ascomycete. Since heat-resistant molds cannot be completely killed by pasteurization, it is desirable to be able to confirm the presence of these molds in foods and the environment.

(probe)
The probe for detecting the target mold of this embodiment is a nucleic acid fragment having a base sequence identified by SEQ ID NOs: 1 to 47 shown in FIGS.
The probe consisting of the base sequence shown in SEQ ID NO: 1 is a probe selected from the ITS region for detecting Talaromyces macrosporus, and the probe consisting of the base sequence shown in SEQ ID NO: 2 is used to detect the same mold A probe selected from the β-tubulin gene.

  The probe consisting of the base sequence shown in SEQ ID NO: 3 is a probe selected from the ITS region for detecting Talaromyces traxpelmus, and the probe consisting of the base sequence shown in SEQ ID NO: 4 is for detecting the same mold A probe selected from the β-tubulin gene.

  The probe consisting of the base sequence shown in SEQ ID NO: 5 is a probe selected from the ITS region for detecting Eupenicillium clusterceum, and the probe consisting of the base sequence shown in SEQ ID NOs: 6 to 7 detects the same mold A probe selected from the β-tubulin gene.

  The probe consisting of the base sequence shown in SEQ ID NO: 8 is a probe selected from the ITS region for detecting Eupenicillium jabanicum, and the probe consisting of the base sequence shown in SEQ ID NO: 9 is used to detect the same mold A probe selected from the β-tubulin gene.

  The probe consisting of the base sequence shown in SEQ ID NO: 8 is also a probe selected from the ITS region for detecting Eupenicillium brefeldinum, and the probe consisting of the base sequence shown in SEQ ID NO: 10 to 15 is the same mold. A probe selected from the β-tubulin gene for detection.

  The probe consisting of the base sequence shown in SEQ ID NO: 16 is a probe selected from the ITS region for detecting Eupenicillium rapidsum, and the probe consisting of the base sequence shown in SEQ ID NOs: 17 to 19 detects the same mold A probe selected from the β-tubulin gene.

The probes consisting of the nucleotide sequences shown in SEQ ID NOs: 20 to 26 are probes selected from the β-tubulin gene for detecting B. clamis fulva.
Moreover, the probe which consists of a base sequence shown to sequence number 27-33 is a probe selected from the (beta) -tubulin gene for detecting Biso clamis nivea.

  The probe consisting of the base sequence shown in SEQ ID NO: 34 is a probe selected from the ITS region for detecting Bisoclamis fulva or Bisoclamis nivea, and the probe consisting of the base sequence shown in SEQ ID NO: 35 detects the same mold It is a probe selected from the β-tubulin gene.

  The probes consisting of the base sequences shown in SEQ ID NOs: 36 to 38 are probes selected from the ITS region for detecting Talaromyces wortmany, and the probes consisting of the base sequences shown in SEQ ID NOs: 39 to 42 are the same molds. A probe selected from the β-tubulin gene for detection.

  The probe consisting of the base sequence shown in SEQ ID NO: 43 is a probe selected from the ITS region for detecting Hamigella abernea, and the probe consisting of the base sequence shown in SEQ ID NO: 44 is β for detecting the same mold A probe selected from the tubulin gene.

  The probe consisting of the base sequence shown in SEQ ID NOs: 45 to 46 is a probe selected from the ITS region for detecting Hamigella striata, and the probe consisting of the base sequence shown in SEQ ID NO: 47 is for detecting the same mold A probe selected from the β-tubulin gene.

  The probe consisting of the base sequence shown in SEQ ID NO: 48 shown in FIG. 4 is a probe selected from the ITS region for detecting Neosartorya fischeri, and consists of the base sequence shown in SEQ ID NO: 49. The probe is a probe selected from the β-tubulin gene for detecting the same mold. The probe consisting of the base sequence shown in SEQ ID NO: 50 shown in the figure is a probe selected from the ITS region for detecting Eurotium chevalieri, and the probe consisting of the base sequence shown in SEQ ID NO: 51 Is a probe selected from the β-tubulin gene for detecting the mold. Further, the probe consisting of the base sequence shown in SEQ ID NO: 52 shown in the figure is selected from the ITS region for detecting Paecilomyces variotii or its teleomorph type Talaromyces spectablis. The probe consisting of the base sequence shown in SEQ ID NO: 53 is a probe selected from the β-tubulin gene for detecting the mold. The probe consisting of the base sequence shown in SEQ ID NO: 54 shown in the figure is a probe selected from the ITS region for detecting Paecilomyces lilacinus, and the probe consisting of the base sequence shown in SEQ ID NO: 55 Is a probe selected from the β-tubulin gene for detecting the mold. These probes may be immobilized in the mold detection carrier of this embodiment.

In the mold detection carrier of this embodiment, as described above, a probe selected from the ITS region and / or the β-tubulin gene in the mold genomic DNA is used.
Specifically, for the following detection type (A) mold, the probe was selected only when a positive reaction was obtained in both of the ITS region and the probe selected from the β-tubulin gene. It is determined that mold has been detected. With respect to these molds, it is possible to reduce the risk of erroneous judgment based on false positive reactions.

Detection type (A): Talaromyces macrosporus, Talaromyces trakispermus, Eupenicilium clusteraceum, Eupenicilium jabanicam, Eupenicilium brefeldinum, Eupenicilium rapidum

  As for the mold of the detection type (B), when a positive reaction is obtained with a probe selected from only the β-tubulin gene, it can be determined that the mold exists. Probes selected from these fungal β-tubulin genes have high specificity and are relatively unlikely to produce false positive reactions. For these fungi, probes selected only from the β-tubulin gene were used. Detection can be performed.

Detection type (B): The following two types: Bisoclamis Fulba, Bisoclamis Nivea

  For mold of detection type (C), it can be determined that the mold is present when a positive reaction is obtained in at least one of the probes selected from the ITS region or the β-tubulin gene. . These molds include those that may be detected only by one of the probe selected from the ITS region or the probe selected from the β-tubulin gene in the mold detection carrier of the present embodiment. The probe has high specificity and can be detected without causing a false positive reaction.

Detection type (C): The following 5 types: Bisoclamis fulba, Bisoclamis nivea, Talaromyces waltmany, Hamiguera abernea, Hamiguera striata

  As described above, according to the mold detection carrier of the present embodiment, it is possible to further improve detection accuracy by appropriately setting the combination of probes used for detection for each type of mold. As for Bisoclamis fulva and Bisoclamis nivea, probes used for detection type C as well as probes used for detection type B were prepared as described above.

  Therefore, according to the mold detection carrier of the present embodiment, for the detection type A mold, both the ITS region and the probe selected from the β-tubulin gene are immobilized, and both probes show positive. If it is detected, it can be determined that the mold to be detected exists. In addition, for molds of detection type B, when a probe selected from only the β-tubulin gene is immobilized and the probe shows positive, it can be determined that mold to be detected exists. Furthermore, for molds of detection type C, it is preferable to immobilize both the ITS region and the probe selected from the β-tubulin gene, and when at least one of the probes is positive, It can be determined that there is mold.

(Probe synthesis)
Each of the probes immobilized on the mold detection carrier of this embodiment has a length of about 21 to 34 bases and can be synthesized by a general DNA synthesizer. As the probes consisting of the base sequences shown in SEQ ID NOs: 1 to 55 immobilized on a mold detection carrier in Examples described later, those synthesized by a DNA synthesizer were used. In addition, the primer which consists of a base sequence shown to sequence number 56-59 used for PCR in the Example mentioned later is also about 19-26 bases in length, and what was synthesize | combined with the DNA synthesizer was used.

  Further, the probe consisting of the base sequence shown in SEQ ID NO: 1 to 55 in this embodiment is not limited to the sequence itself, and one or several bases are deleted, substituted or substituted in the base sequence shown in SEQ ID NO: 1 to 55. Added probes can be used. A probe that can hybridize under stringent conditions to a nucleic acid fragment consisting of a base sequence complementary to the base sequences shown in SEQ ID NOs: 1 to 55 can also be used. In addition, these probes and probes having a base sequence complementary to the probes consisting of the base sequences shown in SEQ ID NOs: 1 to 55 can also be used.

  Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed. For example, a DNA having high homology to the DNA consisting of the base sequence shown in SEQ ID NO: 1 (homology is 90% or more, preferably 95% or more) is complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1. The conditions for hybridizing with DNA consisting of a simple base sequence are mentioned. Usually, it means a case where hybridization occurs at a temperature about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the melting temperature (Tm) of the complete hybrid. For stringent conditions, see J.M. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), especially in section 11.45 “Conditions for Hybridization”.

  Similarly, each primer (SEQ ID NO: 56 and 57 primer set, SEQ ID NO: 58 and 59 primer set) consisting of the nucleotide sequences of SEQ ID NOS: 56 to 59 shown in FIG. A primer in which one or several bases are deleted, substituted or added in the base sequences shown in SEQ ID NOs: 56 to 59, and a primer consisting of the base sequences shown in SEQ ID NOs: 56 to 59 is used. Those capable of amplifying the same region can also be used. A primer comprising a nucleic acid fragment that can specifically anneal to a nucleic acid fragment comprising a base sequence complementary to the base sequence represented by SEQ ID NOs: 56 to 59, wherein the primer comprises a nucleotide sequence represented by SEQ ID NOs: 56 to 59 A primer capable of amplifying the same region as that using a primer can be used.

(Creation of mold detection carrier)
The mold detection carrier of this embodiment can be produced by an existing general method using a probe having the base sequence shown in SEQ ID NOs: 1 to 47.
For example, when an affixed type DNA chip is prepared as the mold detection carrier of the present embodiment, the probe is immobilized on a glass substrate by a DNA spotter, and a spot corresponding to each probe is formed. be able to. When a synthetic DNA chip is produced, it can be produced by synthesizing a single-stranded oligo DNA having the above sequence on a glass substrate by a photolithography technique. Furthermore, the substrate is not limited to glass, and a plastic substrate, a silicon wafer, or the like can also be used. Further, the shape of the substrate is not limited to a flat plate shape, and may be various three-dimensional shapes, and a substrate having a functional group introduced so that a chemical reaction can be performed on the surface can be used. .

The mold detection carrier of the present embodiment can be obtained by immobilizing all or part of the probes having the base sequences shown in SEQ ID NOs: 1 to 47.
In addition, the mold detection carrier of the present embodiment may have a probe for detecting mold of each group immobilized for each of the detection types (A), (B), and (C). . In addition, for each group of several other types of molds, a probe for detecting the molds of each group may be fixed.
Furthermore, the mold detection carrier of the present embodiment is obtained by immobilizing all or part of a probe in which one or several bases are deleted, substituted, or added in the base sequences shown in SEQ ID NOs: 1 to 47. This means that all or part of a probe that can hybridize under stringent conditions to a nucleic acid fragment consisting of a base sequence complementary to the base sequence shown in SEQ ID NOs: 1 to 47 is immobilized. It is also possible to immobilize all or part of these probes or probes having a base sequence complementary to the probes consisting of the base sequences shown in SEQ ID NOs: 1 to 47.

[How to detect mold]
The mold detection method of the present embodiment is a mold detection method comprising a step of amplifying a target region in DNA of a mold to be detected by PCR and confirming the presence or absence of an amplification product, wherein the target region is amplified. A primer set for amplifying the ITS region and / or a primer set for amplifying the β-tubulin gene and a sample, and the sample is a mold to be detected. When at least one of the above DNAs is contained, a PCR reaction solution is used to amplify the target region in the DNA contained in the sample, and the obtained amplification product is dropped onto the mold detection carrier described above. Any method may be used as long as the amplified product is bound to a probe having a complementary base sequence, and the method is not limited to the specific embodiments and examples. Bur, for example, (1) collection of molds, (2) extraction of DNA, can be made including the confirmation of the (3) amplification of the target region, and (4) the amplification product.

(1) Collecting mold For example, when collecting airborne mold, collect indoor or outdoor air in the environment to be inspected. And the extract | collected air is sprayed on an exclusive culture medium and the mold | fungi contained in the air are cultured. As the medium, it is preferable to use a predetermined agar medium or the like formed into a strip shape for an air sampler. As culture conditions, it is preferable to stand at 25 ° C in a dark place for 65 hours or more.
Next, mold colonies cultured on the strip are individually collected for each colony, or two or more colonies are collected at once, frozen by liquid nitrogen, etc., and mold cells contained in the colonies Crush.
For example, when collecting a mold of a plant specimen, a diseased tissue piece or a healthy tissue piece to be examined is collected using scissors or a cutter. The collected plant tissue pieces are surface sterilized with hypochlorous acid or ethanol and then placed on a dedicated medium to culture mold contained in the plant tissue. As culture conditions, it is preferable to stand still within a range of 20 ° C to 25 ° C for 65 hours or more.

(2) Extraction of DNA Next, genomic DNA is extracted from the crushed product of mold cells thus obtained. Genomic DNA can be extracted by a general method such as a method using a CTAB method (Cetyl trimethyl ammonium bromide) or a method using a DNA extraction apparatus.
It is also possible to extract mold DNA directly from the soil. DNA extraction can be performed by a general method such as a method using a direct lysis method or a method using a soil DNA extraction kit.

(3) Amplification of target region Next, the target region in the extracted genomic DNA is amplified. That is, a DNA fragment containing a target region in genomic DNA is amplified. The method for amplifying the target region is not particularly limited, but the PCR method can be suitably used. In the PCR method, a target region is amplified using a PCR reaction solution containing a primer set for amplifying the target region. A general thermal cycler or the like can be used as the PCR apparatus.
In the mold detection method of this embodiment, for example, by performing PCR under the following reaction conditions, target regions of various molds can be suitably amplified.
(A) 95 ° C. 10 minutes, (b) 95 ° C. (DNA denaturation step) 30 seconds, (c) 56 ° C. (annealing step) 30 seconds, (d) 72 ° C. (DNA synthesis step) 60 seconds ((b) to (D) 40 cycles), (e) 72 ° C. 10 minutes

  As a reaction solution for PCR, for example, a solution having the following composition is preferably used. That is, a nucleic acid synthesis substrate (such as dNTPmixture (dCTP, dATP, dTTP, dGTP)), primer set, nucleic acid synthase (such as Nova Taq HotStart DNA polymerase), fluorescent labeling reagent (such as Cy5-dCTP), sample genomic DNA, buffer A PCR reaction solution containing water and water as the remaining component can be preferably used. As the buffer solution, for example, Ampdirect (registered trademark) (manufactured by Shimadzu Corporation) can be used.

In this embodiment, amplification of DNA fragments is performed using the ITS region and / or the β-tubulin gene in the genomic DNA of mold as a target region.
At this time, it is preferable to use a primer set for amplifying the ITS region consisting of the base sequences of SEQ ID NOs: 56 and 57 shown in FIG. 5 (SEQ ID NO: 56: forward primer, SEQ ID NO: 57: reverse primer). .
In addition, as a primer set for amplifying the β-tubulin gene, one consisting of the nucleotide sequences of SEQ ID NOs: 58 and 59 shown in the same figure (SEQ ID NO: 58: forward primer, SEQ ID NO: 59: reverse primer) should be used. Is preferred.

(4) Confirmation of amplification product Next, the amplification product is dropped onto the mold detection carrier of the present embodiment, and by detecting the label of the amplification product hybridized to the probe immobilized on this mold detection carrier, Check for amplification products. As a result, molds present in the environment to be inspected can be identified.
The detection of the label can be performed using a general label detection device such as a fluorescence scanning device, for example, by measuring the fluorescence intensity of the amplification product using BIOSHOT (registered trademark) of Toyo Kohan Co., Ltd. be able to. The measurement result is preferably obtained as an S / N ratio (Signal to Noise ratio) value. This is because it is possible to accurately determine whether the measurement result is positive or negative based on the S / N ratio value. Generally, when the S / N ratio value is 3 or more, it is determined to be positive. Can do. The S / N ratio value described in the present specification is calculated by (median fluorescence intensity value−background value) ÷ background value. The label is not limited to fluorescence, and other labels can also be used.

[Mold detection kit]
The mold detection kit of this embodiment includes a primer set for amplifying a nucleic acid fragment containing a target region in mold DNA, and a mold detection carrier on which a probe for confirming the presence or absence of an amplification product is fixed. included.
It is preferable that the primer set and the mold detection carrier have the following configurations for each of the mold detection types (A), (B), and (C) described above.

Detection type (A):
When the mold to be detected is at least one of Tallaromyces macrosporus, Tallaromyces trakispermus, Eupenicilium clusterceum, Eupenicilium jabanicam, Eupenicilium brefeldinum, Eupenicilium rapidum, A primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 56 for amplifying the ITS region and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a sequence number for amplifying the β-tubulin gene It is preferable to use a primer set comprising a forward primer consisting of the base sequence shown in 58 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59.

When the mold to be detected is the detection type (A), specifically, the mold detection carrier preferably has the following configuration.
A probe having the base sequence shown in SEQ ID NO: 1 selected from the ITS region and the base sequence shown in SEQ ID NO: 2 selected from the β-tubulin gene for detecting Talaromyces macrosporus A first probe group comprising probes having,
A probe having the nucleotide sequence shown in SEQ ID NO: 3 selected from the ITS region and the nucleotide sequence shown in SEQ ID NO: 4 selected from the β-tubulin gene for detecting Talaromyces trachyspermus A second probe group consisting of probes having
A probe having a base sequence shown in SEQ ID NO: 5 selected from the ITS region and a base shown in SEQ ID NOs: 6-7 selected from the β-tubulin gene for detecting Eupenicillium crustaceum A third probe group consisting of at least one of probes having a sequence;
A probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region for detecting Eupenicillium javanicum, and the base sequence shown in SEQ ID NO: 9 selected from the β-tubulin gene A fourth group of probes comprising probes having,
In order to detect Eupenicillium brefeldianum, a probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region, and SEQ ID NOS: 10-15 selected from the β-tubulin gene A fifth probe group consisting of at least one of the probes having the base sequence shown, and
A probe having the base sequence shown in SEQ ID NO: 16 selected from the ITS region and the bases shown in SEQ ID NOs: 17-19 selected from the β-tubulin gene for detecting Eupenicillium lapidosum A mold detection carrier on which a probe of a sixth probe group comprising at least one of probes having a sequence is immobilized.

  That is, the mold detection kit for mold of detection type (A) is a probe having a complementary base sequence by dropping an amplification product amplified using the above primer set onto the mold detection carrier. Used to detect mold by binding to.

Detection type (B):
When the mold to be detected is at least one of Bisoclamis fulba and Bisoclamis nivea, the primer set includes a forward primer consisting of the base sequence shown in SEQ ID NO: 58 for amplifying the β-tubulin gene and SEQ ID NO: 59 It is preferable to use a primer set provided with a reverse primer comprising the base sequence shown in FIG.

In addition, when the mold to be detected is of the detection type (B), specifically, the mold detection carrier preferably has the following configuration.
A seventh probe group consisting of at least one of probes having the base sequences shown in SEQ ID NOs: 20 to 26 selected from the β-tubulin gene for detecting Byssochlamys fulva; and
Immobilizing probes of the eighth probe group consisting of at least one of the probes having the base sequences shown in SEQ ID NOs: 27 to 33 selected from the β-tubulin gene for detecting Byssochlamys nivea Mold detection carrier.

  That is, the mold detection kit for mold of detection type (B) is a probe having a complementary base sequence by dropping an amplification product amplified using the above primer set onto the mold detection carrier. Used to detect mold by binding to.

Detection type (C)
When the mold to be detected is at least one of Bisoclamis fulba, Bisoclamis nivea, Talaromyces wortmany, Hamiguera abernea, Hamiguera striata, the primer set is shown in SEQ ID NO: 56 for amplifying the ITS region. Primer set comprising a forward primer consisting of a base sequence and a reverse primer consisting of a base sequence shown in SEQ ID NO: 57, and a forward primer consisting of a base sequence shown in SEQ ID NO: 58 for amplifying the β-tubulin gene and SEQ ID NO: It is preferable to use a primer set provided with a reverse primer consisting of the base sequence shown in 59.

In addition, when the mold to be detected is of the detection type (C), specifically, the mold detection carrier preferably has the following configuration.
A probe having the base sequence shown in SEQ ID NO: 34 selected from the ITS region and / or a β-tubulin gene for detecting B. bisvamis (Byssochlamys fulva) or B. schilamys nivea A ninth probe group consisting of probes having the base sequence shown in SEQ ID NO: 35,
In order to detect Talaromyces wortmannii, at least one of the probes having the nucleotide sequences shown in SEQ ID NOs: 36 to 38 selected from the ITS region and / or selected from the β-tubulin gene A tenth probe group comprising at least one of the probes having the base sequences represented by SEQ ID NOs: 39 to 42;
A probe having the base sequence shown in SEQ ID NO: 43 selected from the ITS region and / or the base sequence shown in SEQ ID NO: 44 selected from the β-tubulin gene for detecting Hamigera avellanea An eleventh probe group comprising probes having:
A sequence selected from at least one of probes having a base sequence shown in SEQ ID NOs: 45 to 46 selected from the ITS region and / or a β-tubulin gene for detecting Hamigera striata A mold detection carrier on which a probe of a twelfth probe group comprising a probe having the base sequence shown in No. 47 is immobilized.

  That is, the mold detection kit for mold detection type (C) includes a probe having a complementary base sequence by dropping an amplification product amplified using the above primer set onto the mold detection carrier. Used to detect mold by binding to.

  Further, as a mold detection kit of this embodiment, a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57 for amplifying the ITS region, and Using a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 58 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59 for amplifying the β-tubulin gene, and mold detection type (A), It is also preferable to use a mold detection carrier on which all the groups of probes used in (B) and (C) are immobilized.

  According to the mold detection carrier, the mold detection method, and the mold detection kit of this embodiment, these molds are appropriately detected by using a probe that can specifically identify the 11 types of heat-resistant molds. be able to. For this reason, according to the present embodiment, it is possible to quickly and accurately detect these molds in an environmental inspection or the like for confirming the presence or absence of molds.

  Hereinafter, the tests conducted for confirming the effects of the fungus detection carrier, the fungus detection method, and the fungus detection kit according to the embodiments of the present invention will be specifically described.

[Test 1]
As the mold detection carrier, Gene Silicon (registered trademark) (manufactured by Toyo Kohan Co., Ltd.) was used, and a probe having the probes of SEQ ID NOS: 1 to 55 shown in FIGS. Each probe was synthesized by Sigma Aldrich Japan Co., Ltd. In addition, each probe was immobilized on the substrate by microarrayer.

  The molds to be detected were obtained from the RIKEN BioResource Center, Microbial Materials Development Office (Japan Collection of Microorganisms), and the National Institute for Product Evaluation Technology Biotechnology Headquarters, Biological Resource Center (NITE Biological Resource Center). A strain was used. Specifically, 12 types of strains were used for the following 11 types of molds.

(1) Talaromyces macrosporus JCM22818T
(2) Talaromyces trakispermus NBRC31360
(3) Eupenicillium cluster cerium NBRC6094T
(4) Eupenicillium jabanicam NBRC6095T
(5) Eupenicillium brefeldinum NBRC31730T, NBRC31731T,
(6) Eupenicillium rapidum NBRC6100T
(7) Bisoclamis Fulva NBRC31767T
(8) Bisoclamis Nivea JCM12806
(9) Talalomyces Waltmany NBRC7738
(10) Hamiguera Avelanea NBRC31667T
(11) Hamiguera Strelia NBRC6106T

These molds were cultured for each strain. Culturing was carried out using a PDA medium or an M40Y medium and allowed to stand for 7 days in the dark at 25 ° C.
Further, cultured mold colonies were collected, individually placed in vials containing φ0.5 mm zirconia beads, immersed in liquid nitrogen to freeze the samples, and then mold cells were crushed using a shaking device.
Subsequently, mold genomic DNA was extracted with a DNA extraction apparatus, and the ITS region and β-tubulin gene of each mold were amplified for each strain by PCR.

Here, for each mold of detection type (A), each mold of detection type (B), and each mold of detection type (C), an ITS region amplification primer set and a β-tube are added to the PCR reaction solution. The amplification reaction of these target regions was carried out by containing both primer sets for phosphogene amplification.
At this time, a forward primer consisting of the base sequence of SEQ ID NO: 56 and a reverse primer consisting of the base sequence of SEQ ID NO: 57 shown in FIG. 5 were used as the ITS region amplification primer set. Moreover, the forward primer which consists of a base sequence of sequence number 58 shown in the same figure and the reverse primer which consists of a base sequence of sequence number 59 shown in the figure were used as a primer set for β-tubulin gene amplification. Those primers synthesized by Sigma Aldrich Japan Co., Ltd. were used.
In addition, about the mold of a detection type (B), when a positive reaction is obtained in the probe selected only from the β-tubulin gene, it can be determined that the mold exists. Therefore, when only the mold is to be detected, the PCR reaction solution contains the β-tubulin gene amplification primer set, and the target region amplification reaction is performed without including the ITS region amplification primer set. It is of course possible to do so.

As a reaction solution for PCR, Ampdirect (manufactured by Shimadzu Corporation) was used, and 20 μl of the following composition was prepared for each of 12 types of strains.
1. Ampdirect (G / Crich) 4.0μl
2. Ampdirect (addition-4) 4.0μl
3. dNTPmix 1.0μl
4). Cy-5dCTP 0.2μl
5. ITS1-Fw primer (SEQ ID NO: 56) (2.5μM) 1.0μl
6). ITS1-Rv primer (SEQ ID NO: 57) (2.5μM) 1.0μl
7). BtF primer (SEQ ID NO: 58) (10μM) 1.0μl
8). BtR primer (SEQ ID NO: 59) (10 μM) 1.0 μl
9. Template DNA 1.0μl
10. NovaTaq HotStart DNA polymerase 0.2μl
11. Water (water until 20.0 μl total)

Using the PCR reaction solution, DNA amplification was performed under the following conditions using a nucleic acid amplification apparatus (TaKaRa PCR Thermal Cycler Dice (registered trademark) Gradient Takara Bio Inc.).
(A) 95 ° C. 10 minutes (b) 95 ° C. 30 seconds (c) 56 ° C. 30 seconds (d) 72 ° C. 60 seconds (40 cycles of (b) to (d))
(E) 72 ° C 10 minutes

Next, a buffer solution (3 × SSC citrate-saline + 0.3% SDS) is mixed with the PCR amplification product, heated at 94 ° C. for 5 minutes, and dropped onto the mold detection carrier (DNA chip). did. The mold detection carrier was allowed to stand at 45 ° C. for 1 hour, and the PCR product that had not hybridized using the buffer was washed away from the DNA chip.
Then, using a label detection apparatus (BIOSHOT, manufactured by Toyo Kohan Co., Ltd.), the fluorescence intensity (fluorescence intensity of the amplification product bound to the probe) in each probe immobilized on the mold detection carrier is measured. The S / N ratio value in the probe was calculated. The results are shown in FIGS.

  As shown in these figures, the S / N ratio values of the probes each having the base sequences shown in SEQ ID NOs: 1 to 47 are all 3 or more, and can be judged as positive. Therefore, it can be seen that these probes function effectively in order to detect each target mold.

Specifically, for each mold of the detection type (A), when both the probe selected from the ITS region and the probe selected from the β-tubulin gene gave a positive reaction, It is determined that it exists.
For these molds, as shown in FIG. 6, the S / N ratio values of the probe selected from the ITS region and the probe selected from the β-tubulin gene were both 3 or more, indicating a positive reaction. Further, for each mold, no false positive reaction was observed in other probes. Therefore, it can be seen that all the probes that detect these molds function effectively.

For each mold of the detection type (B), when a positive reaction is obtained with a probe selected from only the β-tubulin gene, it is determined that the mold exists.
About these mold | types, as shown in FIG. 7, the S / N ratio value in the probe selected from (beta) -tubulin gene was 3 or more, and showed the positive reaction. Further, for each mold, no false positive reaction was observed in other probes. Therefore, it can be seen that all the probes that detect these molds function effectively.

For each mold of detection type (C), it is determined that the mold is present when a positive reaction is obtained in at least one of the probes selected from the ITS region or the β-tubulin gene.
For these molds, as shown in FIG. 8, the S / N ratio values of the probe selected from the ITS region and the probe selected from the β-tubulin gene were 3 or more, indicating a positive reaction. Further, for each mold, no false positive reaction was observed in other probes. Therefore, it can be seen that all the probes that detect these molds function effectively.

4 and FIG. 9 are used in the detection type (A), and the probes selected from the ITS region are the probes of Neosartoria Fischeri, Eurotium Cevaerieri, Pesilomyces barriotti, and Pesilomyces ribassinas shown in FIGS. In the probe selected from the .beta.-tubulin gene and both, when the positive reaction is obtained, it can be determined that the mold is present.
These molds were tested in the same manner as the detection type (A) mold described above, and as a result, the S / N ratio value of the probe selected from the ITS region and the probe selected from the β-tubulin gene was 3 This is the positive result. Further, for each mold, no false positive reaction was observed in other probes. Therefore, it can be seen that all the probes that detect these molds function effectively.

[Test 2]
When performing multiplex PCR that simultaneously amplifies target regions in a plurality of mold DNAs to be detected, a test for confirming whether the probe immobilized on the mold detection carrier of this embodiment functions effectively went.

The same mold detection carrier as used in Test 1 was used. In addition, as for the fungi to be detected, 12 types of strains were used for 11 types of fungi as in Test 1.
In this test, as shown in FIG. 10, five types of these molds were randomly combined to prepare the following five groups of mixed DNA samples. Therefore, some molds are included in a plurality of samples. Culturing was carried out using a PDA medium or an M40Y medium and allowed to stand for 7 days in the dark at 25 ° C.

(Sample 1)
(1) Talaromyces macrosporus JCM22818T
(3) Eupenicillium cluster cerium NBRC6094T
(5) Eupenicillium brefeldinum NBRC31730T
(7) Bisoclamis Fulva NBRC31767T
(10) Hamiguera Avelanea NBRC31667T

(Sample 2)
(2) Talaromyces trakispermus NBRC31360
(4) Eupenicillium jabanicam NBRC6095T
(6) Eupenicillium rapidum NBRC6100T
(8) Bisoclamis Nivea JCM12806
(9) Talalomyces Waltmany NBRC7738

(Sample 3)
(1) Talaromyces macrosporus JCM22818T
(5) Eupenicillium brefeldinum NBRC31731T
(6) Eupenicillium rapidum NBRC6100T
(10) Hamiguera Avelanea NBRC31667T
(11) Hamiguera Strelia NBRC6106T

(Sample 4)
(1) Talaromyces macrosporus JCM22818T
(5) Eupenicillium brefeldinum NBRC31730T
(8) Bisoclamis Nivea JCM12806
(9) Talalomyces Waltmany NBRC7738
(11) Hamiguera Strelia NBRC6106T

(Sample 5)
(2) Talaromyces trakispermus NBRC31360
(3) Eupenicillium cluster cerium NBRC6094T
(5) Eupenicillium brefeldinum NBRC31731T
(7) Bisoclamis Fulva NBRC31767T
(10) Hamiguera Avelanea NBRC31667T

The cultured mold colonies were collected, placed in a vial containing φ0.5 mm zirconia beads, immersed in liquid nitrogen to freeze the colonies, and then the mold cells were crushed using a shaker.
Next, samples obtained by extracting the genomic DNA of each mold using a DNA extraction apparatus were mixed as in samples 1-5. Then, the ITS region and / or β-tubulin gene of each mold was simultaneously amplified for each sample by the PCR method. The primer set used in the PCR method is the same as that in Test 1, and primers having the nucleotide sequences shown in SEQ ID NOs: 56 and 57 are used as a primer set for amplifying the ITS region. As a primer set for amplifying the gene, primers having the nucleotide sequences shown in SEQ ID NOs: 58 and 59 were used.

As a reaction solution for PCR, Ampdirect (manufactured by Shimadzu Corporation) was used, and 20 μl of the following composition was prepared for each sample.
1. Ampdirect (G / Crich) 4.0μl
2. Ampdirect (addition-4) 4.0μl
3. dNTPmix 1.0μl
4). Cy-5dCTP 0.2μl
5. ITS1-Fw primer (2.5μM) 1.0μl
6). ITS1-Rv primer (2.5μM) 1.0μl
7). BtF primer (10μM) 1.0μl
8). BtR primer (10μM) 1.0μl
9. Template DNA (1.0 μl / strain) 1.0 μl × number of strains10. NovaTaq HotStart DNA polymerase 0.2μl
11. Water (water until 20.0 μl total)

Using each of the PCR reaction solutions, DNA was amplified under the following conditions in the same manner as in Test 1 using a nucleic acid amplification apparatus (TaKaRa PCR Thermal Cycler Dice Gradient manufactured by Takara Bio Inc.).
(A) 95 ° C. 10 minutes (b) 95 ° C. 30 seconds (c) 56 ° C. 30 seconds (d) 72 ° C. 60 seconds (40 cycles of (b) to (d))
(E) 72 ° C 10 minutes

Next, a buffer solution (3 × SSC citrate-saline solution + 0.3% SDS) was mixed with the PCR amplification product, heated at 94 ° C. for 5 minutes, and dropped onto a mold detection carrier (DNA chip). . The mold detection carrier was allowed to stand at 45 ° C. for 1 hour, and the PCR product that had not hybridized using the buffer was washed away from the DNA chip.
Then, using a label detection device (BIOSHOT, manufactured by Toyo Kohan Co., Ltd.), the fluorescence intensity of each probe immobilized on the mold detection carrier (the fluorescence intensity of the amplification product bound to the probe) is measured. The S / N ratio value at was obtained. The result is shown in FIG.

Probes for detecting mold contained in the sample 1 are shown in SEQ ID NOs: 1, 2, 5-7, 8, 10-12, 20-26, 34, 35, 43, 44 as shown in FIG. A probe having a base sequence.
As shown in FIG. 1, the probe having the nucleotide sequence shown in SEQ ID NOs: 1 and 2 is a probe for detecting Talaromyces macrosporus of detection type (A), and a probe selected from the ITS region and β- When both the probes selected from the tubulin gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NOs: 5 to 7 is a probe for detecting detection type (A) Eupenicillium clusterceum, a probe selected from the ITS region, and β- When both the probes selected from the tubulin gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  The probe having the base sequence shown in SEQ ID NOs: 8, 10 to 12, as shown in FIG. 1 and FIG. 6, is a probe for detecting the detection type (A) Eupenicillium brefeldinum (NBRC31730T), When the probe selected from the ITS region and the probe selected from the β-tubulin gene both show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  As shown in FIG. 2, the probe having the nucleotide sequence shown in SEQ ID NOs: 20 to 26 is a probe for detecting Bisoclamis fulva of detection type (B), and is a probe selected only from the β-tubulin gene Is positive, it is determined that mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.

  As shown in FIG. 3, the probe having the base sequence shown in SEQ ID NOs: 34 and 35 is a probe for detecting the detection type (C) bisoclamis fluva or bisoclamis nivea, and is selected from the ITS region. When at least one of the probes selected from the β-tubulin gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.

As shown in FIG. 3, the probes having the nucleotide sequences shown in SEQ ID NOs: 43 and 44 are probes for detecting detection type (C) Hamigella abernea, probes selected from the ITS region and β-tubes When at least one of the probes selected from the phosphorus gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.
In addition, no positive reaction is observed for probes other than these. That is, no false positive reaction has occurred for the probes other than these, and according to each probe for detecting the mold contained in the sample 1, it is shown that mold other than the mold to be detected is not detected by mistake. ing.

As shown in FIG. 10, the probe for detecting mold contained in the sample 2 is a probe having a base sequence shown in SEQ ID NOs: 3, 4, 8, 9, 16 to 19, 27 to 35, 36 to 42. is there.
As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NOs: 3 and 4 is a probe for detecting Talaromyces traxpelmas of group (A), and a probe selected from the ITS region and β- When both the probes selected from the tubulin gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NOs: 8 and 9 is a probe for detecting detection type (A) Eupenicillium jabnicum, and a probe selected from the ITS region and β- When both the probes selected from the tubulin gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  As shown in FIG. 1, the probes having the nucleotide sequences shown in SEQ ID NOs: 16 to 19 are probes for detecting the detection type (A) Eupenicillium rapidsum, and probes selected from the ITS region and β- When both the probes selected from the tubulin gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  As shown in FIG. 2, the probe having the base sequence shown in SEQ ID NOs: 27 to 33 is a probe for detecting a detection type (B) bisoclamis nivea, and is a probe selected only from the β-tubulin gene. Is positive, it is determined that mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.

  As shown in FIG. 3, the probe having the base sequence shown in SEQ ID NOs: 34 and 35 is a probe for detecting the detection type (C) bisoclamis fluva or bisoclamis nivea, and is selected from the ITS region. When at least one of the probes selected from the β-tubulin gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.

As shown in FIG. 3, the probe having the base sequence shown in SEQ ID NOs: 36 to 42 is a probe for detecting Talaromyces wort Many of detection type (C), and a probe selected from the ITS region and β- When at least one of the probes selected from the tubulin gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, the S / N ratio value was 3 or more except for the probe of SEQ ID NO: 37, indicating a positive reaction.
In addition, no positive reaction is observed for probes other than these. That is, no false positive reaction has occurred for the probes other than these, and each probe for detecting mold contained in the sample 2 indicates that mold other than the mold to be detected is not detected by mistake. ing.

As shown in FIG. 10, the probes for detecting mold contained in the sample 3 are base sequences represented by SEQ ID NOs: 1, 2, 8, 10, 13-15, 16-19, 43, 44, 45-47. It is a probe which has.
As shown in FIG. 1, the probe having the nucleotide sequence shown in SEQ ID NOs: 1 and 2 is a probe for detecting Talaromyces macrosporus of group (A), a probe selected from the ITS region and a β-tube When both the probes selected from the phosphorus gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  Probes having the nucleotide sequences shown in SEQ ID NOs: 8, 10, 13 to 15 are probes for detecting the detection type (A) Eupenicillium brefeldinum (NBRC31731T) as shown in FIGS. Yes, when the probe selected from the ITS region and the probe selected from the β-tubulin gene both show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  As shown in FIG. 1, the probes having the nucleotide sequences shown in SEQ ID NOs: 16 to 19 are probes for detecting the detection type (A) Eupenicillium rapidsum, and probes selected from the ITS region and β- When both the probes selected from the tubulin gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  As shown in FIG. 3, the probes having the nucleotide sequences shown in SEQ ID NOs: 43 and 44 are probes for detecting detection type (C) Hamigella abernea, probes selected from the ITS region and β-tubes When at least one of the probes selected from the phosphorus gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.

As shown in FIG. 3, the probe having the base sequence shown in SEQ ID NOs: 45 to 47 is a probe for detecting a detection type (C) Hamigella striata, a probe selected from the ITS region and a β-tube When at least one of the probes selected from the phosphorus gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.
In addition, no positive reaction is observed for probes other than these. That is, no false positive reaction has occurred for the probes other than these, and each probe for detecting mold contained in the sample 3 indicates that mold other than the mold to be detected is not detected by mistake. ing.

As shown in FIG. 10, the probe for detecting mold contained in the sample 4 has a base sequence shown in SEQ ID NOs: 1, 2, 8, 10-12, 27-35, 36-42, 45-47. It is a probe.
As shown in FIG. 1, the probe having the nucleotide sequence shown in SEQ ID NOs: 1 and 2 is a probe for detecting Talaromyces macrosporus of group (A), a probe selected from the ITS region and a β-tube When both the probes selected from the phosphorus gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  The probe having the base sequence shown in SEQ ID NOs: 8, 10 to 12, as shown in FIG. 1 and FIG. 6, is a probe for detecting the detection type (A) Eupenicillium brefeldinum (NBRC31730T), When the probe selected from the ITS region and the probe selected from the β-tubulin gene both show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  As shown in FIG. 2, the probe having the base sequence shown in SEQ ID NOs: 27 to 33 is a probe for detecting a detection type (B) bisoclamis nivea, and is a probe selected only from the β-tubulin gene. Is positive, it is determined that mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.

  As shown in FIG. 3, the probe having the base sequence shown in SEQ ID NOs: 34 and 35 is a probe for detecting the detection type (C) bisoclamis fluva or bisoclamis nivea, and is selected from the ITS region. When at least one of the probes selected from the β-tubulin gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.

  As shown in FIG. 3, the probe having the base sequence shown in SEQ ID NOs: 36 to 42 is a probe for detecting Talaromyces wort Many of detection type (C), and a probe selected from the ITS region and β- When at least one of the probes selected from the tubulin gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, in each of SEQ ID NOS: 36 to 38, the S / N ratio value was 3 or more, indicating a positive reaction.

As shown in FIG. 3, the probe having the base sequence shown in SEQ ID NOs: 45 to 47 is a probe for detecting a detection type (C) Hamigella striata, a probe selected from the ITS region and a β-tube When at least one of the probes selected from the phosphorus gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.
In addition, no positive reaction is observed for probes other than these. That is, no false positive reaction has occurred for the probes other than these, and according to each probe for detecting mold contained in the sample 4, it is shown that mold other than the mold to be detected is not detected by mistake. ing.

As shown in FIG. 10, probes for detecting mold contained in the sample 5 are SEQ ID NOS: 3, 4, 5 to 7, 8, 10, 13 to 15, 20 to 26, 34, 35, 43, 44. A probe having the base sequence shown in FIG.
As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NOs: 3 and 4 is a probe for detecting Talaromyces traxpelmas of group (A), and a probe selected from the ITS region and β- When both the probes selected from the tubulin gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NOs: 5 to 7 is a probe for detecting detection type (A) Eupenicillium clusterceum, a probe selected from the ITS region, and β- When both the probes selected from the tubulin gene show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were both 3 or more, indicating a positive reaction.

  Probes having the nucleotide sequences shown in SEQ ID NOs: 8, 10, 13 to 15 are probes for detecting the detection type (A) Eupenicillium brefeldinum (NBRC31731T) as shown in FIGS. Yes, when the probe selected from the ITS region and the probe selected from the β-tubulin gene both show a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, the S / N ratio values in SEQ ID NO: 8 and SEQ ID NOs: 14-15 were both 3 or more, indicating a positive reaction.

  As shown in FIG. 2, the probe having the nucleotide sequence shown in SEQ ID NOs: 20 to 26 is a probe for detecting Bisoclamis fulva of detection type (B), and is a probe selected only from the β-tubulin gene Is positive, it is determined that mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.

  As shown in FIG. 3, the probe having the base sequence shown in SEQ ID NOs: 34 and 35 is a probe for detecting the detection type (C) bisoclamis fluva or bisoclamis nivea, and is selected from the ITS region. When at least one of the probes selected from the β-tubulin gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.

As shown in FIG. 3, the probes having the nucleotide sequences shown in SEQ ID NOs: 43 and 44 are probes for detecting detection type (C) Hamigella abernea, probes selected from the ITS region and β-tubes When at least one of the probes selected from the phosphorus gene shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 11, these S / N ratio values were all 3 or more, indicating a positive reaction.
In addition, no positive reaction is observed for probes other than these. That is, no false positive reaction has occurred for the probes other than these, and each probe for detecting mold contained in the sample 5 indicates that mold other than the mold to be detected is not detected by mistake. ing.

  As described above, it is clear that the probe immobilized on the mold detection carrier according to the present embodiment functions effectively even when performing multiplex PCR that simultaneously amplifies the target region in the plurality of mold DNAs to be detected. It became. Moreover, it was also revealed that these probes have high specificity and no false positive reaction is observed.

The present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention.
For example, the probe to be immobilized on the mold detection carrier of this embodiment is not limited to one spot per type, and each probe may be immobilized in a plurality of spots. Further, other probes for detecting molds other than the detection target mold of the present embodiment may be immobilized on the mold detection carrier of the present embodiment together with the probes in the present embodiment, and may be changed as appropriate. Is possible.
In addition, anamorph-type, teleomorph-type, and mysterious molds related to the target mold in the present invention are naturally detectable, and the detection-target mold name conforms to the latest phylogenetic research.

  The present invention relates to heat-resistant molds Talaromyces macrosporus, Talaromyces traxpelmas, Eupenicillium clusterceum, Eupenicillium in environmental inspection, food inspection, epidemiological environmental inspection, clinical test, livestock hygiene, plant disease diagnosis Suitable for specific and multiple detection of Javanicum, Eupenicillium brefeldinum, Eupenicillium rapidsum, Bisoclamis fulba, Bisoclamis nivea, Talaromyces wortmany, Hamigella abernea, and Hamigella striata It is possible.

Claims (13)

A mold detection carrier, wherein two or more probes selected from the probe group having the base sequence described in the following (a), (b), or (c) are immobilized.
(A) A probe having the base sequence shown in SEQ ID NOs: 1 to 47.
(B) A probe capable of hybridizing under stringent conditions to a nucleic acid fragment consisting of a base sequence complementary to the base sequences shown in SEQ ID NOs: 1 to 47.
(C) A probe having a base sequence complementary to the probe of (a) or (b). A probe having the base sequence shown in SEQ ID NO: 1 selected from the ITS region and the base sequence shown in SEQ ID NO: 2 selected from the β-tubulin gene for detecting Talaromyces macrosporus A first probe group comprising probes having,
A probe having the nucleotide sequence shown in SEQ ID NO: 3 selected from the ITS region and the nucleotide sequence shown in SEQ ID NO: 4 selected from the β-tubulin gene for detecting Talaromyces trachyspermus A second probe group consisting of probes having
A probe having a base sequence shown in SEQ ID NO: 5 selected from the ITS region and a base shown in SEQ ID NOs: 6-7 selected from the β-tubulin gene for detecting Eupenicillium crustaceum A third probe group consisting of at least one of probes having a sequence;
A probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region for detecting Eupenicillium javanicum, and the base sequence shown in SEQ ID NO: 9 selected from the β-tubulin gene A fourth group of probes comprising probes having,
In order to detect Eupenicillium brefeldianum, a probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region, and SEQ ID NOS: 10-15 selected from the β-tubulin gene A fifth probe group consisting of at least one of the probes having the base sequence shown, and
A probe having the base sequence shown in SEQ ID NO: 16 selected from the ITS region and the bases shown in SEQ ID NOs: 17-19 selected from the β-tubulin gene for detecting Eupenicillium lapidosum The mold detection carrier according to claim 1, wherein the probe of the sixth probe group comprising at least one of the probes having the sequence is immobilized. A seventh probe group consisting of at least one of probes having the base sequences shown in SEQ ID NOs: 20 to 26 selected from the β-tubulin gene for detecting Byssochlamys fulva; and
Immobilizing probes of the eighth probe group consisting of at least one of the probes having the base sequences shown in SEQ ID NOs: 27 to 33 selected from the β-tubulin gene for detecting Byssochlamys nivea The mold detecting carrier according to claim 1, wherein A probe having the base sequence shown in SEQ ID NO: 34 selected from the ITS region and / or a β-tubulin gene for detecting B. bisvamis (Byssochlamys fulva) or B. schilamys nivea A ninth probe group consisting of probes having the base sequence shown in SEQ ID NO: 35,
In order to detect Talaromyces wortmannii, at least one of the probes having the nucleotide sequences shown in SEQ ID NOs: 36 to 38 selected from the ITS region and / or selected from the β-tubulin gene A tenth probe group comprising at least one of the probes having the base sequences represented by SEQ ID NOs: 39 to 42;
A probe having the base sequence shown in SEQ ID NO: 43 selected from the ITS region and / or the base sequence shown in SEQ ID NO: 44 selected from the β-tubulin gene for detecting Hamigera avellanea An eleventh probe group comprising probes having:
A sequence selected from at least one of probes having a base sequence shown in SEQ ID NOs: 45 to 46 selected from the ITS region and / or a β-tubulin gene for detecting Hamigera striata The mold detection carrier according to claim 1, wherein the probe of the twelfth probe group consisting of probes having the base sequence shown in No. 47 is immobilized.   A probe of the first to sixth probe groups according to claim 2, a probe of the seventh to eighth probe groups of claim 3, and a probe of the ninth to twelfth probe groups of claim 4. A mold-detecting carrier characterized by immobilizing a mold. A method for detecting mold, comprising a step of amplifying a target region in DNA of a mold to be detected by PCR and confirming the presence or absence of an amplification product,
In the PCR reaction solution, a primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a base sequence shown in SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59, and a sample,
The samples include Talaromyces macrosporus, Talaromyces trachyspermus, Eupenicillium crustaceum, Eupenicillium javanicum, Eupenicillium feldum ), And at least one DNA of Eupenicillium lapidosum,
Using the PCR reaction solution, the target region in the DNA contained in the sample is amplified,
6. The mold detection method, wherein the obtained amplification product is dropped on a mold detection carrier according to claim 2 or 5 and bound to a probe having a complementary base sequence. A method for detecting mold, comprising a step of amplifying a target region in DNA of a mold to be detected by PCR and confirming the presence or absence of an amplification product,
A PCR reaction solution contains a primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 58 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59, and a sample. ,
When the sample contains at least one DNA of Byssochlamys fulva and Byssochlamys nivea,
Using the PCR reaction solution, the target region in the DNA contained in the sample is amplified,
6. The mold detection method, wherein the obtained amplification product is dropped onto the mold detection carrier according to claim 3 or 5 and bound to a probe having a complementary base sequence. A method for detecting mold, comprising a step of amplifying a target region in DNA of a mold to be detected by PCR and confirming the presence or absence of an amplification product,
In the PCR reaction solution, a primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a base sequence shown in SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59, and a sample,
The samples include Byssochlamys fulva, Byssochlamys nivea, Talaromyces wortmannii, Hamigera avellanea, and at least one of the Hamigera striata If DNA is contained,
Using the PCR reaction solution, the target region in the DNA contained in the sample is amplified,
6. The mold detection method, wherein the obtained amplification product is dropped on the mold detection carrier according to claim 4 or 5 and bound to a probe having a complementary base sequence. A method for detecting mold, comprising a step of amplifying a target region in DNA of a mold to be detected by PCR and confirming the presence or absence of an amplification product,
In the PCR reaction solution, a primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a base sequence shown in SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59, and a sample,
The samples include Talaromyces macrosporus, Talaromyces trachyspermus, Eupenicillium crustaceum, Eupenicillium javanicum, Eupenicillium feldum ), Eupenicillium lapidosum, Byssochlamys fulva, Byssochlamys nivea, Talaromyces wortmannii, Hamiguera agera, Hamigera gera striata) at least any DNA of
Using the PCR reaction solution, the target region in the DNA contained in the sample is amplified,
6. The mold detection method, wherein the obtained amplification product is dropped onto the mold detection carrier according to claim 5 and bound to a probe having a complementary base sequence. A mold detection kit comprising a primer set for amplifying a target region in a mold DNA to be detected and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed,
The primer set is
A primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a forward primer and a sequence consisting of the base sequence shown in SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a reverse primer consisting of the base sequence shown in No. 59;
The carrier is
A probe having the base sequence shown in SEQ ID NO: 1 selected from the ITS region and the base sequence shown in SEQ ID NO: 2 selected from the β-tubulin gene for detecting Talaromyces macrosporus A first probe group comprising probes having,
A probe having the nucleotide sequence shown in SEQ ID NO: 3 selected from the ITS region and the nucleotide sequence shown in SEQ ID NO: 4 selected from the β-tubulin gene for detecting Talaromyces trachyspermus A second probe group consisting of probes having
A probe having a base sequence shown in SEQ ID NO: 5 selected from the ITS region and a base shown in SEQ ID NOs: 6-7 selected from the β-tubulin gene for detecting Eupenicillium crustaceum A third probe group consisting of at least one of probes having a sequence;
A probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region for detecting Eupenicillium javanicum, and the base sequence shown in SEQ ID NO: 9 selected from the β-tubulin gene A fourth group of probes comprising probes having,
In order to detect Eupenicillium brefeldianum, a probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region, and SEQ ID NOS: 10-15 selected from the β-tubulin gene A fifth probe group consisting of at least one of the probes having the base sequence shown, and
A probe having the base sequence shown in SEQ ID NO: 16 selected from the ITS region and the bases shown in SEQ ID NOs: 17-19 selected from the β-tubulin gene for detecting Eupenicillium lapidosum A fungus detection kit, wherein a probe of a sixth probe group consisting of at least one of probes having a sequence is immobilized. A mold detection kit comprising a primer set for amplifying a target region in a mold DNA to be detected and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed,
The primer set is
A primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 58 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 59;
The carrier is
A seventh probe group consisting of at least one of probes having the base sequences shown in SEQ ID NOs: 20 to 26 selected from the β-tubulin gene for detecting Byssochlamys fulva; and
Immobilizing probes of the eighth probe group consisting of at least one of the probes having the base sequences shown in SEQ ID NOs: 27 to 33 selected from the β-tubulin gene for detecting Byssochlamys nivea A fungus detection kit characterized by that. A mold detection kit comprising a primer set for amplifying a target region in a mold DNA to be detected and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed,
The primer set is
A primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a forward primer and a sequence consisting of the base sequence shown in SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a reverse primer consisting of the base sequence shown in No. 59;
The carrier is
A probe having the base sequence shown in SEQ ID NO: 34 selected from the ITS region and / or a β-tubulin gene for detecting B. bisvamis (Byssochlamys fulva) or B. schilamys nivea A ninth probe group consisting of probes having the base sequence shown in SEQ ID NO: 35,
In order to detect Talaromyces wortmannii, at least one of the probes having the nucleotide sequences shown in SEQ ID NOs: 36 to 38 selected from the ITS region and / or selected from the β-tubulin gene A tenth probe group comprising at least one of the probes having the base sequences represented by SEQ ID NOs: 39 to 42;
A probe having the base sequence shown in SEQ ID NO: 43 selected from the ITS region and / or the base sequence shown in SEQ ID NO: 44 selected from the β-tubulin gene for detecting Hamigera avellanea An eleventh probe group comprising probes having:
A sequence selected from at least one of probes having a base sequence shown in SEQ ID NOs: 45 to 46 selected from the ITS region and / or a β-tubulin gene for detecting Hamigera striata A fungus detection kit, wherein a probe of a twelfth probe group consisting of probes having the base sequence shown in No. 47 is immobilized. A mold detection kit comprising a primer set for amplifying a target region in a mold DNA to be detected and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed,
The primer set is
A primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 56 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 57, and a forward primer and a sequence consisting of the base sequence shown in SEQ ID NO: 58 A primer set for amplifying a β-tubulin gene consisting of a reverse primer consisting of the base sequence shown in No. 59;
The carrier is
A probe having the base sequence shown in SEQ ID NO: 1 selected from the ITS region and the base sequence shown in SEQ ID NO: 2 selected from the β-tubulin gene for detecting Talaromyces macrosporus A first probe group comprising probes having,
A probe having the nucleotide sequence shown in SEQ ID NO: 3 selected from the ITS region and the nucleotide sequence shown in SEQ ID NO: 4 selected from the β-tubulin gene for detecting Talaromyces trachyspermus A second probe group consisting of probes having
A probe having a base sequence shown in SEQ ID NO: 5 selected from the ITS region and a base shown in SEQ ID NOs: 6-7 selected from the β-tubulin gene for detecting Eupenicillium crustaceum A third probe group consisting of at least one of probes having a sequence;
A probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region for detecting Eupenicillium javanicum, and the base sequence shown in SEQ ID NO: 9 selected from the β-tubulin gene A fourth group of probes comprising probes having,
In order to detect Eupenicillium brefeldianum, a probe having the base sequence shown in SEQ ID NO: 8 selected from the ITS region, and SEQ ID NOS: 10-15 selected from the β-tubulin gene A fifth probe group consisting of at least one of the probes having the base sequence shown;
A probe having the base sequence shown in SEQ ID NO: 16 selected from the ITS region and the bases shown in SEQ ID NOs: 17-19 selected from the β-tubulin gene for detecting Eupenicillium lapidosum A sixth probe group comprising at least one of probes having a sequence;
A seventh probe group consisting of at least one of probes having a base sequence shown in SEQ ID NOs: 20 to 26 selected from the β-tubulin gene for detecting Byssochlamys fulva,
An eighth probe group consisting of at least one of probes having a base sequence represented by SEQ ID NOs: 27 to 33 selected from the β-tubulin gene, for detecting Byssochlamys nivea,
A probe having the base sequence shown in SEQ ID NO: 34 selected from the ITS region and / or a β-tubulin gene for detecting B. bisvamis (Byssochlamys fulva) or B. schilamys nivea A ninth probe group consisting of probes having the base sequence shown in SEQ ID NO: 35,
In order to detect Talaromyces wortmannii, at least one of the probes having the nucleotide sequences shown in SEQ ID NOs: 36 to 38 selected from the ITS region and / or selected from the β-tubulin gene A tenth probe group comprising at least one of the probes having the base sequences represented by SEQ ID NOs: 39 to 42;
A probe having the base sequence shown in SEQ ID NO: 43 selected from the ITS region and / or the base sequence shown in SEQ ID NO: 44 selected from the β-tubulin gene for detecting Hamigera avellanea An eleventh probe group comprising probes having:
A sequence selected from at least one of probes having a base sequence shown in SEQ ID NOs: 45 to 46 selected from the ITS region and / or a β-tubulin gene for detecting Hamigera striata A fungus detection kit, wherein a probe of a twelfth probe group consisting of probes having the base sequence shown in No. 47 is immobilized.

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