JP6880554B2 - Mold detection carrier, mold detection method, and mold detection kit - Google Patents

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 manufacturing sites, clinical sites, agricultural sites, cultural property protection environments, etc., it has been possible to inspect the presence of microorganisms such as molds to confirm their safety and soundness and prevent their reproduction. It has become important.
In such a mold test, generally, the mold is collected from the environment, pre-cultured, and then cultured in the optimum medium for each bacterial species for about 20 days, and then the morphological characteristics are observed. Therefore, a morphological observation method (culture 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 each mold species. In addition, since it takes a long time to culture, there is a problem that it is not suitable for inspections that require quickness, such as indoor inspections in which humans live, food inspections, and plant disease diagnosis at agricultural sites. .. Furthermore, there is also a problem that labor may be wasted because identification cannot be performed unless spores representing morphological characteristics are formed.

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

JP-A-2007-195454 Japanese Patent No. 5670623 Japanese Patent No. 5196848 Japanese Unexamined Patent Publication No. 2010-42005 JP-A-2007-174903 Japanese Unexamined Patent Publication No. 2014-3946 Japanese Unexamined Patent Publication No. 2013-99256

When using a DNA chip, first, a probe that specifically binds to a target region in the DNA of the mold to be detected is prepared and immobilized on the DNA chip. Then, the mold collected from the environment or the sample of the product or plant is cultured, the DNA of the mold is extracted, and the target region in the DNA of the mold is amplified by the PCR method or the like. Further, the amplification product is dropped onto a DNA chip, the amplification product is hybridized with a probe, and the fluorescence intensity of the label contained in the amplification product is measured to detect mold.
By the way, since there are various types of molds to be detected in the environment, it is desirable that a single DNA chip can detect a plurality of molds at the same time. For that purpose, it is necessary to immobilize a probe selected from a plurality of molds on the DNA chip.

On the other hand, the amplification product of the target region to hybridize to the probe can be obtained by the PCR method or the like using a predetermined primer set, but depending on the type of mold, a false positive reaction is likely to occur with only a single target region. Therefore, it may be desirable to simultaneously amplify multiple target regions with multiple primer sets. In addition, instead of amplifying the cultured molds individually, if multiple types of molds are simultaneously amplified and the molds are detected by a DNA chip on which a probe selected from these multiple types of molds is immobilized, the test can be expedited. Gender improves.
However, when performing multiplex PCR that simultaneously amplifies a plurality of target regions, a probe that functions with higher accuracy is required. Further, as the number of molds to be detected to be amplified at the same time increases, the accuracy required for the probe becomes higher.

The present inventors are heat-resistant molds, Mucor racemosus, Lichtheimia corymbifera, Monascus ruber, Thermoascus crustaceus, Thermoascus.・ Aurorascus aurantiacus, Thermoascus thermophilus, Nigrospora oryzae, Neurospora crassa, Emericella nidulans, Talaromyces flavus Eleven types of flavus) and Talaromyces bacillisporus were used as detection target molds, and intensive studies were conducted to prepare a mold detection carrier capable of detecting these types.
Of the specific target regions in the DNA of each of these multiple types of molds, a probe is selected by selecting a base sequence that has a base sequence that specifically binds only to each mold and does not bind to other types of molds. In theory, it is possible to specifically detect these multiple types of molds with each probe.

However, in reality, even the probe obtained in this way does not always function effectively. Therefore, in order to prepare each probe, it was necessary to find a probe that functions effectively by repeating trial and error while confirming the effect of the probe by experiment.

Here, Patent Document 4 describes a probe for detecting Mucol Lasemosas and Liquitemia kolimbifera. In addition, Patent Document 5 describes a primer set for amplifying and detecting the target region in the DNA of Monascus ruber and Neurospora classa by the PCR method, and Thermoscus clusterseus and Thermoscus au. Patent Document 6 describes a primer set for amplifying and detecting the target region in the DNA of Lantiacas and Thermoascus thermophilus by the PCR method, and PCR the target region in the DNA of Talaromyces flavus and Talaromyces bacilisporus Patent Document 7 describes a primer set or the like for detecting by amplifying by a method.
However, Mucol Lasemosas, Liquitimia colimbifera, Monascus ruber, Thermoscus clusterseus, Thermoscus aurantiacas, Thermoscus thermophilus, Nigrospora oryzae, Neurospora Classa, Emerisera Nidurance, Talaromyces -There is no known mold detection carrier on which a probe capable of specifically detecting 11 types of molds of Flavas and Talaromyces bacilli sporas 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, Mucol Lasemosas, Lictimia corimbifera, Monascus ruber, Thermoscus clusterseus, Thermoscus aurantiacas, Thermoscus. A mold detection carrier and mold that can detect 11 types of molds, Thermophilus, Nigrospora oryzae, Neurospora Classa, Emerisera Nidurance, Talalomyces flavus, and Talalomyces bacillisporus, quickly, accurately and specifically. It is an object of the present invention to provide a method for detecting mold and a kit for detecting mold.

In order to achieve the above object, the mold detection carrier of the present invention is composed of two or more probe groups selected from the first to seventh probe groups consisting of the base sequences described in (a) to (g) below. It is a carrier for mold detection, which comprises immobilizing a probe and simultaneously detecting two or more molds to be detected.
(A) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 1, 2 and 4 selected from the ITS region for detecting Mucor racemosus, and a probe consisting of a nucleotide sequence complementary to the probe. First probe group consisting of at least one of
(B) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 5 to 7 selected from the ITS region for detecting Lichtheimia corymbifera, and at least a probe consisting of a nucleotide sequence complementary to the probe. A second probe group consisting of either,
(C) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 8 to 9 selected from the ITS region for detecting Monascus ruber, and at least a probe consisting of a nucleotide sequence complementary to the probe. A third probe group consisting of either,
(D) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 10 to 11 selected from the ITS region for detecting Thermoascus crustaceus, and a nucleotide sequence complementary to the probe. A fourth probe group consisting of at least one of the probes,
(E) A probe consisting of the base sequence shown in SEQ ID NO: 12 selected from the ITS region for detecting Thermoascus aurantiacus, and a probe consisting of a base sequence complementary to the probe. Fifth probe group consisting of at least one of
(F) At least a probe consisting of the base sequence shown in SEQ ID NO: 13 selected from the ITS region for detecting Thermoascus thermophilus, and a probe having a base sequence complementary to the probe. A sixth probe group consisting of either, and
(G) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 14 to 15 selected from the ITS region for detecting Nigrospora oryzae, and at least a probe consisting of a nucleotide sequence complementary to the probe. A seventh probe group consisting of either.

Further, the mold detection carrier of the present invention may be configured to immobilize the probes of the first to seventh probe groups and simultaneously detect the mold to be detected according to the above (a) to (g). preferable.

In addition, the mold detection carrier of the present invention immobilizes probes of two or more probe groups selected from the eighth to eleventh probe groups consisting of the base sequences described in the following (h) to (k). , A carrier for detecting mold, which is characterized by simultaneously detecting two or more molds to be detected.
(H) From a probe consisting of the nucleotide sequences shown in SEQ ID NOs: 16 to 17 selected from the β-tubulin gene for detecting Neurospora crassa, and a nucleotide sequence complementary to the probe. Eighth probe group consisting of at least one of the probes,
(I) From a probe consisting of the nucleotide sequences shown in SEQ ID NOs: 18 to 19 selected from the β-tubulin gene for detecting Emmericella nidulans, and a nucleotide sequence complementary to the probe. Ninth probe group consisting of at least one of the probes,
(J) A probe consisting of the nucleotide sequence shown in SEQ ID NO: 20 selected from the β-tubulin gene for detecting Talaromyces flavus, and a probe consisting of a nucleotide sequence complementary to the probe. A tenth probe group consisting of at least one, and
(K) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 21 to 22 selected from the β-tubulin gene for detecting Talaromyces bacillisporus, and a nucleotide sequence complementary to the probe. Eleventh probe group consisting of at least one of the probes.

Further, the mold detection carrier of the present invention has a configuration in which the probes of the eighth to eleventh probe groups are immobilized and the mold to be detected according to the above (h) to (k) is simultaneously detected. Is preferable.
Further, the probes of the first to seventh probe groups and the probes of the eighth to eleventh probe groups are immobilized, and the mold to be detected according to the above (a) to (k) is simultaneously detected. It is preferable that the configuration is such that

Further, the mold detection method of the present invention is a mold detection method including a step of amplifying a target region in the DNA of a mold to be detected by PCR and confirming the presence or absence of an amplification product, and the mold detection method includes ITS as the target region. Using the region, the reaction solution for PCR contains a primer set for amplifying an ITS region consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 28, and a sample. Then, the sample was used as Mucor racemosus, Lichtheimia corymbifera, Monascus ruber, Thermoascus crustaceus, Thermoascus orantiaceus. If at least one of the DNAs of aurantiacus, Thermoascus thermophilus, and Nigrospora oryzae is contained, the reaction solution for PCR is used and contained in the sample. The target region in DNA is amplified, and the obtained amplification product is subjected to the above-mentioned mold detection carrier , the above-mentioned first to the above, to which the probes of two or more probe groups selected from the above-mentioned first to seventh probe groups are immobilized. A complementary base is added dropwise to the mold detection carrier on which the probe of the seventh probe group is immobilized, or the mold detection carrier on which the probes of the first to eleventh probe groups are immobilized. There is a method of binding with a probe having a sequence.

Further, the mold detection method of the present invention is a mold detection method including a step of amplifying a target region in the DNA of a mold to be detected by PCR and confirming the presence or absence of an amplification product, and β as the target region. -A primer set for amplifying the β-tubulin gene consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 29 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 30 in a reaction solution for PCR using the tuberin gene. And a sample, and the sample contains at least one of Neurospora crassa, Emericella nidulans, Talaromyces flavus, and Talaromyces bacillisporus. When the above-mentioned DNA is contained, the target region in the DNA contained in the sample is amplified by using the reaction solution for PCR, and the obtained amplification product is used as the eighth to eleventh probe group. The mold detection carrier on which the probes of two or more probe groups selected from the above are immobilized, the mold detection carrier on which the probes of the eighth to eleventh probe groups are immobilized, or the first to the first to the above. This is a method in which the probe of the eleventh probe group is dropped onto the immobilized carrier for detecting mold and bound to a probe having a complementary base sequence.

Further, the method for detecting mold of the present invention is a method for detecting mold including a step of amplifying a target region in the DNA of the mold to be detected by PCR and confirming the presence or absence of an amplification product, and ITS is used as the target region. A primer set for amplifying an ITS region consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 28 in a PCR reaction solution using a region and a β-tubulin gene. , And a primer set for amplifying the β-tubulin gene consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 29 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 30 and a sample. In addition, Mucor racemosus, Lichtheimia corymbifera, Monascus ruber, Thermoascus crusta
ceus, Thermoascus aurantiacus, Thermoascus thermophilus, Nigrospora oryzae, Neurospora crassa, Emericella nidulans , Talaromyces flavus, and Talaromyces bacillisporus, if at least one of the DNAs is contained, the PCR reaction solution is used to target the target region in the DNA contained in the sample. The obtained amplification product is used as a probe of one or more probe groups selected from the first to seventh probe groups and one or more probes selected from the eighth to eleventh probe groups. group of probes immobilized fungal detection carrier, or, the first through eleventh probe group of the probe is added dropwise to immobilized the mold detection carrier, the probe having a complementary base sequence There is a way to combine with.

Further, the mold detection kit of the present invention is a mold detection kit containing a primer set for amplifying a target region in the DNA of the mold to be detected and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed. The carrier includes a primer set for amplifying an ITS region consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 28. The mold detection carrier on which the probes of two or more probe groups selected from the first to seventh probe groups are immobilized, and the mold detection on which the probes of the first to seventh probe groups are immobilized. The carrier or the carrier for detecting mold is configured in which the probes of the first to eleventh probe groups are immobilized.

Further, the mold detection kit of the present invention is a mold detection kit containing a primer set for amplifying a target region in the DNA of the mold 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 nucleotide sequence shown in SEQ ID NO: 29 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 30. The carrier is the mold detection carrier on which the probes of two or more probe groups selected from the eighth to eleventh probe groups are immobilized, and the probes of the eighth to eleventh probe groups are immobilized. The above-mentioned carrier for detecting mold or the above-mentioned carrier for detecting mold is configured in which the probes of the first to eleventh probe groups are immobilized.

Further, the mold detection kit of the present invention is a mold detection kit containing a primer set for amplifying a target region in the DNA of the mold 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 an ITS region consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 28, and SEQ ID NO: 29. The carrier includes 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 reverse primer shown in SEQ ID NO: 30, and the carrier is from the first to seventh probe groups. selected one or more probe group of probes and the eighth to eleventh probes immobilized fungal detection carrier for one or more probe group selected from probe groups, or, the first through tenth The probe of one probe group is an immobilized carrier for detecting mold.

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

According to the present invention, the heat-resistant molds Mucol Lasemosas, Liquitemia corimbifera, Monascus ruber, Thermoscus clusterseus, Thermoscus aurantiacas, Thermoscus thermophilus, Nigrospora oryzae, Neurospora crassa, Emericella nidulans, Talaromyces flavus, or Talaromyces Bachirisuporasu, it is possible to detect with accuracy higher specific and multiplexing.

Probes immobilized on the mold detection carrier according to the embodiment of the present invention (molds to be detected: Mucol lacemosus (1), Liquitemia corimbifera (2), Monascus ruber (3), Thermolascus clusterseus (4), Thermoscus aurantiacas (5), Thermoscus thermophilus (6), Nigrospora oryzae (7)). Probes immobilized on the mold detection carrier according to the embodiment of the present invention (molds to be detected: Neurospora Classa (8), Emericella nidurance (9), Paecilomyces flavus (10), Paecilomyces bacilomyces (10) 11), Pesiromyces variotti (12), Bisoclamis zoraniae (13)). It is a figure which shows the base sequence of the primer for amplifying the target region of the target mold detected by the mold detection carrier 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: Mucol lacemosus (1), Liquitemia corimbifera (2), Monascus ruber (3), Thermolascus clusterseus ( 4), Thermoscus aurantiacas (5), Thermoscus thermophilus (6), Nigrospora oryzae (7)) are shown to show the S / N ratio values of the detected fluorescence intensities. Probes immobilized on a carrier for detecting mold according to an embodiment of the present invention (molds to be detected: Neurospora Classa (8), Emmericera Nidurance (9), Paecilomyces flavus (10), Paecilomyces bacilomyces (11) ), Paecilomyces variotti (12), Bisoclamis zoraniae (13)), which shows the S / N ratio value of the fluorescence intensity detected. It is a figure which shows the sequence number of the probe corresponding to the mold of each detection target, and the sample containing 5 types of the mold of the target to be detected by the mold detection carrier which concerns on embodiment of this invention. It is a figure which shows the S / N ratio value of the fluorescence intensity for each probe detected about the sample containing 5 kinds of the molds to be detected by the mold detection carrier which concerns on embodiment of this invention.

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

[Mold detection carrier]
(Mold to be detected)
The molds to be detected by the mold detection carrier of the present embodiment are heat-resistant molds such as Mucor racemosus, Lichtheimia corymbifera, Monascus ruber, and Thermoscus ruber. Clusterseus (Thermoascus crustaceus), Thermoascus aurantiacus, Thermoascus thermophilus, Nigrospora oryzae, Neurospora crassa, Emericella durance (Emericella nidulans), Talaromyces flavus, and Talaromyces bacillisporus. Further, it is also preferable to add 13 types of molds, which are also heat-resistant molds, Paecilomyces variotii and Byssochlamys zollerniae.

These heat-resistant molds form highly heat-resistant structures such as ascospores and can survive heat treatment at 75 ° C. for 30 minutes. Since it is difficult to completely kill heat-resistant molds by pasteurization, it is desirable to be able to confirm the presence or absence of these molds in foods and the environment.

(probe)
The probe for detecting the target mold of the present embodiment is a nucleic acid fragment consisting of the base sequences specified by SEQ ID NOs: 1 to 22 shown in FIGS. 1 to 2.

The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 4 is a probe selected from the ITS region for detecting Mucol lasemosas.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 5 to 7 is a probe selected from the ITS region for detecting Liquitimia colimbifera.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 8 to 9 is a probe selected from the ITS region for detecting Monascus ruber.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 10 to 11 is a probe selected from the ITS region for detecting Thermoscus clusterseus.
The probe consisting of the nucleotide sequence shown in SEQ ID NO: 12 is a probe selected from the ITS region for detecting Thermoscus orrantiacus.
The probe consisting of the nucleotide sequence shown in SEQ ID NO: 13 is a probe selected from the ITS region for detecting Thermoscus thermophilus.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 14 to 15 is a probe selected from the ITS region for detecting nigrospora oryzae.

The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 16 to 17 is a probe selected from the β-tubulin gene for detecting neurospora classa.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 18 to 19 is a probe selected from the β-tubulin gene for detecting emerisera nidurance.
The probe consisting of the nucleotide sequence shown in SEQ ID NO: 20 is a probe selected from the β-tubulin gene for detecting Talaromyces flavus.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 21 to 22 is a probe selected from the β-tubulin gene for detecting Talaromyces bacillispulus.

Further, the probe consisting of the nucleotide sequence shown in SEQ ID NO: 23 shown in FIG. 2 is a probe selected from the ITS region for detecting Talaromyces bacillispirus, and the probe consisting of the nucleotide sequence shown in SEQ ID NOs: 24 to 25 is A probe selected from the β-tubulin gene for detecting pesilomyces variotti, and a probe consisting of the nucleotide sequence shown in SEQ ID NO: 26 is selected from the β-tubulin gene for detecting Bisoclamis zolaniae. It is a probe. These probes may be immobilized on the mold detection carrier of the present embodiment.

In the mold detection carrier of the present embodiment, as described above, a probe selected from the ITS region and / or the β-tubulin gene in the genomic DNA of the mold is used.
Specifically, the seven types shown in Fig. 1 are Mucol Lasemosas, Liquitimia corimbifera, Monascus ruber, Thermoscus clusterseus, Thermoscus aurantiacas, Thermoscus thermophilus, and Nigrospora oryzae. , The specificity of the probe selected from the ITS region is high, and false positive reactions are relatively unlikely to occur. Therefore, when a positive reaction is obtained with these probes, it can be determined that the mold is present. ..
In addition, for the four types shown in FIG. 2, Neurospora classa, Emerisera nidurance, Talaromyces flavus, and Talaromyces bacillisporus, the probe selected from the β-tubulin gene has high specificity and false positive reactions occur. Since it is relatively unlikely to occur, it can be determined that the mold is present when a positive reaction is obtained with these probes.

Regarding the taralomyces bacillispulus shown in FIG. 2, a probe consisting of the nucleotide sequence shown in SEQ ID NO: 23 selected from the ITS region can also be used for determining the presence or absence of the mold. In addition, regarding the two types of Paecilomyces bariotti and Bisoclamis zoraniae shown in the figure, if a positive reaction is obtained with a probe selected from the β-tubulin gene, it is determined that the mold is present. be able to.

(Probe synthesis)
Each of the above probes immobilized on the carrier for detecting mold of the present embodiment has a length of about 18 to 30 bases and can be synthesized by a general DNA synthesizer. As the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 26 immobilized on the carrier for detecting mold in the examples described later, those synthesized by a DNA synthesizer were used. The primers consisting of the base sequences shown in SEQ ID NOs: 27 to 30 used for PCR in the examples described later also had a length of about 19 to 26 bases, and those synthesized by a DNA synthesizer were used.

Further, the probe consisting of the base sequences shown in SEQ ID NOs: 1 to 26 in the present embodiment is not limited to the sequence itself, and one or several bases are deleted, substituted or deleted in the base sequences shown in SEQ ID NOs: 1 to 26. An added probe can be used. It is also possible to use 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 26. Further, these probes and probes having a base sequence complementary to the probes having the base sequences shown in SEQ ID NOs: 1 to 26 can also be used.

The stringent condition means a condition in which a specific hybrid is formed and a non-specific hybrid is not formed. For example, a DNA having high homology (homology of 90% or more, preferably 95% or more) with respect to the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 is complementary to the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1. Conditions for hybridizing with a DNA having a different base sequence can be mentioned. Usually, it refers to the case where hybridization occurs at a temperature of about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the dissolution temperature (Tm) of the complete hybrid. For stringent conditions, see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), in particular, Section 11.45, "Conditions for Hybrid."

Similarly, each primer consisting of the nucleotide sequences of SEQ ID NOs: 27 to 30 shown in FIG. 3 (primer sets of SEQ ID NOs: 27 and 28 and each primer in the primer sets of SEQ ID NOs: 29 and 30) is also limited to the sequence itself. However, when one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NOs: 27 to 30, and a primer consisting of the base sequence shown in SEQ ID NOs: 27 to 30 is used. Those capable of amplifying the same region can also be used. Further, it is a primer composed of a nucleic acid fragment that can be specifically annealed to a nucleic acid fragment consisting of a base sequence complementary to the base sequence shown in SEQ ID NOs: 27 to 30, and is derived from the base sequence shown in SEQ ID NOs: 27 to 30. It is also possible to use a primer capable of amplifying the same region as when the primer is used.

(Creation of carrier for mold detection)
The carrier for detecting mold of the present embodiment can be produced by an existing general method using a probe consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 26.
For example, when a paste-type DNA chip is prepared as a carrier for detecting mold of the present embodiment, the probe is immobilized on a glass substrate by a DNA spotter to form a spot corresponding to each probe. be able to. Further, 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 photolithography technology. Further, the substrate is not limited to glass, and a plastic substrate, a silicon wafer, or the like can be used. Further, the shape of the substrate is not limited to a flat plate shape, and various three-dimensional shapes can be used, and a substrate having a functional group introduced so as to enable a chemical reaction can be used. ..

The mold detection carrier of the present embodiment may have all or a part of the probe consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 26 immobilized therein. Further, the mold detection carrier of the present embodiment is assumed to have all or a part of the probe in which one or several bases have been deleted, substituted or added in the base sequences shown in SEQ ID NOs: 1 to 26 immobilized therein. Also, all or part of the 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: 1-26 shall be immobilized. It is also possible to immobilize all or part of these probes or probes having a base sequence complementary to the probes having the base sequences shown in SEQ ID NOs: 1 to 26.
Further, in the mold detection carrier of the present embodiment, a probe for detecting mold in each group is immobilized on each group of probes selected from the ITS region and probes selected from the β-tubulin gene. Can be assumed to be.

[Mold detection method]
The mold detection method of the present embodiment is a mold detection method including a step of amplifying a target region in the DNA of a mold to be detected by PCR and confirming the presence or absence of an amplification product, and amplifies the target region. In the PCR reaction solution for this purpose, a primer set for amplifying the above-mentioned ITS region and / or a primer set for amplifying the β-tubulin gene and a sample are contained in the sample, and the mold to be detected is contained in the sample. When at least one of the DNAs of the above is contained, the reaction solution for PCR is used to amplify the target region in the DNA contained in the sample, and the obtained amplification product is added dropwise to the above-mentioned mold detection carrier. The method may be any method in which the amplification product is bound to a probe having a complementary base sequence, and is not limited to the specific embodiments and configurations of the examples. It can include 2) extraction of DNA, (3) amplification of the target region, and (4) confirmation of the amplification product.

(1) Collection of mold For example, when collecting airborne mold, indoor or outdoor air in the environment to be inspected is collected. Then, the collected air is blown onto a special medium to culture the mold contained in the air. As the medium, it is preferable to use a predetermined agar medium or the like in a strip shape for an air sampler. As the culture conditions, it is preferable to leave the cells at 25 ° C. in a dark place for 65 hours or more.
Next, the mold colonies cultured on the strip are collected individually for each colony, or two or more colonies are collected together, and the colonies are frozen with liquid nitrogen or the like to freeze the mold cells contained in the colonies. Crush.
For example, when collecting mold from a plant sample, a diseased tissue piece or a healthy tissue piece to be inspected is collected using scissors or a cutter. Then, after surface sterilizing the collected plant tissue pieces with hypochlorous acid or ethanol, the collected plant tissue pieces are placed on a special medium to culture the mold contained in the plant tissue. As the culture conditions, it is preferable to leave the cells in the range of 20 ° C. to 25 ° C. for 65 hours or more.

(2) Extraction of DNA Next, genomic DNA is extracted from the crushed mold cells thus obtained. Genomic DNA can be extracted by a general method such as a method by the CTAB method (Cetyl trimethyl ammonium bromide) or a method using a DNA extraction device.
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 by 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, it amplifies a DNA fragment containing a target region in genomic DNA. The method for amplifying the target region is not particularly limited, but the PCR method can be preferably used. In the PCR method, the target region is amplified by using a PCR reaction solution containing a primer set for amplifying the target region. As the PCR device, a general thermal cycler or the like can be used.
In the mold detection method of the present embodiment, for example, by performing PCR under the following reaction conditions, the 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) ~ (D) for 40 cycles), (e) 72 ° C. for 10 minutes

As the reaction solution for PCR, for example, it is preferable to use a reaction solution having the following composition. That is, nucleic acid synthesis substrate (dNTP mixture (dCTP, dATP, dTTP, dGTP), etc.), primer set, nucleic acid synthase (Nova Taq HotStart DNA polymerase, etc.), fluorescent labeling reagent (Cy5-dCTP, etc.), genomic DNA of sample, buffer. A solution and a PCR reaction solution containing 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, the DNA fragment is amplified by targeting the ITS region and / or the β-tubulin gene in the genomic DNA of the mold as a target region.
At this time, as a primer set for amplifying the ITS region, it is preferable to use one consisting of the nucleotide sequences of SEQ ID NOs: 27 and 28 shown in FIG. 3 (SEQ ID NO: 27: forward primer, SEQ ID NO: 28: reverse primer). ..
As a primer set for amplifying the β-tubulin gene, a primer set consisting of the nucleotide sequences of SEQ ID NOs: 29 and 30 shown in the figure (SEQ ID NO: 29: forward primer, SEQ ID NO: 30: reverse primer) should be used. Is preferable.

(4) Confirmation of Amplification Product Next, the amplification product is dropped onto the mold detection carrier of the present embodiment, and the label of the amplification product hybridized to the probe immobilized on the mold detection carrier is detected. Check for the presence of amplification products. This makes it possible to identify the mold existing in the environment to be inspected.
The label can be detected by using a general label detection device such as a fluorescence scanning device, for example, by measuring the fluorescence intensity of the amplified 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, and generally, when the S / N ratio value is 3 or more, it is determined to be positive. Can be done. 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 may be used.

[Mold detection kit]
The mold detection kit of the present 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. The mold detection kit of the present embodiment detects mold by dropping an amplification product amplified using a primer set onto a mold detection carrier and binding the amplification product to a probe having a complementary base sequence. Used for
The primer set and the carrier for detecting mold can have the following configurations for each group of probes selected from the above-mentioned ITS region and probes selected from the β-tubulin gene.

Group of probes selected from the ITS area:
Primer if the mold to be detected is at least one of Mucol Lasemosas, Liquitimia colimbifera, Monascus ruber, Thermoscus clusterseus, Thermoscus aurantiacas, Thermoscus thermophilus, Nigrospora oryzae. As the set, it is preferable to use a primer set including a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 28 for amplifying the ITS region.

Specifically, the carrier for detecting mold preferably has the following configuration.
A first probe group consisting of at least one of the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 4 selected from the ITS region for detecting Mucor racemosus.
A second probe group consisting of at least one of the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 5 to 7 selected from the ITS region for detecting Lichtheimia corymbifera,
A third probe group consisting of at least one of the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 8 to 9 selected from the ITS region for detecting Monascus ruber,
A fourth probe group consisting of at least one of the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 10 to 11 selected from the ITS region for detecting Thermoascus crustaceus.
A fifth probe group consisting of probes consisting of the nucleotide sequence shown in SEQ ID NO: 12 selected from the ITS region for detecting Thermoascus aurantiacus,
A sixth probe group consisting of probes consisting of the nucleotide sequence shown in SEQ ID NO: 13 selected from the ITS region for detecting Thermoascus thermophilus, and
Mold detection in which the probe of the seventh probe group consisting of at least one of the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 14 to 15 selected from the ITS region for detecting Nigrospora oryzae is immobilized. For carrier.

Group of probes selected from the β-tubulin gene:
When the mold to be detected is at least one of Neurospora Classa, Emerisera Nidurance, Talaromyces flavus, and Talaromyces bacilisporus, the primer set is SEQ ID NO: 29 for amplifying the β-tubulin gene. It is preferable to use a primer set including a forward primer consisting of the base sequence shown and a reverse primer consisting of the base sequence shown in SEQ ID NO: 30.

Specifically, the carrier for detecting mold preferably has the following configuration.
Eighth probe group consisting of at least one of the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 16 to 17 selected from the β-tubulin gene for detecting Neurospora crassa,
A ninth probe group consisting of at least one of the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 18 to 19 selected from the β-tubulin gene for detecting Emmericella nidulans,
A tenth probe group consisting of a probe consisting of the nucleotide sequence shown in SEQ ID NO: 20 selected from the β-tubulin gene for detecting Talaromyces flavus, and a group of tenth probes.
Fix the probe of the eleventh probe group consisting of at least one of the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 21 to 22 selected from the β-tubulin gene for detecting Talaromyces bacillisporus. A carrier for detecting mold.

Further, as the mold detection kit of the present embodiment, a primer set including a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 28 for amplifying the ITS region, and a primer set. A primer set including a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 29 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 30 for amplifying the β-tubulin gene is used, and the probes of all the above groups are fixed. It is also preferable to use a modified carrier for detecting mold.

According to the mold detection carrier, the mold detection method, and the mold detection kit of the present embodiment, these molds are appropriately detected by using a probe capable of specifically identifying the above 11 types of heat-resistant molds. be able to. Therefore, according to the present embodiment, it is possible to detect these molds quickly and with high accuracy in an environmental inspection or the like for confirming the presence or absence of molds.

Hereinafter, a test conducted for confirming the effects of the mold detection carrier, the mold detection method, and the examples of the mold detection kit according to the embodiment 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 the probes of SEQ ID NOs: 1 to 26 shown in FIGS. Each probe used was synthesized by Sigma-Aldrich Japan Co., Ltd. In addition, each probe was immobilized on a substrate by a microarray.

The molds to be detected were obtained from the Japan Collection of Microorganisms, RIKEN BioResource Center, and the NITE Biological Resource Center, Biotechnology Headquarters, National Institute of Technology and Evaluation. The strain was used. Specifically, 13 types of strains were used for the following 13 types of mold.

(1) Mucol Racemosus NBRC4555
(2) Rictemia Kolim Bifera NBRC4009
(3) Monascus Luber NBRC4483
(4) Thermo Asuka Clusterseus NBRC9129
(5) Thermo Asuka Aurantiakas NBRC6766
(6) Thermo Asuka Thermophilus NBRC9643
(7) Nigrospora Orize NBRC32860
(8) Neurospora Classa NBRC6067
(9) Emerisera Nidurance NBRC4340
(10) Talaromyces flavus NBRC7232
(11) Talaromyces Bachirisporus NBRC31150
(12) Paecilomyces Variotti NBRC100534
(13) Bisoclamis Zoraniae JCM12808

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

The PCR reaction solution contained both the ITS region amplification primer set and the β-tubulin gene amplification primer set, and the amplification reaction of these target regions was carried out.
As the primer set for ITS region amplification, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 28 shown in FIG. 3 were used. As the β-tubulin gene amplification primer set, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 29 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 30 shown in the figure were used. As these primers, those synthesized by Sigma-Aldrich Japan Co., Ltd. were used.

Regarding the mold shown in FIG. 1, when a positive reaction is obtained with the probe selected from the ITS region, it can be determined that the mold is present, so that the ITS region is amplified in the reaction solution for PCR. It is also possible to include the primer set for β-tubulin gene amplification and to carry out the amplification reaction of the target region without containing the primer set for amplifying the β-tubulin gene. Similarly, with respect to the mold shown in FIG. 2, when a positive reaction is obtained with a probe selected from the β-tubulin gene, it can be determined that the mold is present, and thus a reaction solution for PCR. Can also contain a primer set for amplifying the β-tubulin gene, and the amplification reaction of the target region can be carried out without containing the primer set for amplifying the ITS region.

Ampdirect (manufactured by Shimadzu Corporation) was used as the reaction solution for PCR, and 20 μl of the following composition was prepared for each of the 13 types of strains.
1. 1. Ampdirect (G / Crich) 4.0 μl
2. Ampdirect (addition-4) 4.0 μl
3. 3. dNTPmix 1.0 μl
4. Cy-5dCTP 0.2 μl
5. ITS1-Fw primer (SEQ ID NO: 27) (2.5 μM) 1.0 μl
6. ITS1-Rv primer (SEQ ID NO: 28) (2.5 μM) 1.0 μl
7. BtF primer (SEQ ID NO: 29) (10 μM) 1.0 μl
8. BtR primer (SEQ ID NO: 30) (10 μM) 1.0 μl
9. Template DNA 1.0 μl
10. NovaTaq HotStart DNA polymerase 0.2 μl
11. Water (water until the whole is 20.0 μl)

Using the above PCR reaction solution, DNA was amplified by a nucleic acid amplification device (TaKaRa PCR Thermal Cycler Disk (registered trademark) manufactured by Gradient Takara Bio Inc.) under the following conditions.
(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 for 10 minutes

Next, a buffer solution (3 × SSC citric acid-physiological saline + 0.3% SDS) is mixed with the PCR amplification product, heated at 94 ° C. for 5 minutes, and added dropwise to 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 did not hybridize was washed away from the DNA chip using the above buffer solution.
Then, using a label detection device (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 was measured, and each was measured. The S / N ratio value in the probe was calculated. The results are shown in FIGS. 4 to 5.

As shown in these figures, the S / N ratio values of the probes having the nucleotide sequences shown in SEQ ID NOs: 1 to 26 are all 3 or more, and can be judged to be positive. Therefore, it can be seen that these probes are effectively functioning to detect each target mold.

[Test 2]
A test for confirming whether the probe immobilized on the mold detection carrier of the present embodiment functions effectively when multiplex PCR is performed to simultaneously amplify the target region in the DNA of a plurality of molds to be detected. went.

The same carrier for mold detection as in Test 1 was used. As for the molds to be detected, 13 types of strains were used for 13 types of molds as in Test 1.
In this test, as shown in FIG. 6, five types of these molds were randomly combined to prepare a mixed DNA sample of the following five groups. Therefore, some molds are duplicated in a plurality of samples. Culturing was carried out using PDA medium or M40Y medium in a dark place at 25 ° C. for 7 days.

(Sample 1)
(1) Mucol Racemosus NBRC4555
(4) Thermo Asuka Clusterseus NBRC9129
(6) Thermo Asuka Thermophilus NBRC9643
(9) Emerisera Nidurance NBRC4340
(11) Talaromyces Bachirisporus NBRC31150

(Sample 2)
(2) Rictemia Kolim Bifera NBRC4009
(5) Thermo Asuka Aurantiakas NBRC6766
(8) Neurospora Classa NBRC6067
(10) Talaromyces flavus NBRC7232
(13) Bisoclamis Zoraniae JCM12808

(Sample 3)
(3) Monascus Luber NBRC4483
(6) Thermo Asuka Thermophilus NBRC9643
(7) Nigrospora Orize NBRC32860
(12) Paecilomyces Variotti NBRC100534
(13) Bisoclamis Zoraniae JCM12808

Then, 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 shaking device.
Next, the genomic DNA of each mold was extracted by a DNA extraction device and mixed as shown in Samples 1 to 3 above. 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 as a primer set for amplifying the ITS region, a primer consisting of the nucleotide sequences shown in SEQ ID NOs: 27 and 28 was used, and β-tubulin was used. As a primer set for amplifying the gene, a primer consisting of the nucleotide sequences shown in SEQ ID NOs: 29 and 30 was used.

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

Using each of the above PCR reaction solutions, DNA was amplified by a nucleic acid amplification device (TaKaRa PCR Thermal Cycler Dice Gradient, manufactured by Takara Bio Inc.) under the following conditions in the same manner as in Test 1.
(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 for 10 minutes

Next, a buffer solution (3 × SSC citric acid-physiological saline + 0.3% SDS) was mixed with the PCR amplification product, heated at 94 ° C. for 5 minutes, and added dropwise to 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 did not hybridize was washed away from the DNA chip using the above buffer solution.
Then, using a label detection device (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 was measured, and each probe was measured. The S / N ratio value in. The result is shown in FIG.

As shown in FIG. 6, the probe for detecting the mold contained in the sample 1 is a probe having the base sequence shown in SEQ ID NOs: 1 to 4,10,11,13,18,19,21-23.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 4 is a probe for detecting Mucol lasemosas. Of these, for SEQ ID NOs: 1, 2 and 4, the S / N ratio values are all 3 or more, indicating a positive reaction. Regarding SEQ ID NO: 3, a positive reaction was not shown in Test 2 in which multiplex PCR containing 5 types of molds was performed, but a positive reaction was shown in Test 1 in which only one type of mold was individually amplified. ..
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 10 and 11 is a probe for detecting Thermoscus clusterseus. All of these have S / N ratio values of 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequence shown in SEQ ID NO: 13 is a probe for detecting Thermoscus thermophilus. Regarding this, the S / N ratio value is 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 18 and 19 is a probe for detecting emerisera nidurance. All of these have S / N ratio values of 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 21 to 23 is a probe for detecting Talaromyces bacillispulus. All of these have S / N ratio values of 3 or more, indicating a positive reaction.
In addition, no positive reaction was observed for probes other than these. That is, no false positive reaction occurred for the probes other than these, and it was shown that the molds other than the mold to be detected were not erroneously detected according to each probe for detecting the mold contained in the sample 1. ing.

As shown in FIG. 6, the probe for detecting the mold contained in the sample 2 is a probe having the base sequence shown in SEQ ID NOs: 5 to 7, 12, 16, 17, 20, and 26.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 5 to 7 is a probe for detecting Liquitimia colimbifera. All of these have S / N ratio values of 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequence shown in SEQ ID NO: 12 is a probe for detecting Thermoscus orrantiacus. Regarding this, the S / N ratio value is 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 16 and 17 is a probe for detecting neurospora classa. All of these have S / N ratio values of 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequence shown in SEQ ID NO: 20 is a probe for detecting Talaromyces flavus. Regarding this, the S / N ratio value is 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequence shown in SEQ ID NO: 26 is a probe for detecting Bisoclamis zolaniae. Regarding this, the S / N ratio value is 3 or more, indicating a positive reaction.
In addition, no positive reaction was observed for probes other than these. That is, no false positive reaction occurred for the probes other than these, and it was shown that the molds other than the mold to be detected were not erroneously detected according to each probe for detecting the mold contained in the sample 2. ing.

As shown in FIG. 6, the probe for detecting the mold contained in the sample 3 is a probe having the base sequence shown in SEQ ID NO: 8, 9, 13, 14, 15, 24, 25, 26.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 8 and 9 is a probe for detecting Monascus ruber. All of these have S / N ratio values of 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequence shown in SEQ ID NO: 13 is a probe for detecting Thermoscus thermophilus. Regarding this, the S / N ratio value is 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 14 and 15 is a probe for detecting nigrospora oryzae. All of these have S / N ratio values of 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequences shown in SEQ ID NOs: 24 and 25 is a probe for detecting Paecilomyces variotti. All of these have S / N ratio values of 3 or more, indicating a positive reaction.
The probe consisting of the nucleotide sequence shown in SEQ ID NO: 26 is a probe for detecting Bisoclamis zolaniae. Regarding this, the S / N ratio value is 3 or more, indicating a positive reaction.
In addition, no positive reaction was observed for probes other than these. That is, no false positive reaction occurred for the probes other than these, and it was shown that the molds other than the mold to be detected were not erroneously detected according to each probe for detecting the mold contained in the sample 3. ing.

As described above, the probe immobilized on the mold detection carrier of the present embodiment can function almost effectively even when multiplex PCR is performed to simultaneously amplify the target region in the DNA of a plurality of molds to be detected. It became clear. It was also revealed that these probes are highly specific and do not show false positive reactions.

It goes without saying that the present invention is not limited to the above embodiments and examples, and 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 the present embodiment is not limited to one spot for each type, and each probe may be immobilized for a plurality of spots. Further, other probes for detecting mold other than the mold to be detected of the present embodiment may be immobilized on the mold detection carrier of the present embodiment together with the probe of the present embodiment, and may be appropriately changed. It is possible.
In addition, the anamorph type, the teleomorph type, and the mold having a synonym related to the target mold in the present invention can be naturally detected, and the detection target mold name shall be based on the latest phylogenetic nomenclature research.

INDUSTRIAL APPLICABILITY The present invention relates to heat-resistant molds Mucol Lasemosas, Liquitemia kolimbifera, Monascus ruber, Thermoascus clusterseus in environmental inspections, food inspections, epidemiological environmental inspections, clinical trials, livestock hygiene, plant disease diagnosis, etc. , Thermoscus aurantiacas, Thermoscus thermophilus, Nigrospora oryzae, Neurospora classa, Emericella nidurance, Talalomyces flavus, and Talalomyces bacillisporus, suitable for specific and multiple detection. It is possible to do.

Claims (11)

The probes of two or more probe groups selected from the first to seventh probe groups consisting of the base sequences described in the following (a) to (g) are immobilized, and two or more molds to be detected are detected at the same time. A carrier for detecting mold.
(A) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 1, 2 and 4 selected from the ITS region for detecting Mucor racemosus, and a probe consisting of a nucleotide sequence complementary to the probe. First probe group consisting of at least one of
(B) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 5 to 7 selected from the ITS region for detecting Lichtheimia corymbifera, and at least a probe consisting of a nucleotide sequence complementary to the probe. A second probe group consisting of either,
(C) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 8 to 9 selected from the ITS region for detecting Monascus ruber, and at least a probe consisting of a nucleotide sequence complementary to the probe. A third probe group consisting of either,
(D) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 10 to 11 selected from the ITS region for detecting Thermoascus crustaceus, and a nucleotide sequence complementary to the probe. A fourth probe group consisting of at least one of the probes,
(E) A probe consisting of the base sequence shown in SEQ ID NO: 12 selected from the ITS region for detecting Thermoascus aurantiacus, and a probe consisting of a base sequence complementary to the probe. Fifth probe group consisting of at least one of
(F) At least a probe consisting of the base sequence shown in SEQ ID NO: 13 selected from the ITS region for detecting Thermoascus thermophilus, and a probe having a base sequence complementary to the probe. A sixth probe group consisting of either, and
(G) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 14 to 15 selected from the ITS region for detecting Nigrospora oryzae, and at least a probe consisting of a nucleotide sequence complementary to the probe. A seventh probe group consisting of either. A carrier for detecting mold, which comprises immobilizing the probes of the first to seventh probe groups according to claim 1 and simultaneously detecting the mold to be detected according to the above (a) to (g). The probes of two or more probe groups selected from the eighth to eleventh probe groups consisting of the base sequences described in the following (h) to (k) are immobilized, and two or more molds to be detected are detected at the same time. A carrier for detecting mold, which is characterized by being used.
(H) From a probe consisting of the nucleotide sequences shown in SEQ ID NOs: 16 to 17 selected from the β-tubulin gene for detecting Neurospora crassa, and a nucleotide sequence complementary to the probe. Eighth probe group consisting of at least one of the probes,
(I) From a probe consisting of the nucleotide sequences shown in SEQ ID NOs: 18 to 19 selected from the β-tubulin gene for detecting Emmericella nidulans, and a nucleotide sequence complementary to the probe. Ninth probe group consisting of at least one of the probes,
(J) A probe consisting of the nucleotide sequence shown in SEQ ID NO: 20 selected from the β-tubulin gene for detecting Talaromyces flavus, and a probe consisting of a nucleotide sequence complementary to the probe. A tenth probe group consisting of at least one, and
(K) A probe consisting of the nucleotide sequences shown in SEQ ID NOs: 21 to 22 selected from the β-tubulin gene for detecting Talaromyces bacillisporus, and a nucleotide sequence complementary to the probe. Eleventh probe group consisting of at least one of the probes. A carrier for detecting mold, which comprises immobilizing the probes of the eighth to eleventh probe groups according to claim 3 and simultaneously detecting the mold to be detected according to the above (h) to (k). The probes of the first to seventh probe groups according to claim 1 and the probes of the eighth to eleventh probe groups according to claim 3 are immobilized, and the detection according to the above (a) to (k). A carrier for detecting mold, which comprises simultaneously detecting a target mold. A method for detecting mold, which comprises a step of amplifying a target region in the DNA of the mold to be detected by PCR and confirming the presence or absence of an amplification product.
Using the ITS region as the target region,
The PCR reaction solution contains a primer set for amplifying an ITS region consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 28, and a sample.
In the sample, Mucor racemosus, Lichtheimia corymbifera, Monascus ruber, Thermoascus crustaceus, Thermoascus aurant , Thermoascus thermophilus, and Nigrospora oryzae, if at least one of the DNAs is contained.
The PCR reaction solution was used to amplify the target region in the DNA contained in the sample.
A method for detecting mold, which comprises dropping the obtained amplification product onto the carrier for detecting mold according to claim 1, 2 or 5, and binding the obtained amplification product to a probe having a complementary base sequence. A method for detecting mold, which comprises a step of amplifying a target region in the DNA of the mold to be detected by PCR and confirming the presence or absence of an amplification product.
Using the β-tubulin gene as the target region,
The PCR reaction solution contains a primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 29 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 30, and a sample. ,
When the sample contains at least one of the DNAs of Neurospora crassa, Emericella nidulans, Talaromyces flavus, and Talaromyces bacillisporus. ,
The PCR reaction solution was used to amplify the target region in the DNA contained in the sample.
A method for detecting mold, which comprises dropping the obtained amplification product onto the carrier for detecting mold according to claim 3, 4 or 5, and binding the obtained amplification product to a probe having a complementary base sequence. A method for detecting mold, which comprises a step of amplifying a target region in the DNA of the mold to be detected by PCR and confirming the presence or absence of an amplification product.
Using the ITS region and the β-tubulin gene as the target region,
A primer set for amplifying an ITS region consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 28, and a base sequence shown in SEQ ID NO: 29 in a PCR reaction solution. A primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of a forward primer and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 30 and a sample are contained.
The samples include Mucor racemosus, Lichtheimia corymbifera, Monascus ruber, Thermoascus crustaceus, Thermoascus aurant. , Thermoascus thermophilus, Nigrospora oryzae, Neurospora crassa, Emericella nidulans, Taralomyces flavus, and Taralomyces flavus. If at least one of the DNAs of Talaromyces bacillisporus) is contained
The PCR reaction solution was used to amplify the target region in the DNA contained in the sample.
The resulting amplification products are selected from claims 1 Symbol placement first to seventh eighth to eleventh probe group selected one or more probe group of probes and claim 3, wherein the probe group of one or more probe groups of probes immobilized fungal detection carrier was, or was dropped on mold detection carrier according to claim 5, characterized in that for coupling a probe having a base sequence complementary How to detect mold. A mold detection kit containing a primer set for amplifying a target region in the DNA of a mold 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
A primer set for amplifying an ITS region consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 28 is included.
A mold detection kit, wherein the carrier is the mold detection carrier according to claim 1, 2 or 5. A mold detection kit containing a primer set for amplifying a target region in the DNA of a mold 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
It contains a primer set for amplifying a β-tubulin gene consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 29 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 30.
A mold detection kit, wherein the carrier is the mold detection carrier according to claim 3, 4 or 5. A mold detection kit containing a primer set for amplifying a target region in the DNA of a mold 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
A primer set for amplifying the ITS region consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 28, and a forward primer and sequence consisting of the nucleotide sequence shown in SEQ ID NO: 29. It contains a primer set for amplifying a β-tubulin gene consisting of a reverse primer consisting of the nucleotide sequence shown in No. 30.
Wherein said carrier, one or more selected from claims 1 Symbol placement first to seventh eighth to eleventh probe group selected one or more probe group of probes and claim 3, wherein the probe group of mold detection carrier probe groups of the probe is immobilized, or a kit for mold detection, which is a fungus detection carrier according to claim 5, wherein.

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