JP2014217374A - Carriers for mold detection, method for detecting molds and kit for detecting molds - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a wide range of molds with accuracy and specificity in an assay to confirm presence or absence of the molds, and to provide a carrier for detecting molds and a kit for detecting molds.SOLUTION: The carrier for detecting molds comprises more than two immobilized probes selected from the group consisting of probes defined in the following (a), (b) and (c), where (a) probes having specified base sequences; (b) probes capable of hybridizing under a stringent condition to nucleic acid fragments of which base sequences are complementary to the specified base sequences; (c) probes of which base sequences are complementary to the probes of (a) or (b).

Description

  The present invention relates to a mold detection carrier for detecting a wide variety of molds, 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 checked whether microorganisms such as mold exist to confirm safety and soundness, and prevent their reproduction It is important.
In such mold inspection, generally, a sample 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). To 4).

JP 2007-195454 A JP 2008-278848 A JP 2008-278861 A JP 2010-4881 A

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. Then, 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 target area of the cultured mold individually, if multiple types of mold are simultaneously amplified, and mold detection is performed with a DNA chip on which a probe selected from these multiple types of mold is immobilized, The speed of inspection is improved.
However, when performing multiplex that simultaneously amplifies a plurality of target regions, probes that function with higher accuracy are also required. In addition, the accuracy required for the probe becomes higher as the number of molds to be detected that are amplified simultaneously increases.

Here, the present inventors include Acremonium sp., Aureobasidium sp., Alternaria sp., Aspergillus Section Circumdati, Aspergillus Section Circumdati, Aspergillus Section Nigri, Aspergillus Section Terrei, Ketomium sp., Colletotrichum sp., Curvularia sp., Fusarium・ Fusarium graminiarum, Fusarium oxysporum, Paecilomyces sp., Trichophyton sp., Verticillium sp., Earthrinium (Arthrinium sp.), 17 of Forma sp. (Phoma sp.), Pythium sp. We conducted diligent research to create molds for detection of molds that can be detected as the types of molds to be detected.
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. In theory, it is considered that each of these types of molds can be specifically detected by each probe.

However, actually, even a probe obtained in this way 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. So far, no mold detection carrier has been known in which a probe capable of specifically detecting each of these 17 types of molds with high accuracy is immobilized.
The present invention has been made in view of the above circumstances, and a mold detection carrier, a mold detection method, and a mold detection method capable of specifically detecting a wide variety of molds more quickly and accurately than in the past. The purpose is to provide a 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 39.
(B) A probe capable of hybridizing 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 39.
(C) A probe having a base sequence complementary to the probe of (a) or (b).

  Further, the mold detection carrier of the present invention includes a probe having a base sequence shown in SEQ ID NO: 4 selected from the ITS region and β-tubulin for detecting Aureobasidium sp. SEQ ID NO: 7 or 8 selected from the ITS region for detecting a first probe group consisting of probes having the base sequence shown in SEQ ID NO: 5 selected from the gene, Alternaria sp. A second probe group consisting of a probe having a base sequence and a probe having a base sequence represented by SEQ ID NO: 9 selected from the β-tubulin gene, for detecting Aspergillus Section Circumdati From the probe having the base sequence shown in SEQ ID NO: 10 selected from the ITS region and the β-tubulin gene A third probe group consisting of probes having the selected base sequence shown in SEQ ID NO: 11, the base sequence shown in SEQ ID NO: 12 selected from the ITS region for detecting Aspergillus Section Nigri And a fourth group of probes consisting of a probe having the nucleotide sequence shown in SEQ ID NO: 13 selected from the β-tubulin gene, and Aspergillus Section Terrei for detecting Aspergillus Section Terrei , Selected from the fifth probe group consisting of a probe having the base sequence shown in SEQ ID NO: 14 selected from the ITS region and a probe having the base sequence shown in SEQ ID NO: 15 selected from the β-tubulin gene A configuration in which two or more groups of probes are immobilized is preferable.

  The mold detection carrier of the present invention includes a probe having the base sequence shown in SEQ ID NO: 20 selected from the ITS region for detecting Fusarium graminiarum, and a β-tubulin gene. A sixth probe group consisting of probes having the base sequence shown in SEQ ID NO: 21 has the base sequence shown in SEQ ID NO: 22 selected from the ITS region for detecting Fusarium oxysporum. From an ITS region for detecting a seventh probe group consisting of a probe and a probe having the base sequence shown in SEQ ID NO: 23 selected from the β-tubulin gene, and Paecilomyces sp. At least one of the probes having the selected nucleotide sequence shown in SEQ ID NO: 24 or 25, And it is set as the structure which fixed 2 or more groups of probes selected from the 8th probe group which consists of at least any one of the probes which have the base sequence shown to sequence number 26 or 27 selected from the beta-tubulin gene. It is preferable.

  In addition, the mold detection carrier of the present invention includes a probe having a base sequence shown in SEQ ID NO: 28 selected from the ITS region for detecting Trichophyton sp., And a β-tubulin gene A ninth probe group consisting of probes having the base sequence shown in SEQ ID NO: 29 selected from the above, the base shown in SEQ ID NO: 30 selected from the ITS region for detecting Verticillium sp. A tenth probe group consisting of at least one of probes having a sequence and a probe having a base sequence shown in SEQ ID NO: 32 or 33 selected from a β-tubulin gene, and Phoma sp. From the probe having the base sequence shown in SEQ ID NO: 36 selected from the ITS region and the β-tubulin gene It is preferable that the immobilized constituting the eleventh selected two groups or more probes from a probe group composed of probes having the nucleotide sequence shown in-option has been SEQ ID NO: 37.

  The mold detection method of the present invention is a mold detection method comprising a step of amplifying a nucleic acid fragment containing a target region in the mold DNA to be detected and confirming the presence or absence of an amplification product, wherein the target region is As a primer set for amplifying the ITS region in the PCR reaction solution for amplifying the nucleic acid fragment containing the target region using both the ITS region and the β-tubulin gene, SEQ ID NO: 40 A base set shown in SEQ ID NO: 42 as a primer set for amplifying the β-tubulin gene using a primer set comprising a forward primer consisting of the indicated base sequence and a reverse primer consisting of the base sequence shown in SEQ ID NO: 41 Forward primer consisting of the sequence and reverse ply consisting of the base sequence shown in SEQ ID NO: 43 In this method, the nucleic acid fragment is amplified using a primer set equipped with a mer, and the obtained amplification product is dropped onto the mold detection carrier to bind to a probe having a complementary base sequence.

The mold detection kit of the present invention includes a primer set for amplifying a nucleic acid fragment containing a target region in the mold DNA to be detected, and a carrier on which a probe for confirming the presence or absence of the amplification product is fixed. A fungus detection kit, the mold to be detected is Aureobasidium sp., Alternaria sp., Aspergillus Section Circumdati, Aspergillus nigri (Aspergillus Section Nigri), Aspergillus Section Terrei, Fusarium graminiarum, Fusarium oxysporum, Paecilomyces sp., Trichophyton sp. sp.), Verticillium sp., Forma (Phoma sp.) And a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 40 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 41 for amplifying the ITS region And a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 42 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 43 for amplifying the β-tubulin gene, A probe having a base sequence shown in SEQ ID NO: 4 selected from the ITS region and a base sequence shown in SEQ ID NO: 5 selected from the β-tubulin gene for detecting Aureobasidium sp. The first group of probes consisting of probes possessed by the genus Alternaria (Al ternaria sp.) for detecting at least one of the probes having the base sequence shown in SEQ ID NO: 7 or 8 selected from the ITS region and the base shown in SEQ ID NO: 9 selected from the β-tubulin gene A second probe group comprising probes having a sequence, a probe having a base sequence shown in SEQ ID NO: 10 selected from the ITS region for detecting Aspergillus Section Circumdati, and a β-tube SEQ ID NO: 12 selected from the ITS region for detecting a third probe group consisting of probes having the base sequence shown in SEQ ID NO: 11 selected from the phosphorus gene, Aspergillus Section Nigri SEQ ID NO: 13 selected from the probe having the nucleotide sequence shown and the β-tubulin gene A probe group having a base sequence shown in SEQ ID NO: 14 selected from the ITS region for detecting Aspergillus Section Terrei, SEQ ID NO: 20 selected from the ITS region for detecting a fifth probe group consisting of probes having the base sequence shown in SEQ ID NO: 15 selected from the β-tubulin gene, Fusarium graminiarum And a sixth probe group consisting of a probe having the base sequence shown in FIG. 5 and a probe having the base sequence shown in SEQ ID NO: 21 selected from the β-tubulin gene, for detecting Fusarium oxysporum A probe having the base sequence shown in SEQ ID NO: 22 selected from the ITS region And a seventh probe group consisting of probes having the base sequence shown in SEQ ID NO: 23 selected from the β-tubulin gene, selected from the ITS region for detecting Paecilomyces sp. An eighth probe comprising at least one of the probes having the base sequence shown in SEQ ID NO: 24 or 25 and at least one of the probes having the base sequence shown in SEQ ID NO: 26 or 27 selected from the β-tubulin gene Group, a probe having the base sequence shown in SEQ ID NO: 28 selected from the ITS region, and SEQ ID NO: 29 selected from the β-tubulin gene for detecting Trichophyton sp. Detects the ninth group of probes consisting of probes with base sequences, Verticillium sp. Therefore, the probe has at least one of a probe having the base sequence shown in SEQ ID NO: 30 selected from the ITS region and a probe having the base sequence shown in SEQ ID NO: 32 or 33 selected from the β-tubulin gene. A tenth probe group, a probe having the base sequence shown in SEQ ID NO: 36 selected from the ITS region, and a β-tubulin gene for detecting a genus Phoma sp. Two or more groups of probes selected from the eleventh probe group consisting of probes having the base sequence shown in SEQ ID NO: 37 are immobilized, and a nucleic acid fragment is amplified using a primer set and obtained. The amplification product thus obtained is preferably dropped onto a carrier and bound to a probe having a complementary base sequence to detect mold.
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, it is possible to specifically detect a wide variety of molds quickly and with high accuracy.

It is a figure which shows the base sequence of the probe fix | immobilized by the support | carrier for a mold detection which concerns on embodiment of this invention. It is a figure which shows the base sequence of the primer set 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. It is a figure which shows the fluorescence intensity (S / N ratio value) detected about the probe fix | immobilized by the support | carrier for mold detection which concerns on embodiment of this invention. Amplification of the target region was performed for each strain. It is a figure which shows the mold | type, strain, and corresponding sequence number which are contained in the sample for the test | inspection of the support | carrier for mold detection which concerns on embodiment of this invention. It is a figure which shows the fluorescence intensity (S / N ratio value) detected about the probe fix | immobilized by the support | carrier for mold detection which concerns on embodiment of this invention. Amplification of the target region was performed by a multiplex that amplifies a plurality of strains simultaneously.

  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]
The mold detection carrier of the present embodiment is particularly limited as long as at least one of the probes (SEQ ID NOs: 1 to 39) for detecting the specific mold shown in FIG. 1 is immobilized on the substrate. For example, a spot type or synthetic type DNA chip or DNA microarray can be used.

(Target mold)
Molds to be detected by the mold detection carrier of the present embodiment include Acremonium sp., Aureobasidium sp., Alternaria sp., Aspergillus silk mudati Aspergillus Section Circumdati, Aspergillus Section Nigri, Aspergillus Section Terrei, Chaetomium sp., Colletotrichum sp., Carbraria (Curvularia sp.), Fusarium graminiarum, Fusarium oxysporum, Paecilomyces sp., Trichophyton sp., Verticillium sp.), Arthrinium sp., Phoma sp. , It is a 17 kind of Pythium spp. (Pythium sp.).

(probe)
The probe for detecting the target mold of this embodiment is a nucleic acid fragment having a base sequence specified by SEQ ID NOs: 1 to 39, respectively.
Among these, the probe having the base sequence shown in SEQ ID NO: 1 to 3 is a probe selected from the ITS region for detecting Acremonium sp. In particular, SEQ ID NO: 1 shows high specificity for Acremonium butyri, SEQ ID NO: 2 shows high specificity for Acremonium strictum, and SEQ ID NO: 3 High specificity for Acremonium chaticola.
In addition, the probe having the base sequence shown in SEQ ID NO: 4 is a probe selected from the ITS region for detecting Aureobasidium sp. And has the base sequence shown in SEQ ID NO: 5. Is a probe selected from the β-tubulin gene for detecting the same bacteria.

  Moreover, what has the base sequence shown to sequence number 6-8 is a probe selected from the ITS area | region for detecting Alternaria sp. (Alternaria sp.), And has the base sequence shown to sequence number 9 Is a probe selected from the β-tubulin gene for detecting the same bacteria. In particular, those having the base sequence shown in SEQ ID NO: 6 exhibit high specificity against Alternaria alternata, and SEQ ID NOs: 7 and 9 are high against Alternaria brassicae. Shows specificity, SEQ ID NOs: 8 and 9 show high specificity for Alternaria brassicicola.

A probe having the base sequence shown in SEQ ID NO: 10 is a probe selected from the ITS region for detecting Aspergillus Section Circumdati and has the base sequence shown in SEQ ID NO: 11. Is a probe selected from the β-tubulin gene for detecting the same bacteria.
A probe having the base sequence shown in SEQ ID NO: 12 is a probe selected from the ITS region for detecting Aspergillus Section Nigri and has the base sequence shown in SEQ ID NO: 13. Is a probe selected from the β-tubulin gene for detecting the same bacteria.
In addition, the probe having the base sequence shown in SEQ ID NO: 14 is a probe selected from the ITS region for detecting Aspergillus Section Terrei and has the base sequence shown in SEQ ID NO: 15. Is a probe selected from the β-tubulin gene for detecting the same bacteria.

Moreover, what has a base sequence shown to sequence number 16 is the probe selected from the ITS area | region for detecting a genus Ketomium (Chaetomium sp.).
Further, the probe having the base sequence shown in SEQ ID NO: 17 is a probe selected from the ITS region for detecting Colletotrichum sp., And having the base sequence shown in SEQ ID NO: 18, A probe selected from the β-tubulin gene for detecting the same bacterium.

Moreover, what has the base sequence shown to sequence number 19 is the probe selected from the ITS area | region for detecting Curvularia sp. (Curvularia sp.).
Moreover, what has the base sequence shown to sequence number 20 is a probe selected from the ITS area | region for detecting Fusarium graminearum (Fusarium graminiarum), and has the base sequence shown to sequence number 21, A probe selected from the β-tubulin gene for detecting the same bacterium.
Further, the probe having the base sequence shown in SEQ ID NO: 22 is a probe selected from the ITS region for detecting Fusarium oxysporum, and has the base sequence shown in SEQ ID NO: 23. A probe selected from the β-tubulin gene for detecting the same bacterium.

In addition, the probes having the nucleotide sequences shown in SEQ ID NOs: 24 and 25 are probes selected from the ITS region for detecting Paecilomyces sp. What has is a probe selected from the β-tubulin gene for detecting the same bacterium. In particular, SEQ ID NO: 24 and SEQ ID NO: 26 show high specificity for Paecilomyces variotii, and SEQ ID NO: 25 and SEQ ID NO: 27 show high specificity for Paecilomyces linacinus. Indicates.
Further, a probe having the base sequence shown in SEQ ID NO: 28 is a probe selected from the ITS region for detecting Trichophyton sp., And has a base sequence shown in SEQ ID NO: 29 , A probe selected from the β-tubulin gene for detecting the same bacterium.

  Moreover, what has the base sequence shown to sequence number 30 and 31 is a probe selected from the ITS area | region for detecting Verticillium sp. (Verticillium sp.), The base shown to sequence number 32 and 33 Those having the sequence are probes selected from the β-tubulin gene for detecting the same bacteria. In particular, SEQ ID NO: 30 exhibits high specificity for Verticillium dahiae.

Further, the probe having the base sequence shown in SEQ ID NO: 34 is a probe selected from the ITS region for detecting Arthrinium sp., And has the base sequence shown in SEQ ID NO: 35. A probe selected from the β-tubulin gene for detecting the same bacterium.
Further, the probe having the base sequence shown in SEQ ID NO: 36 is a probe selected from the ITS region for detecting Phoma sp., And having the base sequence shown in SEQ ID NO: 37, A probe selected from the β-tubulin gene for detecting the same bacterium.
Further, the probe having the base sequence shown in SEQ ID NO: 38 is a probe selected from the ITS region for detecting Pythium sp., And having the base sequence shown in SEQ ID NO: 39, A probe selected from the β-tubulin gene for detecting the same bacterium.

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.
The ITS region is a portion that is spliced into RNA after transcription. For this reason, compared with the coding region, the storage stability is low and changes are abundant, but the similarity between the types of molds is high, and a false positive reaction occurs relatively often. For this reason, when many types of mold probes are immobilized on a DNA chip and used for mold identification, depending on the type of mold, if only a probe selected from the ITS region is used, the detection accuracy decreases. There is a case. On the other hand, the β-tubulin gene has a relatively large number of unique sequences for each type of mold. Therefore, in the mold detection carrier of the present embodiment, depending on the type of mold, it is possible to reduce false positive reaction by using both the ITS region and the β-tubulin gene as target regions.

  Specifically, the mold of the following group (A) is used only when a positive reaction is obtained in both of the ITS region and the probe selected from the β-tubulin gene. It is preferable to determine that has been detected. With respect to these molds, it is possible to reduce the risk of erroneous judgment based on false positive reactions.

Group (A): When a probe selected from the ITS region and a probe selected from the β tubulin gene both show a positive reaction, it is determined that the mold to be detected exists. Aureobasidium spp. Fungus, Aspergillus silk mudati, Aspergillus nigri, Aspergillus terre, Fusarium graminearum, Fusarium oxysporum, Pesilomyces, Trichophyton, Verticillium, Forma

  As for the mold of group (B), when a positive reaction is obtained with a probe selected only from the ITS region, it can be determined that the mold exists. For these molds, the β tubulin gene database is not yet established, or probes that function effectively in the β tubulin gene could not be found. However, since probes selected from the ITS region of these molds have high specificity and are relatively unlikely to produce a false positive reaction, these molds should be detected using a probe selected only from the ITS region. Is possible.

Group (B): A probe selected only from the ITS region shows a positive reaction, and it is determined that the mold to be detected exists Acremonium, Altanaria, Ketomium, Carbaria, Verti Silium genus

  About the mold of group (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 molds of a type that can be detected only by one of the probes selected from the ITS region or the probe selected from the β-tubulin gene in the mold detection carrier of the present embodiment. The specificity is high, and it is possible to detect without causing a false positive reaction.

Group (C): a group that determines that mold to be detected is present when at least one of a probe selected from the ITS region and a probe selected from the β-tubulin gene is positive. Genus, Psium

  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. In addition, about Alternaria genus bacteria and Verticillium genus bacteria, the probe applicable to group (B) was created besides the probe applicable to group (A).

  Therefore, the mold detection carrier of the present embodiment, when the mold selected from the ITS region and β-tubulin gene is immobilized together for the mold of group (A), and both probes show positive, It is preferable to determine that mold to be detected exists. As for the mold of group (B), when a probe selected only from the ITS region is immobilized and the probe shows a positive result, it can be determined that the mold to be detected exists. For the mold of group (C), it is preferable to immobilize both the ITS region and the probe selected from the β-tubulin gene, but when at least one of the probes is positive, Can be determined to exist.

(Probe synthesis)
Each of the probes immobilized on the mold detection carrier of this embodiment has a length of about 20 to 44 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 39 immobilized on a mold detection carrier in the 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 40-45 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 39 in the present 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 39. 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 39 can also be used. Moreover, the probe which has a base sequence complementary to the probe which consists of these probes and the base sequence shown to sequence number 1-39 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”.

  In addition, each primer (The primer set of sequence number 40 and 41, the primer set of sequence number 42 and 43, the primer set of sequence number 44 and 41, and sequence number 45 which consists of a base sequence of sequence number 40-45 shown in FIG. Similarly, each primer in the primer set of No. 43 and No. 43 is not limited to the sequence itself, but is a primer in which one or several bases are deleted, substituted or added in the base sequences shown in SEQ ID NOs: 40 to 45. In addition, those capable of amplifying the same region as in the case of using the primer having the base sequence shown in SEQ ID NOs: 40 to 45 can also be used. A primer comprising a nucleic acid fragment capable of hybridizing under stringent conditions to a nucleic acid fragment comprising a base sequence complementary to the base sequence shown in SEQ ID NOs: 40 to 45, comprising: Those capable of amplifying the same region as in the case of using a primer having the base sequence shown can also 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 39.
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 this embodiment can be obtained by immobilizing all or part of the probes having the base sequences shown in SEQ ID NOs: 1 to 39.
In addition, the mold detection carrier of the present embodiment may have a probe for detecting mold of each group immobilized on each of the groups (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 39. In other words, 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 39 is immobilized. It is also possible to immobilize all or a 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 39.

[How to detect mold]
The mold detection method of the present embodiment is a mold detection method including a step of amplifying a nucleic acid fragment containing a target region in one or two or more molds to be detected and confirming the presence or absence of an amplification product, A primer set for amplifying the β-tubulin gene in the PCR reaction solution for amplifying the target region using the ITS region and / or the β-tubulin gene as the target region, and / or The primer set for amplifying the ITS region is used to amplify the nucleic acid fragment, and the obtained amplification product is dropped on the above-described mold detection carrier, and the amplified product is used as a probe having a complementary base sequence. Any method can be used as long as it is a binding method, and the present invention is not limited to the specific embodiments and examples. For example, (1) fungal collection, (2) DNA extraction, (3 And) (4) amplification of the target region, and (4) confirmation of 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 in 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, nucleic acid synthesis substrate (dNTPmixture (dCTP, dATP, dTTP, dGTP)), primer set, nucleic acid synthase (Nova Taq HotStart DNA polymerase, etc.), fluorescent labeling reagent (Cy5-dCTP, etc.), sample genomic DNA, buffer solution , And a PCR reaction solution containing water as the remaining component can be preferably used. For example, Ampdirect (R) (manufactured by Shimadzu Corporation) can be used as the buffer solution.

In this embodiment, DNA fragments are amplified using the ITS region and / or 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: 40 and 41 shown in FIG. 2 (SEQ ID NO: 40: forward primer, SEQ ID NO: 41: reverse primer). .
Further, as a primer set for amplifying the β-tubulin gene, a primer set consisting of the nucleotide sequences of SEQ ID NOs: 42 and 43 shown in the same figure (SEQ ID NO: 42: forward primer, SEQ ID NO: 43: reverse primer) may be used. preferable.

However, these primer sets cannot amplify the ITS region and β-tubulin gene of Pythium sp.
Therefore, the primer set for amplifying the ITS region of Pythium sp. Is composed of the base sequences shown in SEQ ID NOs: 44 and 41 (SEQ ID NO: 44: forward primer, SEQ ID NO: 41: reverse primer). Is preferably used.
The primer set for amplifying the β-tubulin gene of Pythium sp. Is composed of the nucleotide sequences shown in SEQ ID NOs: 45 and 43 (SEQ ID NO: 45: forward primer, SEQ ID NO: 43: reverse) It is preferable to use a primer.

(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 by 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 (R) of Toyo Kohan Co., Ltd. Can do. The measurement result is preferably obtained as an S / N ratio (Signal to Noise ratio) value. 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.
The primer set and the mold detection carrier preferably have the following configurations for each of the mold groups (A), (B), and (C) described above.

Group (A): When a probe selected from the ITS region and a probe selected from the β-tubulin gene both show a positive reaction, it is determined that the mold to be detected is present. Aureobasidium sp., Alternaria sp., Aspergillus Section Circumdati, Aspergillus Section Nigri, Aspergillus Section Terrei , Fusarium graminiarum, Fusarium oxysporum, Paecilomyces sp., Trichophyton sp., Verticillium sp., Former Is at least one of the genus (Phoma sp.) As an Immer set, a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 40 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 41 for amplifying the ITS region, and a β-tubulin gene are amplified. It is preferable to use a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 42 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 43.

  In addition, when the fungi to be detected are group (A), as the fungus detection carrier, a probe on which a probe for detecting at least one of these fungi is immobilized is used. Specifically, the following configuration is preferable.

A probe having a base sequence shown in SEQ ID NO: 4 selected from the ITS region and a base shown in SEQ ID NO: 5 selected from the β-tubulin gene for detecting Aureobasidium sp. A first probe group comprising probes having a sequence;
A probe having a base sequence shown in SEQ ID NO: 7 or 8 selected from the ITS region and a base shown in SEQ ID NO: 9 selected from the β-tubulin gene for detecting Alternaria sp. A second group of probes comprising probes having sequences,
A probe having the base sequence shown in SEQ ID NO: 10 selected from the ITS region and a base shown in SEQ ID NO: 11 selected from the β-tubulin gene for detecting Aspergillus Section Circumdati A third probe group consisting of probes having sequences;
A probe having the base sequence shown in SEQ ID NO: 12 selected from the ITS region and the base shown in SEQ ID NO: 13 selected from the β-tubulin gene for detecting Aspergillus Section Nigri A fourth group of probes consisting of probes having sequences;
A probe having a base sequence shown in SEQ ID NO: 14 selected from the ITS region and a base shown in SEQ ID NO: 15 selected from the β-tubulin gene for detecting Aspergillus Section Terrei A fifth group of probes comprising probes having sequences;
A probe having the base sequence shown in SEQ ID NO: 20 selected from the ITS region and the base sequence shown in SEQ ID NO: 21 selected from the β-tubulin gene for detecting Fusarium graminiarum. A sixth probe group comprising probes having,
A probe having the base sequence shown in SEQ ID NO: 22 selected from the ITS region and the base sequence shown in SEQ ID NO: 23 selected from the β-tubulin gene for detecting Fusarium oxysporum A seventh probe group comprising probes having,
At least one of probes having the nucleotide sequence shown in SEQ ID NO: 24 or 25 selected from the ITS region and SEQ ID NO: selected from the β-tubulin gene, for detecting Paecilomyces sp. An eighth probe group comprising at least one of the probes having the base sequence shown in 26 or 27,
A probe having the base sequence shown in SEQ ID NO: 28 selected from the ITS region and the base sequence shown in SEQ ID NO: 29 selected from the β-tubulin gene for detecting Trichophyton sp. A ninth probe group consisting of probes having
In order to detect Verticillium sp., A probe having the base sequence shown in SEQ ID NO: 30 selected from the ITS region and SEQ ID NO: 32 or 33 selected from the β-tubulin gene A tenth probe group consisting of at least one of the probes having the base sequence shown, and
A probe having a base sequence shown in SEQ ID NO: 36 selected from the ITS region and a base sequence shown in SEQ ID NO: 37 selected from the β-tubulin gene for detecting a Forma sp. A mold detection carrier on which one or more groups of probes selected from the eleventh probe group comprising probes are immobilized.
The “probe group” means a set of probes, and does not mean only a case of a plurality of probes, but includes a single probe. The same applies to the following.

  That is, the mold detection kit for mold of group (A) drops the amplification product amplified using the above primer set onto the mold detection carrier and binds the amplification product to a probe having a complementary base sequence. Is used to detect mold.

Group (B): When a probe selected only from the ITS region shows a positive reaction, it is determined that the mold to be detected exists. The mold to be detected is Acremonium sp., Alternaria sp. It is a case of at least any one of Alternaria sp., Chaetomium sp., Curvularia sp., And Verticillium sp. The primer set is ITS It is preferable to use a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 40 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 41 for amplifying the region.

In addition, when the mold to be detected is group (B), as the mold detection carrier, a probe on which a probe for detecting at least one of these molds is immobilized is used. Specifically, the following configuration is preferable.
A twelfth probe group comprising at least one of probes having a base sequence represented by SEQ ID NOs: 1 to 3 selected from the ITS region, for detecting Acremonium sp.
A thirteenth probe group comprising probes having the base sequence shown in SEQ ID NO: 6 selected from the ITS region, for detecting Alternaria sp.
A fourteenth probe group comprising probes having a base sequence represented by SEQ ID NO: 16 selected from the ITS region, for detecting a genus Chaetomium sp.
A fifteenth probe group comprising a probe having a base sequence represented by SEQ ID NO: 19 selected from the ITS region for detecting Curvularia sp., And
One or two or more groups selected from the sixteenth probe group consisting of probes having the base sequence shown in SEQ ID NO: 31 selected from the ITS region for detecting Verticillium sp. A mold detection carrier on which a probe is immobilized.
That is, the mold detection kit for mold of group (B) drops the amplification product amplified using the above primer set onto the mold detection carrier and binds the amplification product to a probe having a complementary base sequence. Is used to detect mold.

Group (C): When the detection target mold is present when at least one of the probe selected from the ITS region and the probe selected from the β-tubulin gene is positive, the detection target mold is , At least one of the genus Colletotrichum sp., Arthrinium sp., And Pythium sp.
As a primer set, when the fungus to be detected is a genus Colletotrichum or a genus Earthlinium, it consists of a forward primer consisting of the base sequence shown in SEQ ID NO: 40 for amplifying the ITS region and the base sequence shown in SEQ ID NO: 41 Primer set with a reverse primer and / or a primer set with a forward primer consisting of the base sequence shown in SEQ ID NO: 42 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 43 for amplifying the β-tubulin gene Is preferably used.
In addition, when the fungus to be detected is a genus Psium, a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 44 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 41 for amplifying the ITS region It is preferable to use a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 45 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 43 for amplifying the β-tubulin gene.

In addition, when the fungi to be detected are group (C), as the fungus detection carrier, a probe on which a probe for detecting at least one of these fungi is immobilized is used. Specifically, the following configuration is preferable.
A probe having the base sequence shown in SEQ ID NO: 17 selected from the ITS region and / or the base shown in SEQ ID NO: 18 selected from the β-tubulin gene for detecting Colletotrichum sp. A seventeenth probe group comprising probes having a sequence;
A probe having a base sequence shown in SEQ ID NO: 34 selected from the ITS region and / or a base shown in SEQ ID NO: 35 selected from the β-tubulin gene for detecting Arthrinium sp. An eighteenth probe group comprising probes having a sequence; and
A probe having a base sequence shown in SEQ ID NO: 38 selected from the ITS region and / or a base shown in SEQ ID NO: 39 selected from the β-tubulin gene for detecting Pythium sp. A mold detection carrier on which one or more groups of probes selected from the nineteenth probe group consisting of probes having sequences are immobilized.
That is, the mold detection kit for the mold group (C) drops the amplification product amplified using the above primer set onto the mold detection support and binds the amplification product to a probe having a complementary base sequence. Is used to detect mold.

  According to the mold detection carrier, the mold detection method, and the mold detection kit of the present embodiment, these molds can be appropriately detected by using a probe that can specifically identify the 17 types of molds. it can. For this reason, it is possible to detect molds of a wider range of types more quickly and accurately than in the past in environmental inspections for confirming the presence or absence of mold.

  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 detecting carrier, Gene Silicon (R) (manufactured by Toyo Kohan Co., Ltd.) was used, and the one having the probes of SEQ ID NOS: 1 to 39 shown in FIG. 1 immobilized thereon was used. Each probe was synthesized by Operon Technology 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, as shown in FIG. 3, 24 types of strains were used for the following 17 types of molds.

(1) Acremonium sp. NBRC6615, NBRC102318, NBRC8952
(2) Aureobasidium sp. NBRC100716
(3) Alternaria sp. NBRC4026, NBRC9766, NBRC31226
(4) Aspergillus Section Circumdati NBRC31221
(5) Aspergillus Section Nigri JCM10254
(6) Aspergillus Section Terrei NBRC6346
(7) Chaetomium sp. NBRC6347
(8) Colletotrichum sp. NBRC104617
(9) Curvularia sp. NBRC100165
(10) Fusarium graminiarum NBRC9873
(11) Fusarium oxysporum JCM11502
(12) Paecilomyces sp. NBRC32038, NBRC5752
(13) Trichophyton sp. NBRC5807
(14) Verticillium sp. NBRC9435, NBRC9470
(15) Arthrinium sp. NBRC100562, NBRC30798
(16) Forma sp. NBRC104600
(17) Pythium sp. NBRC100125

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 / or β-tubulin gene of each mold was amplified for each strain by PCR.

Here, each mold of group (A) (Aureobasidium, Alternaria, Aspergillus silk mudati, Aspergillus nigri, Aspergillus terre, Fusarium graminearum, Fusarium oxysporum, Pesilomyces For the genera, Trichophyton, Verticillium, Forma, the ITS region and the β-tubulin gene were amplified simultaneously.
At this time, a forward primer consisting of the base sequence of SEQ ID NO: 40 and a reverse primer consisting of the base sequence of SEQ ID NO: 41 shown in FIG. 2 were used as the ITS region amplification primer set. Moreover, as a primer set for β-tubulin gene amplification, a forward primer consisting of the base sequence of SEQ ID NO: 42 and a reverse primer consisting of the base sequence of SEQ ID NO: 43 shown in the figure were used.

In addition, for each mold of group (B) (Acremonium genus, Alternaria genus (SEQ ID NO: 6), Ketomium genus, Carbaria genus, Verticillium genus (SEQ ID NO: 31)), only the ITS region is used. Amplified.
At this time, a forward primer consisting of the base sequence of SEQ ID NO: 40 and a reverse primer consisting of the base sequence of SEQ ID NO: 41 shown in FIG. 2 were used as the ITS region amplification primer set.

In addition, for each mold of the group (C) (Colletotricum spp., Earthrinium spp., Psium spp.), The ITS region and / or the β-tubulin gene was amplified.
At this time, the forward primer consisting of the base sequence of SEQ ID NO: 40 shown in FIG. 2 and the reverse primer consisting of the base sequence of SEQ ID NO: 41 shown in FIG. Moreover, as a primer set for β-tubulin gene amplification, a forward primer consisting of the base sequence of SEQ ID NO: 42 and a reverse primer consisting of the base sequence of SEQ ID NO: 43 shown in the figure were used.
For Psium, the target region cannot be amplified using these primer sets. For this reason, the test for SEQ ID NO: 38 of the genus Psium was carried out by amplifying only the ITS region using primers consisting of the nucleotide sequences shown in SEQ ID NOs: 44 and 41 as an ITS region amplification primer set. In addition, the test for SEQ ID NO: 39 of the genus Psium was carried out by amplifying only the β tubulin gene using primers consisting of the nucleotide sequences shown in SEQ ID NOs: 45 and 43 as a primer set for amplifying β tubulin gene. .
The above primers were synthesized by Operon Technology Co., Ltd.

As a reaction solution for PCR, Ampdirect (R) (manufactured by Shimadzu Corporation) is used for each of 22 types of strains except for a strain of the genus Verticillium (NBRC9470) and a strain of the genus Psium (NBRC100125). 20 μl of the following composition was prepared.
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: 40) (2.5μM) 1.0μl
6). ITS1-Rv primer (SEQ ID NO: 41) (2.5μM) 1.0μl
7). BtF primer (SEQ ID NO: 42) (10 μM) 1.0 μl
8). BtR primer (SEQ ID NO: 43) (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)

A PCR reaction solution for amplifying a strain of the genus Verticillium (NBRC9470) was prepared with the following composition.
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: 40) (10μM) 1.0μl
6). ITS1-Rv primer (SEQ ID NO: 41) (10μM) 1.0μl
7). Template DNA 1.0μl
8). NovaTaq HotStart DNA polymerase 0.2μl
9. Water (water until 20.0 μl total)

As a reaction solution for PCR for amplifying the ITS region of the strain of Psium (NBRC100125), the following composition was prepared.
1. Ampdirect (G / Crich) 4.0μl
2. Ampdirect (addition-4) 4.0μl
3. dNTPmix 1.0μl
4). Cy-5dCTP 0.2μl
5. Pyu1-1 primer (SEQ ID NO: 44) (10μM) 1.0μl
6). ITS1-Rv primer (SEQ ID NO: 41) (10μM) 1.0μl
7). Template DNA 1.0μl
8). NovaTaq HotStart DNA polymerase 0.2μl
9. Water (water until 20.0 μl total)

As a reaction solution for PCR for amplifying the β-tubulin gene of a strain of the genus Psium (NBRC100125), one having the following composition was prepared.
1. Ampdirect (G / Crich) 4.0μl
2. Ampdirect (addition-4) 4.0μl
3. dNTPmix 1.0μl
4). Cy-5dCTP 0.2μl
5. Pu-bete1 primer (SEQ ID NO: 45) (10μM) 1.0μl
6). BtR primer (SEQ ID NO: 43) (10 μM) 1.0 μl
7). Template DNA 1.0μl
8). NovaTaq HotStart DNA polymerase 0.2μl
9. Water (water until 20.0 μl total)

Using each of the PCR reaction solutions, DNA was amplified using the nucleic acid amplification apparatus (TaKaRa PCR Thermal Cycler Dice (R) Gradient manufactured by 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 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 device (BIOSHOT (R), 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. Then, the S / N ratio value ((median fluorescence intensity value−background value) ÷ background value) in each probe was obtained. The result is shown in FIG.

  As shown in the figure, the S / N ratio values of the probes having the nucleotide sequences shown in SEQ ID NOs: 1 to 39 are all 3 or more, and can be judged as positive. Therefore, it can be seen that all of these probes are judged to be positive and function effectively in order to detect each target mold.

Specifically, each mold of group (A) (Aureobasidium, Alternaria, Aspergillus silk mudati, Aspergillus nigri, Aspergillus terre, Fusarium graminearum, Fusarium oxysporum , Pecilomyces spp., Trichophyton spp., Verticillium spp., Forma spp.) Were positive for both the probe selected from the ITS region and the probe selected from the β-tubulin gene. In this case, it is determined that the mold exists.
As for these molds, as shown in FIG. 3, the S / N ratio values of both the probe selected from the ITS region and the probe selected from the β-tubulin gene show a positive reaction. Therefore, it can be seen that all the probes that detect these molds function effectively.

For each group (B) mold (Acremonium spp, Altanaria spp, Ketomium spp, Carbaria spp, Verticillium spp) when a positive reaction is obtained with a probe selected only from the ITS region It is determined that the mold is present.
As for these molds, as shown in FIG. 3, the S / N ratio value in the probe selected from the ITS region shows a positive reaction, and all the probes for detecting these molds are effective. You can see that it works.

For each mold of the group (C) (Choletotricum spp., Earthurinium spp., Psium spp.), When a positive reaction is obtained in at least one of the probes selected from the ITS region or the β-tubulin gene, It is determined that the mold is present.
As for these molds, as shown in FIG. 3, the S / N ratio values of both the probe selected from the ITS region and the probe selected from the β-tubulin gene showed positive reactions in the genus Coretolicum. ing. In addition, regarding the genus Arsilinium, the S / N ratio value in the probe selected from the ITS region showed a positive reaction in the arsolinium faeospam strain, and was selected from the β tubulin gene in the arsolinium japonicum strain. The S / N ratio value in the probe indicates a positive reaction. Furthermore, in the genus Psium, the S / N ratio values of both the probe selected from the ITS region and the probe selected from the β-tubulin gene show positive reactions, respectively. 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 is performed to confirm 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 mold to be detected, 24 types of strains were used for 17 types of mold as in Test 1.
In this test, as shown in FIG. 4, 4 or 5 of these molds were randomly combined to prepare the following 6 groups of samples, and molds were cultured for each sample. 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)
Acremonium charticola NBRC8952
Alternaria alternata NBRC4026
Arthrinium phaeospermum NBRC100562
Aureobasidium pullulans NBRC100716
Colletotrichum gloeosporioides NBRC104617

(Sample 2)
Acremonium butyri NBRC6615
Ketotomium globosum NBRC6347
Paecilomyces variotii NBRC32038
Forma exigua NBRC104600

(Sample 3)
Acremonium strictum NBRC102318
Arthrinium phaeospermum NBRC100562
Aspergillus niger JCM10254
Aspergillus terreus NBRC6346
Trichophyton rubrum NBRC5807

(Sample 4)
Acremonium butyri NBRC6615
Alternaria alternata NBRC4026
Aspergillus ochraceus NBRC31221
Aureobasidium pullulans NBRC100716
Ketotomium globosum NBRC6347

(Sample 5)
Alternaria barassicae NBRC9766
Alternaria brassicicola NBRC31226
Pythium ultimum NBRC100125
Verticillium dahiae NBRC9435

(Sample 6)
Arthrinium japonicum NBRC30798
Carvularia lunata NBRC100165
Fusarium oxysporum JCM11502
Fusarium Graminiarum (NBRC9873)
Paecilomyces lilacinus NBRC5752

Then, mold colonies cultured for each sample were collected in a lump, put into each vial containing φ0.5 mm zirconia beads, and immersed in liquid nitrogen to freeze the sample, then using a shaking device, Mold cells were crushed.
Subsequently, mold genomic DNA was extracted by a DNA extraction apparatus, and the ITS region and / or β-tubulin gene of each mold was simultaneously amplified for each sample by PCR. The primer set used in the PCR method is the same as in Test 1. That is, for samples 1 to 4, 6, a primer set for amplifying the tubulin gene using primers having the nucleotide sequences shown in SEQ ID NOs: 40 and 41 as a primer set for amplifying the ITS region. As the primers, primers having the nucleotide sequences shown in SEQ ID NOs: 42 and 43 were used. Further, for sample 5 containing Psium Ultimum bacteria, in addition to these primer sets, there is also a primer having the base sequence shown in SEQ ID NO: 45 as a dedicated forward primer for amplifying the β-tubulin gene. Contained. In this test, a primer having the base sequence shown in SEQ ID NO: 44 was not used as a dedicated forward primer for amplifying the ITS region of Pythium Ultimum. Therefore, the ITS region of P. ultimum is not amplified, and as for SEQ ID NO: 38, the effect of multiplex has not been confirmed by this test.

As a reaction solution for PCR, Ampdirect (R) (manufactured by Shimadzu Corporation) was used, and 20 μl of the following composition was prepared for each sample. However, only Sample 5 also contained 1.0 μl of Pu-beta1 primer (10 μM) for amplifying the β-tubulin gene of the genus Psium.
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 the above 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 (R) 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 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 (GenePix4100A Molecular Devices), 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, and the S / N ratio values were obtained. The result is shown in FIG.

As shown in FIG. 4, the probe for detecting mold contained in the sample 1 is a probe having the base sequences shown in SEQ ID NOs: 3 to 6, 17, 18, and 34.
As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NOs: 3 and 6 is a probe for detecting mold of group (B), and a probe selected only from the ITS region showed a positive result. In this case, it is determined that the mold to be detected exists. As shown in FIG. 5, all of these have a S / N ratio value of 3 or more, indicating a positive reaction.
As shown in FIG. 1, the probes having the base sequences shown in SEQ ID NOs: 4 and 5 are probes for detecting the mold of group (A), and are selected from probes selected from the ITS region and β-tubulin gene. When both of the selected probes show a positive reaction, it is determined that mold to be detected exists. As shown in FIG. 5, both of these have a S / N ratio value of 3 or more, indicating a positive reaction.

As shown in FIG. 1, the probes having the base sequences shown in SEQ ID NOs: 17, 18, and 34 are probes for detecting mold of group (C), and probes selected from the ITS region and β-tubulin When at least one of the probes selected from the genes shows a positive reaction, it is determined that the mold to be detected exists. As shown in FIG. 5, the probe having the base sequence shown in SEQ ID NO: 17 has an S / N ratio value of 3 or more, indicating a positive reaction. The probe having the base sequence shown in SEQ ID NO: 18 has an S / N ratio value of less than 3. Therefore, the probe did not function sufficiently in multiplex PCR that simultaneously amplifies a plurality of mold DNAs. However, the S / N ratio value is 0.55, and it can be seen that the reaction is weak although it is compared with other S / N ratio values that have not reacted at all. The probe having the base sequence shown in SEQ ID NO: 34 has a S / N ratio value of 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 the probe for detecting mold contained in the sample 1 indicates that mold other than the mold to be detected is not erroneously detected. Yes.

As shown in FIG. 4, the probe for detecting mold contained in the sample 2 is a probe having the base sequences shown in SEQ ID NOs: 1, 16, 24, 26, 36, and 37.
As shown in FIG. 1, the probes having the nucleotide sequences shown in SEQ ID NOs: 1 and 16 are probes for detecting mold of group (B), and probes selected only from the ITS region show a positive reaction. If the detection target mold exists, it is determined that the detection target mold exists. As shown in FIG. 5, all of these have a S / N ratio value of 3 or more, indicating a positive reaction.
The probes having the base sequences shown in SEQ ID NOs: 24 and 26 and the probes having the base sequences shown in SEQ ID NOs: 36 and 37 are probes for detecting mold of group (A), as shown in FIG. 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. 5, each of these has a S / N ratio value of 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 the probe for detecting the mold contained in the sample 2 indicates that mold other than the mold to be detected is not erroneously detected. Yes.

As shown in FIG. 4, the probe for detecting mold contained in the sample 3 is a probe having the base sequence shown in SEQ ID NOs: 2, 12-15, 28, 29, and 34.
The probe having the base sequence shown in SEQ ID NO: 2 is a probe for detecting mold of group (B) as shown in FIG. 1, and a probe selected only from the ITS region shows a positive reaction In addition, it is determined that the mold to be detected exists. As shown in FIG. 5, the S / N ratio value of this probe is 3 or more, indicating a positive reaction.
As shown in FIG. 1, the probes having the base sequences shown in SEQ ID NOs: 12 and 13, the probes having the base sequences shown in SEQ ID NOs: 14 and 15, and the probes having the base sequences shown in SEQ ID NOs: 28 and 29 are groups. (A) A probe for detecting mold, and when both the probe selected from the ITS region and the probe selected from the β-tubulin gene show a positive reaction, it is determined that the mold to be detected exists. Is. As shown in FIG. 5, each of these has a S / N ratio value of 3 or more, indicating a positive reaction.

As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NO: 34 is a probe for detecting mold of group (C), and is selected from a probe selected from the ITS region and a β-tubulin gene. When at least one of the probes shows a positive reaction, it is determined that mold to be detected exists. As shown in FIG. 5, the S / N ratio value of this probe is 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 the probe for detecting mold contained in the sample 3 indicates that mold other than the mold to be detected is not erroneously detected. Yes.

As shown in FIG. 4, the probe for detecting mold contained in the sample 4 is a probe having the base sequences shown in SEQ ID NOs: 1, 4-6, 10, 11, and 16.
As shown in FIG. 1, the probes having the nucleotide sequences shown in SEQ ID NOs: 1, 6 and 16 are probes for detecting mold of group (B), and a probe selected only from the ITS region is positively reacted. Is determined that there is mold to be detected. As shown in FIG. 5, all of these have a S / N ratio value of 3 or more, indicating a positive reaction.
The probes having the base sequences shown in SEQ ID NOs: 4 and 5 and the probes having the base sequences shown in SEQ ID NOs: 10 and 11 are probes for detecting mold of group (A) as shown in FIG. 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. 5, each of these has a S / N ratio value of 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 the probe for detecting mold contained in the sample 4 indicates that mold other than the mold to be detected is not detected by mistake. Yes.

As shown in FIG. 4, the probe for detecting mold contained in the sample 5 is a probe having a base sequence shown in SEQ ID NOs: 7-9, 30-33, 38, and 39.
A probe having a base sequence shown in SEQ ID NOs: 7 and 9, a probe having a base sequence shown in SEQ ID NOs: 8 and 9, a probe having a base sequence shown in SEQ ID NOs: 30 and 32, and a base sequence shown in SEQ ID NOs: 30 and 33 As shown in FIG. 1, the probe is a probe for detecting the mold of group (A), and both the probe selected from the ITS region and the probe selected from the β-tubulin gene showed a positive reaction. If the detection target mold exists, it is determined that the detection target mold exists. As shown in FIG. 5, each of these has a S / N ratio value of 3 or more, indicating a positive reaction.
As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NO: 31 is a probe for detecting mold of group (B), and a probe selected only from the ITS region shows a positive reaction In addition, it is determined that the mold to be detected exists. As shown in FIG. 5, the S / N ratio value of this probe is less than 3. Therefore, the probe did not function sufficiently in multiplex PCR that simultaneously amplifies a plurality of mold DNAs. However, the S / N ratio value is 1.74, and it can be seen that the reaction is weak although it is compared with other S / N ratio values that have not reacted at all.

As shown in FIG. 1, the probes having the nucleotide sequences shown in SEQ ID NOs: 38 and 39 are probes for detecting mold of group (C), and are selected from probes selected from the ITS region and β-tubulin gene. When at least one of the selected probes shows a positive reaction, it is determined that mold to be detected exists. As shown in FIG. 5, the S / N ratio value of the probe having the base sequence shown in SEQ ID NO: 39 is 3 or more, indicating a positive reaction. In this test, the effect of the probe having the base sequence shown in SEQ ID NO: 38 was not confirmed, but it was confirmed by a separately conducted test that the effect of the multiplex was not sufficient for the probe. . However, as shown in Test 1, it has been found that when the ITS region is amplified alone, it functions effectively. Test 2 shows that the probe does not produce a false positive reaction.
Furthermore, no positive reaction is seen with probes other than these. That is, no false positive reaction has occurred for the probes other than these, and the probe for detecting the mold contained in the sample 5 indicates that mold other than the mold to be detected is not detected by mistake. Yes.

As shown in FIG. 4, the probe for detecting mold contained in the sample 6 is a probe having a base sequence shown in SEQ ID NOs: 19-23, 25, 27, and 35.
As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NO: 19 is a probe for detecting mold of group (B), and a probe selected only from the ITS region shows a positive reaction In addition, it is determined that the mold to be detected exists. As shown in FIG. 5, the S / N ratio value of this probe is 3 or more, indicating a positive reaction.
As shown in FIG. 1, the probes having the base sequences shown in SEQ ID NOs: 20 and 21, the probes having the base sequences shown in SEQ ID NOs: 22 and 23, and the probes having the base sequences shown in SEQ ID NOs: 25 and 27 are groups. (A) Probe for detecting mold, and when both the probe selected from the ITS region and the probe selected from the β-tubulin gene show a positive reaction, it is determined that the mold to be detected exists. Is. As shown in FIG. 5, each of these has a S / N ratio value of 3 or more, indicating a positive reaction.

As shown in FIG. 1, the probe having the base sequence shown in SEQ ID NO: 35 is a probe for detecting mold of group (C), and is selected from a probe selected from the ITS region and a β-tubulin gene. When at least one of the detected probes shows a positive reaction, it is determined that the detection target mold exists. As shown in FIG. 5, the S / N ratio value of this probe is 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 the probe for detecting the mold contained in the sample 6 indicates that mold other than the mold to be detected is not detected by mistake. Yes.

  As described above, the probe immobilized on the mold detection carrier of the present embodiment, except for some, even when performing multiplex PCR that simultaneously amplifies the target region in a plurality of mold DNAs to be detected, It became clear that it functions effectively. 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.

  INDUSTRIAL APPLICABILITY The present invention can be suitably used when a plurality of molds are specifically and multiplely detected in environmental inspections, food inspections, epidemiological environmental inspections, clinical tests, livestock hygiene, plant disease diagnosis, and the like.

Claims (12)

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 39.
(B) A probe capable of hybridizing 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 39.
(C) A probe having a base sequence complementary to the probe of (a) or (b). A probe having a base sequence shown in SEQ ID NO: 4 selected from the ITS region and a base shown in SEQ ID NO: 5 selected from the β-tubulin gene for detecting Aureobasidium sp. A first probe group comprising probes having a sequence;
A probe having a base sequence shown in SEQ ID NO: 7 or 8 selected from the ITS region and a base shown in SEQ ID NO: 9 selected from the β-tubulin gene for detecting Alternaria sp. A second group of probes comprising probes having sequences,
A probe having the base sequence shown in SEQ ID NO: 10 selected from the ITS region and a base shown in SEQ ID NO: 11 selected from the β-tubulin gene for detecting Aspergillus Section Circumdati A third probe group consisting of probes having sequences;
A probe having the base sequence shown in SEQ ID NO: 12 selected from the ITS region and the base shown in SEQ ID NO: 13 selected from the β-tubulin gene for detecting Aspergillus Section Nigri A fourth group of probes comprising probes having sequences; and
A probe having a base sequence shown in SEQ ID NO: 14 selected from the ITS region and a base shown in SEQ ID NO: 15 selected from the β-tubulin gene for detecting Aspergillus Section Terrei The mold detection carrier according to claim 1, wherein two or more groups of probes selected from the fifth probe group comprising probes having sequences are immobilized. A probe having the base sequence shown in SEQ ID NO: 20 selected from the ITS region and the base sequence shown in SEQ ID NO: 21 selected from the β-tubulin gene for detecting Fusarium graminiarum. A sixth probe group comprising probes having,
A probe having the base sequence shown in SEQ ID NO: 22 selected from the ITS region and the base sequence shown in SEQ ID NO: 23 selected from the β-tubulin gene for detecting Fusarium oxysporum A seventh group of probes comprising probes having, and
At least one of probes having the nucleotide sequence shown in SEQ ID NO: 24 or 25 selected from the ITS region and SEQ ID NO: selected from the β-tubulin gene, for detecting Paecilomyces sp. The mold detection carrier according to claim 1, wherein two or more groups of probes selected from the eighth probe group consisting of at least one of the probes having the base sequence shown in 26 or 27 are immobilized. A probe having the base sequence shown in SEQ ID NO: 28 selected from the ITS region and the base sequence shown in SEQ ID NO: 29 selected from the β-tubulin gene for detecting Trichophyton sp. A ninth probe group consisting of probes having
In order to detect Verticillium sp., A probe having the base sequence shown in SEQ ID NO: 30 selected from the ITS region and SEQ ID NO: 32 or 33 selected from the β-tubulin gene A tenth probe group consisting of at least one of the probes having the base sequence shown, and
A probe having a base sequence shown in SEQ ID NO: 36 selected from the ITS region and a base sequence shown in SEQ ID NO: 37 selected from the β-tubulin gene for detecting a Forma sp. 2. The mold detection carrier according to claim 1, wherein two or more groups of probes selected from the eleventh probe group comprising probes are immobilized. A twelfth probe group comprising at least one of probes having a base sequence represented by SEQ ID NOs: 1 to 3 selected from the ITS region, for detecting Acremonium sp.
A thirteenth probe group comprising probes having the base sequence shown in SEQ ID NO: 6 selected from the ITS region, for detecting Alternaria sp.
A fourteenth probe group comprising probes having a base sequence represented by SEQ ID NO: 16 selected from the ITS region, for detecting a genus Chaetomium sp.
A fifteenth probe group comprising a probe having a base sequence represented by SEQ ID NO: 19 selected from the ITS region for detecting Curvularia sp., And
In order to detect Verticillium sp., Two or more probes selected from the sixteenth probe group consisting of probes having the base sequence shown in SEQ ID NO: 31 selected from the ITS region are used. The mold detecting carrier according to claim 1, which is immobilized. A probe having the base sequence shown in SEQ ID NO: 17 selected from the ITS region and / or the base shown in SEQ ID NO: 18 selected from the β-tubulin gene for detecting Colletotrichum sp. A seventeenth probe group comprising probes having a sequence;
A probe having a base sequence shown in SEQ ID NO: 34 selected from the ITS region and / or a base shown in SEQ ID NO: 35 selected from the β-tubulin gene for detecting Arthrinium sp. An eighteenth probe group comprising probes having a sequence; and
A probe having a base sequence shown in SEQ ID NO: 38 selected from the ITS region and / or a base shown in SEQ ID NO: 39 selected from the β-tubulin gene for detecting Pythium sp. 2. The mold detection carrier according to claim 1, wherein two or more groups of probes selected from the nineteenth probe group consisting of probes having sequences are immobilized. A method for detecting mold, comprising a step of amplifying a nucleic acid fragment containing a target region in the DNA of a mold to be detected, and confirming the presence or absence of the amplification product,
Using both the ITS region and the β-tubulin gene as the target region,
In a PCR reaction solution for amplifying a nucleic acid fragment containing the target region, as a primer set for amplifying the ITS region, a forward primer consisting of the base sequence shown in SEQ ID NO: 40 and a base sequence shown in SEQ ID NO: 41 As a primer set for amplifying the β-tubulin gene, a reverse primer consisting of the base sequence shown in SEQ ID NO: 42 and a reverse sequence consisting of the base sequence shown in SEQ ID NO: 43 is used. Amplifying the nucleic acid fragment using a primer set with primers;
The obtained amplification product is dropped onto the mold detection carrier according to any one of claims 1 to 4 and bound to a probe having a complementary base sequence. A method for detecting mold, comprising a step of amplifying a nucleic acid fragment containing a target region in the DNA of a mold to be detected, and confirming the presence or absence of the amplification product,
Use the ITS area as the target area,
In a PCR reaction solution for amplifying a nucleic acid fragment containing the target region, as a primer set for amplifying the ITS region, a forward primer consisting of the base sequence shown in SEQ ID NO: 40 and a base sequence shown in SEQ ID NO: 41 Amplifying the nucleic acid fragment using a primer set comprising a reverse primer
A method for detecting mold, which comprises dropping the obtained amplification product onto the mold detection carrier according to claim 5 and binding it to a probe having a complementary base sequence. A method for detecting mold, comprising a step of amplifying a nucleic acid fragment containing a target region in the DNA of a mold to be detected, and confirming the presence or absence of the amplification product,
As the target region, an ITS region and / or a β-tubulin gene is used,
When the mold to be detected is at least one of the genus Colletotrichum sp. Or Arthrinium sp.
In a PCR reaction solution for amplifying a nucleic acid fragment containing the target region, as a primer set for amplifying the ITS region, a forward primer consisting of the base sequence shown in SEQ ID NO: 40 and a base sequence shown in SEQ ID NO: 41 A primer set comprising a reverse primer and / or a primer set for amplifying the β-tubulin gene, comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 42 and a base sequence shown in SEQ ID NO: 43 Using a primer set with a reverse primer, the nucleic acid fragment is amplified,
If the mold to be detected is Pythium sp.
In the PCR reaction solution for amplifying the nucleic acid fragment containing the target region, as a primer set for amplifying the ITS region, a forward primer consisting of the base sequence shown in SEQ ID NO: 44 and a base sequence shown in SEQ ID NO: 41 A primer set comprising a reverse primer and / or a primer set for amplifying the β-tubulin gene, comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 45 and a base sequence shown in SEQ ID NO: 43 Using a primer set with a reverse primer, the nucleic acid fragment is amplified,
A method for detecting mold, which comprises dropping the obtained amplification product onto the mold detection carrier according to claim 6 and binding it to a probe having a complementary base sequence. A mold detection kit comprising a primer set for amplifying a nucleic acid fragment containing a target region in mold DNA to be detected, and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed,
Molds to be detected are Aureobasidium sp., Alternaria sp., Aspergillus Section Circumdati, Aspergillus Section Nigri, Aspergillus Section Nigri, Aspergillus Aspergillus Section Terrei, Fusarium graminiarum, Fusarium oxysporum, Paecilomyces sp., Trichophyton sp., Verticillium At least one of a fungus (Verticillium sp.) And a Forma sp. (Phoma sp.),
The primer set comprises a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 40 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 41 for amplifying the ITS region, and amplifies the β-tubulin gene A primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 42 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 43,
The carrier is
A probe having a base sequence shown in SEQ ID NO: 4 selected from the ITS region and a base shown in SEQ ID NO: 5 selected from the β-tubulin gene for detecting Aureobasidium sp. A first probe group comprising probes having a sequence;
At least one of probes having a base sequence shown in SEQ ID NO: 7 or 8 selected from the ITS region and SEQ ID NO selected from the β-tubulin gene for detecting Alternaria sp. A second probe group consisting of probes having the base sequence shown in 9,
A probe having the base sequence shown in SEQ ID NO: 10 selected from the ITS region and a base shown in SEQ ID NO: 11 selected from the β-tubulin gene for detecting Aspergillus Section Circumdati A third probe group consisting of probes having sequences;
A probe having the base sequence shown in SEQ ID NO: 12 selected from the ITS region and the base shown in SEQ ID NO: 13 selected from the β-tubulin gene for detecting Aspergillus Section Nigri A fourth group of probes consisting of probes having sequences;
A probe having a base sequence shown in SEQ ID NO: 14 selected from the ITS region and a base shown in SEQ ID NO: 15 selected from the β-tubulin gene for detecting Aspergillus Section Terrei A fifth group of probes comprising probes having sequences;
A probe having the base sequence shown in SEQ ID NO: 20 selected from the ITS region and the base sequence shown in SEQ ID NO: 21 selected from the β-tubulin gene for detecting Fusarium graminiarum. A sixth probe group comprising probes having,
A probe having the base sequence shown in SEQ ID NO: 22 selected from the ITS region and the base sequence shown in SEQ ID NO: 23 selected from the β-tubulin gene for detecting Fusarium oxysporum A seventh probe group comprising probes having,
At least one of probes having the nucleotide sequence shown in SEQ ID NO: 24 or 25 selected from the ITS region and SEQ ID NO: selected from the β-tubulin gene, for detecting Paecilomyces sp. An eighth probe group comprising at least one of the probes having the base sequence shown in 26 or 27,
A probe having the base sequence shown in SEQ ID NO: 28 selected from the ITS region and the base sequence shown in SEQ ID NO: 29 selected from the β-tubulin gene for detecting Trichophyton sp. A ninth probe group consisting of probes having
In order to detect Verticillium sp., A probe having the base sequence shown in SEQ ID NO: 30 selected from the ITS region and SEQ ID NO: 32 or 33 selected from the β-tubulin gene A tenth probe group consisting of at least one of the probes having the base sequence shown, and
A probe having a base sequence shown in SEQ ID NO: 36 selected from the ITS region and a base sequence shown in SEQ ID NO: 37 selected from the β-tubulin gene for detecting a Forma sp. Two or more groups of probes selected from the eleventh probe group consisting of probes having the probes immobilized thereon,
Mold detection kit for detecting mold by amplifying the nucleic acid fragment using the primer set, dropping the obtained amplification product onto the carrier, and binding to a probe having a complementary base sequence . A mold detection kit comprising a primer set for amplifying a nucleic acid fragment containing a target region in mold DNA to be detected, and a carrier on which a probe for confirming the presence or absence of an amplification product is fixed,
Molds to be detected are Acremonium sp., Alternaria sp., Chaetomium sp., Curvularia sp., Verticillium sp. sp.), and
The primer set comprises a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 40 for amplifying the ITS region and a reverse primer consisting of the base sequence shown in SEQ ID NO: 41;
The carrier is
A twelfth probe group comprising at least one of probes having a base sequence represented by SEQ ID NOs: 1 to 3 selected from the ITS region, for detecting Acremonium sp.
A thirteenth probe group comprising probes having the base sequence shown in SEQ ID NO: 6 selected from the ITS region, for detecting Alternaria sp.
A fourteenth probe group comprising probes having a base sequence represented by SEQ ID NO: 16 selected from the ITS region, for detecting a genus Chaetomium sp.
A fifteenth probe group comprising a probe having a base sequence represented by SEQ ID NO: 19 selected from the ITS region for detecting Curvularia sp., And
In order to detect Verticillium sp., Two or more probes selected from the sixteenth probe group consisting of probes having the base sequence shown in SEQ ID NO: 31 selected from the ITS region are used. Is fixed,
Mold detection kit for detecting mold by amplifying the nucleic acid fragment using the primer set, dropping the obtained amplification product onto the carrier, and binding to a probe having a complementary base sequence . A mold detection kit comprising a primer set for amplifying a nucleic acid fragment containing a target region in 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 mold to be detected is at least one of Colletotrichum sp., Arthrinium sp., Pythium sp.
When the mold to be detected is at least one of the genus Colletotrichum sp. And Arthrinium sp., The primer set is based on the nucleotide sequence shown in SEQ ID NO: 40 for amplifying the ITS region. A primer set comprising a forward primer and a reverse primer consisting of the base sequence shown in SEQ ID NO: 41, and / or a forward primer consisting of a base sequence shown in SEQ ID NO: 42 for amplifying the β-tubulin gene and SEQ ID NO: 43 Including a primer set comprising a reverse primer consisting of the base sequence shown in
When the mold to be detected is Pythium sp., The primer set consists of a forward primer consisting of the base sequence shown in SEQ ID NO: 44 for amplifying the ITS region and the base sequence shown in SEQ ID NO: 41 Primer set comprising a reverse primer and / or a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 45 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 43 for amplifying the β-tubulin gene Including
The carrier is
A probe having the base sequence shown in SEQ ID NO: 17 selected from the ITS region and / or the base shown in SEQ ID NO: 18 selected from the β-tubulin gene for detecting Colletotrichum sp. A seventeenth probe group comprising probes having a sequence;
A probe having a base sequence shown in SEQ ID NO: 34 selected from the ITS region and / or a base shown in SEQ ID NO: 35 selected from the β-tubulin gene for detecting Arthrinium sp. An eighteenth probe group comprising probes having a sequence; and
A probe having a base sequence shown in SEQ ID NO: 38 selected from the ITS region and / or a base shown in SEQ ID NO: 39 selected from the β-tubulin gene for detecting Pythium sp. Two groups of probes selected from the nineteenth probe group consisting of probes having sequences are immobilized,
Mold detection kit for detecting mold by amplifying the nucleic acid fragment using the primer set, dropping the obtained amplification product onto the carrier, and binding to a probe having a complementary base sequence .

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