JP2017006012A - Microorganism test method, microorganism test kit, and microarray for microorganism test - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of suitably detecting Plasmodiophora fungus, Pythium fungus. and Phytophthora fungus.SOLUTION: A microorganism testing method comprises: taking a sample from a plant tissue or soil, water, or other environments; extracting DNA from microorganisms contained in the sample; subjecting the extracted DNA to PCR; and determining the presence or absence of a microorganism in the sample based on an amplified product obtained. The PCR is performed using the extracted DNA and a primer set of a forward primer and a revere primer, the forward primer consisting of at least one of specific base sequences for which β-tubulin gene is a target of PCR amplification in DNAs of Plasmodiophora fungus, Pythium fungus and Phytophthora fungus, and the reverse primer consisting of at least one of specific base sequences. The presence or absence of these fungus in the sample is determined based on the amplified product obtained.SELECTED DRAWING: None

Description

  The present invention relates to a genetic testing technique for specifying the type of organism based on a gene sequence, and more particularly to a microorganism testing method, a microorganism testing kit, and a microarray for microorganism testing for detecting pathogenic bacteria related to soil infectious diseases.

In recent years, in food inspections and environmental inspections, in order to identify the types of microorganisms such as molds and food poisoning bacteria present in foods and the environment, nucleic acid amplification methods such as PCR (polymerase chain reaction) method are used as targets. Amplifying DNA in a gene and detecting the amplification product to determine the type of microorganism.
On the other hand, there are some pathogens related to soil infectious diseases present in plant tissues or soil and water that have different boundaries from those to be detected in food inspections and environmental inspections. However, even if PCR was performed, it was difficult to amplify DNA of soil infectious disease bacteria.

JP-A-10-234399 Japanese Patent No. 5522820

Here, among the soil infectious pathogens, the present inventors, in particular, Plasmodiophora spp. That causes clubroot on Chinese cabbage, etc., and Psium spp. That cause seedling blight on cucurbitaceae vegetables, etc. And Phytophthora spp. That cause epidemics in potatoes and the like were selected as detection target bacteria.
Then, the present inventors have intensively researched, a primer set that can suitably amplify the β-tubulin gene in the DNA of these detection target bacteria as a target region, and an rDNA gene (ribosomal RNA gene) ITS region. In addition to developing primer sets that can be suitably amplified as target regions, we have developed microarrays with probes (single-stranded oligo DNA on a DNA chip substrate) that can identify each by-product. It succeeded in detecting specifically.

Here, Patent Document 1 discloses an oligonucleotide for detecting or identifying Pythium spp. In a simple, rapid and sensitive manner. Patent Document 2 discloses a method for detecting pathogens of strawberry important diseases and detection primers capable of detecting Phytophthora spp.
However, these techniques have failed to detect Plasmodiophora spp. In addition, it is desirable that the Plasmodiophora spp., Psium spp., And Phytofutra spp. Can be more specifically and simultaneously detected.

  The present invention has been made in view of the above circumstances, and a microorganism testing method, a microorganism testing kit, and a microorganism capable of suitably detecting Plasmodiophora, Psium, and Phytophthra The purpose is to provide a microarray for inspection.

  In order to achieve the above object, the microorganism testing method of the present invention is a sample obtained by taking a sample from plant tissue or soil, water, or other environment, and extracting DNA from the group of microorganisms contained in the sample. A method for examining microorganisms by performing PCR using the obtained DNA and determining the presence or absence of microorganisms in the sample based on the obtained amplification product, wherein the β-tubulin gene in DNA of Plasmodiophora is obtained by PCR A first primer set consisting of a forward primer consisting of at least one of the base sequences shown in SEQ ID NOs: 1 to 3 to be amplified and a reverse primer consisting of at least one of the base sequences shown in SEQ ID NOs: 4 and 5; PCR is performed using the extracted DNA, and the plasmoid in the sample is determined based on the obtained amplification product. There a method for determining the presence or absence of Fora spp.

  In addition, the microorganism testing kit of the present invention collects a sample from a plant tissue or soil, water, or other environment, extracts DNA from the microorganism group contained in the sample, and uses the extracted DNA to perform PCR. A microorganism testing kit used to determine the presence or absence of microorganisms in the sample based on the obtained amplification product, wherein β- in the DNA of Plasmodiophora, Psium, and Phytophthora It consists of a forward primer consisting of at least one of the base sequences shown in SEQ ID NOs: 1 to 3 and a reverse primer consisting of at least one of the base sequences shown in SEQ ID NOs: 4 and 5 that target the tubulin gene for amplification by PCR. The configuration includes a primer set.

  Furthermore, the microarray for microbiological examination of the present invention collects a sample from a plant tissue or soil, water, or other environment, extracts DNA from a group of microorganisms contained in the sample, and performs PCR using the extracted DNA. A microarray for microbiological examination for determining the presence or absence of microorganisms in the sample based on the obtained amplification product, wherein the DNA of Plasmodiophora and the β-tubulin in the DNA of Plasmodiophora A first primer comprising a forward primer consisting of at least one of the base sequences shown in SEQ ID NOs: 1 to 3 and a reverse primer consisting of at least one of the base sequences shown in SEQ ID NOs: 4 and 5 that target the gene for amplification by PCR A primer that binds complementarily to the first amplification product obtained by PCR using the above primer set. There as immobilized constituting the first probe comprising at least any one of the nucleotide sequences and the complementary sequence shown in ID NO: 12.

  According to the present invention, it is possible to suitably detect Plasmodiophora spp., Psium spp., And Phytofutra spp.

It is a figure which shows the base sequence of the primer used with the microbe test | inspection method, microbe test kit, and microbe microarray for microbe based on embodiment of this invention. It is a figure which shows the base sequence of the probe which targets the beta-tubulin gene used with the microbe test | inspection method, microbe test kit, and microbe test microarray which concern on embodiment of this invention. Results of detecting Plasmodiophora spp., Psium spp., Phytophthra spp., Etc. using a microbiological test method, a microbiological test kit, and a microbiological test microarray according to an embodiment of the present invention (Test Example 1) ). Results of detecting Plasmodiophora spp., Psium spp., Phytophthra spp., Etc. using a microbiological test method, a microbiological test kit, and a microbiological test microarray according to an embodiment of the present invention (Test Example 2) ). Results of detecting Plasmodiophora spp., Psium spp., Phytophthra spp., Etc. using the microorganism testing method, the microorganism testing kit, and the microorganism testing microarray according to the embodiment of the present invention (Test Example 3) ). Results of simultaneous detection of Plasmodiophora spp., Psium spp., Phytophthra spp., Etc. using the microbiological testing method, microbiological testing kit, and microbiological testing microarray according to the embodiment of the present invention (Test Example) It is a figure which shows 4). Results of simultaneous detection of Plasmodiophora spp., Psium spp., Phytophthra spp., Etc. using the microbiological testing method, microbiological testing kit, and microbiological testing microarray according to the embodiment of the present invention (Test Example) It is a figure which shows 5). It is a figure which shows the base sequence of the probe which targets the rDNA gene ITS area | region used with the microbe test | inspection method, microbe test kit, and microbe test microarray which concern on embodiment of this invention. Results of detecting Plasmodiophora spp., Psium spp., And Phytophthora spp. Using the microbiological test method, the microbiological test kit, and the microarray for microbiological testing according to the embodiment of the present invention (Test Example 6) FIG. Results of simultaneous detection of Plasmodiophora spp., Psium spp. Or Phytophthora spp. Using a microbiological test method, a microbiological test kit, and a microarray for microbiological testing according to an embodiment of the present invention (Test Example) It is a figure which shows 7).

Hereinafter, a microorganism testing method, a microorganism testing kit, and a microorganism testing microarray according to embodiments of the present invention will be described in detail.
The microorganism testing method of the present embodiment is a method in which a sample is collected from a plant tissue or soil, water, or other environment, DNA is extracted from a group of microorganisms contained in the sample, and PCR is performed using the extracted DNA. A microorganism testing method for determining the presence or absence of microorganisms in a sample based on the obtained amplification product, wherein the β-tubulin gene in DNA of Plasmodiophora is used as a target for amplification by PCR. PCR using a first primer set consisting of a forward primer consisting of at least one of the base sequences shown in FIG. 3, a reverse primer consisting of at least one of the base sequences shown in SEQ ID NOs: 4 and 5, and the extracted DNA And based on the obtained amplification product, the presence or absence of Plasmodiophora in the sample is determined. To.

In addition, the microorganism testing method of this embodiment is shown in SEQ ID NO: 12 that complementarily binds to the first amplification product obtained by PCR using the DNA of Plasmodiophora and the first primer set. A first amplification product is brought into contact with a microarray on which a first probe consisting of at least one of a base sequence and a complementary sequence is immobilized, and a label of the first amplification product that is complementarily bound to the first probe is labeled. It is characterized by detecting.
Plasmodiophora spp. Are soil-borne pathogens, causing root-knot disease by parasitic on cruciferous vegetables such as Chinese cabbage and cabbage. It is an absolute parasitic fungus that causes disease only by living plant cells.
In this specification, all base sequences are understood from the 5 ′ end to the 3 ′ end.

According to the microorganism testing method of the present embodiment, by performing PCR using the first primer set, the β-tubulin gene in the DNA of the plasmodiaphora is suitably amplified. It is possible.
Further, by using the microarray on which the first probe is immobilized, it is possible to specifically detect Plasmodiophora spp.

  Further, the microorganism testing method of the present embodiment, the β-tubulin gene in the DNA of Plasmodiophora spp., Psium spp., And Phytophthora spp. PCR is performed using the extracted DNA, and based on the obtained amplification product, the presence or absence of Plasmodiophora, Psium, and Phytophthra in the sample is determined at the same time.

In addition, the microorganism testing method of the present embodiment includes the base shown in SEQ ID NO: 13 or 14 that binds complementarily to the second amplification product obtained by PCR using the DNA of the genus Psium and the first primer set. A second amplification product is brought into contact with a microarray on which a second probe consisting of at least one of these sequences and a complementary sequence thereof is immobilized, and a label of the second amplification product that is complementarily bound to the second probe is labeled. It is characterized by detecting.
The genus Psium is a polycytic soil-borne pathogen that causes seedling blight and rot on many vegetables including cucurbitaceae and solanaceous vegetables.

Furthermore, the microorganism testing method of the present embodiment includes a base shown in SEQ ID NO: 15 or 16 that binds complementarily to a third amplification product obtained by PCR using DNA of Phytophthora and a first primer set. The third amplification product is brought into contact with a microarray on which a third probe consisting of at least one of the sequences is immobilized, and the label of the third amplification product that binds complementarily to the third probe is detected. And
Phytophthora spp. Are pathogens that inhabit soil and plants, and are well known for Phytophthora infestans, which caused potato famine in the middle of the 19th century.

According to the microorganism testing method of the present embodiment, by performing PCR using the first primer set described above, the β-tubulin gene in the DNA of such Psium and Phytophthora is suitably amplified. It is possible to make it.
In addition, by using the microarray on which the second probe is immobilized, it is possible to specifically detect the genus Psium, and by using the microarray on which the third probe is immobilized, It is possible to specifically detect the genus fungus.

  Further, according to the microorganism testing method of the present embodiment, by performing PCR using the first primer set described above, β- in the DNA of Plasmodiophora spp., Psium spp., And Phytophthora spp. By using a microarray in which the tubulin gene is suitably amplified and the first probe, the second probe, and the third probe are immobilized, these microorganisms can be specifically detected simultaneously. ing.

Furthermore, the microorganism testing method of the present embodiment is obtained by PCR using DNA of at least one of Plasmodiophora, Psium, Phytophthra, and other microorganisms and a first primer set. The fourth amplification product is brought into contact with a microarray on which a fourth probe comprising at least one of the base sequence shown in SEQ ID NO: 17 and the complementary sequence that binds complementarily to the fourth amplification product is immobilized, It is characterized by detecting the label of the fourth amplification product that is complementarily bound to the four probes.
According to the microorganism testing method of the present embodiment, by using the microarray on which the fourth probe is immobilized, Plasmodiophora spp., Psium spp., Phytophthra spp., And, for example, Aspergillus barge color It is possible to detect other microorganisms.
In addition, other microorganisms are not limited to Aspergillus barge color, You may utilize the microarray which fix | immobilized said 4th probe for common fungi.

Still further, the microorganism testing method of the present embodiment includes a forward primer comprising the base sequence shown in SEQ ID NO: 6 that targets the rDNA gene ITS region in the DNA of Plasmodiophora sp. Perform PCR using the second primer set consisting of the reverse primer consisting of the base sequence shown in FIG. 5 and the extracted DNA, and determine the presence or absence of Plasmodiophora in the sample based on the amplification product obtained It is characterized by that.
In addition, the microorganism testing method of this embodiment is shown in SEQ ID NO: 18 that complementarily binds to the fifth amplification product obtained by PCR using the DNA of Plasmodiophora and the second primer set. A fifth amplification product is brought into contact with a microarray on which a fifth probe consisting of at least one of the base sequence and the complementary sequence is immobilized, and the label of the fifth amplification product that is complementarily bound to the fifth probe is labeled. It is characterized by detecting.

According to the microorganism testing method of the present embodiment, it is possible to suitably amplify the rDNA gene ITS region in the DNA of Plasmodiophora by performing PCR using the second primer set described above. It has become.
In addition, by using a microarray on which the fifth probe is immobilized, it is possible to specifically detect Plasmodiophora spp.

In addition, the microorganism testing method of the present embodiment includes a forward primer comprising at least one of the base sequences shown in SEQ ID NO: 7 or 8, which targets the rDNA gene ITS region in DNA of Psium genus by PCR, Perform PCR using the third primer set consisting of the reverse primer consisting of the base sequence shown in No. 10 and the extracted DNA, and determine the presence or absence of Pythium in the sample based on the obtained amplification product It is characterized by.
In addition, the microorganism testing method of the present embodiment includes a base sequence represented by SEQ ID NO: 19 that binds complementarily to a sixth amplification product obtained by PCR using a DNA of Psium and a third primer set; Contacting a sixth amplification product with a microarray on which a sixth probe comprising at least one of the complementary sequences is immobilized, and detecting a label of the sixth amplification product complementary to the sixth probe; It is characterized by.

According to the microorganism testing method of the present embodiment, it is possible to suitably amplify the rDNA gene ITS region in the DNA of the genus Psium by performing PCR using the above third primer set. .
Further, by using a microarray on which the sixth probe is immobilized, it is possible to specifically detect the genus Psium.

  Further, according to the microorganism testing method of the present embodiment, the rDNA gene ITS in the DNA of Plasmodiophora and Psium is obtained by performing PCR using the second primer set and the third primer set. By suitably amplifying the region and using a microarray in which the fifth probe and the sixth probe are immobilized, it is possible to specifically and simultaneously detect Plasmodiophora spp. And Psium spp. Yes.

Further, the microorganism testing method of the present embodiment includes a forward primer comprising the base sequence shown in SEQ ID NO: 8 that targets the rDNA gene ITS region in the DNA of Psium or Phytophthra spp. Perform PCR using the 4th primer set consisting of the reverse primer consisting of the base sequence shown in the above and the extracted DNA, and determine the presence or absence of Pythium or Phytophthra in the sample based on the obtained amplification product It is characterized by doing.
In addition, the microorganism testing method of the present embodiment includes SEQ ID NO: 20 that complementarily binds to a seventh amplification product obtained by PCR using DNA of the genus Psium or Phytophthora and a fourth primer set. A seventh amplification product is brought into contact with a microarray on which a seventh probe comprising at least one of the base sequence shown in FIG. 21 and a complementary sequence thereof is immobilized, and the seventh amplification product is complementarily bound to the seventh probe. It is characterized by detecting the label of the product.

According to the microorganism testing method of the present embodiment, it is possible to suitably amplify the rDNA gene ITS region in the DNA of the genus Psium and Phytophthora by performing PCR using the fourth primer set described above. It has become.
In addition, by using the microarray on which the seventh probe is immobilized, it is possible to detect Psium and Phytophthora.

Here, in the microorganism testing method of the present embodiment, without using the first primer set targeting the β-tubulin gene as the primer set, when using the primer set targeting the rDNA gene ITS region, A third primer set (SEQ ID NO: 7, 8, 10) and a fourth primer set (SEQ ID NO: 8, 11) are used in combination, and a sixth probe (SEQ ID NO: 19 + complementary sequence) and a seventh probe By using a microarray in which (SEQ ID NO: 20, 21+ complementary sequence) is immobilized, the detection of Pythium and Phytophthora can be determined as follows.
First, when the label of the amplification product is detected for both the sixth probe and the seventh probe, it can be seen that Pythium spp. Exists and the presence or absence of Phytophthora spp. Is unknown. Moreover, when the label of an amplification product is not detected about a 6th probe and the label of an amplification product is detected about a 7th probe, it turns out that a Pythium genus microbe does not exist but a Phytofutra genus microbe exists. Moreover, when the label of an amplification product is not detected about both the 6th probe and the 7th probe, it turns out that neither a Pythium genus bacteria nor a Phytophthra genus bacteria exists.

  In the microorganism testing method of the present embodiment, as described above, when the first primer set targeting the β-tubulin gene is used as the primer set, the first probe (SEQ ID NO: 12 + complementary sequence), By using a microarray on which a second probe (SEQ ID NO: 13, 14+ complementary sequence) and a third probe (SEQ ID NO: 15, 16+ complementary sequence) are immobilized, Plasmodiophora, Psium, and It is possible to specifically and simultaneously detect Phytophyra species.

  The microorganism testing kit of this embodiment collects a sample from a plant tissue or soil, water, or other environment, extracts DNA from a group of microorganisms contained in the sample, and performs PCR using the extracted DNA. , A microorganism testing kit used to determine the presence or absence of microorganisms in a sample based on the obtained amplification product, the β-tubulin gene in DNA of Plasmodiophora, Psium, and Phytophthra A primer set consisting of a forward primer consisting of at least one of the base sequences shown in SEQ ID NOs: 1 to 3 and a reverse primer consisting of at least one of the base sequences shown in SEQ ID NOs: 4 and 5 It is characterized by including.

  According to the microorganism testing kit of this embodiment, by performing PCR using such a primer set, the β-tubulin gene in the DNA of Plasmodiophora spp., Psium spp. It can be suitably amplified.

Further, the microorganism testing kit of the present embodiment includes a forward primer consisting of the base sequence shown in SEQ ID NO: 6, which targets the rDNA gene ITS region in the DNA of Plasmodiophora sp. A primer set comprising a reverse primer comprising the indicated base sequence is included.
According to the microorganism testing kit of the present embodiment, by performing PCR using such a primer set, it is possible to suitably amplify the rDNA gene ITS region in the DNA of the genus Plasmodiophora. .
Further, according to the microorganism testing kit of the present embodiment, by performing PCR using each of the above primer sets, the β-tubulin gene in the DNA of Plasmodiophora spp., Psium spp., And Phytophthra spp. And the rDNA gene ITS region in the DNA of the Plasmodiophora genus can be preferably amplified simultaneously.

In addition, the microorganism testing kit of the present embodiment includes a forward primer comprising at least one of the base sequences shown in SEQ ID NO: 7 or 8, which targets the rDNA gene ITS region in the DNA of the genus Psium by PCR, A primer set consisting of a reverse primer consisting of the base sequence shown in No. 10 is included.
According to the microorganism testing kit of this embodiment, it is possible to suitably amplify the rDNA gene ITS region in the DNA of the genus Psium by performing PCR using such a primer set.
Further, according to the microorganism testing kit of the present embodiment, by performing PCR using each of the above primer sets, the β-tubulin gene in the DNA of Plasmodiophora spp., Psium spp., And Phytophthra spp. The rDNA gene ITS region in the DNA of Plasmodiophora and Psium can be preferably amplified simultaneously.

In addition, the microorganism testing kit of the present embodiment includes a forward primer comprising the base sequence shown in SEQ ID NO: 8 that targets the rDNA gene ITS region in the DNA of Psium or Phytophthra spp. And a primer set comprising a reverse primer comprising the base sequence shown in FIG.
According to the microorganism testing kit of the present embodiment, it is possible to suitably amplify the rDNA gene ITS region in the DNA of the genus Psium and Phytophthora by performing PCR using such a primer set. ing.
Further, according to the microorganism testing kit of the present embodiment, by performing PCR using each of the above primer sets, the β-tubulin gene in the DNA of Plasmodiophora spp., Psium spp., And Phytophthra spp. And the rDNA gene ITS region in the DNA of Plasmodiophora spp., Psium spp., And Phytophthra spp. Can be preferably simultaneously amplified.

In using the microorganism testing method and the microorganism testing kit of the present embodiment, a method for extracting DNA from a sample is not particularly limited, and can be performed, for example, as follows.
First, a colony of a microorganism is collected, the colony is frozen with liquid nitrogen or the like, and the microorganism cell contained in the colony is crushed. Next, genomic DNA is extracted from the disrupted cells of the obtained microorganism. 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. Also in this case, DNA can be extracted by a general method such as a method using a direct lysis method or a method using a soil DNA extraction kit.

  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. As a method for amplifying the target region, a PCR method can be suitably used. A general thermal cycler or the like can be used as the PCR apparatus.

  As a reaction solution for PCR, for example, a solution having the following composition is preferably used. That is, a nucleic acid synthesis substrate (dNTPmixture (dCTP, dATP, dTTP, dGTP)), primer set, nucleic acid synthase (EX Taq HotStart DNA polymerase, etc.), fluorescent labeling reagent (Cy5-dCTP, etc.), sample DNA, buffer solution, 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 using the microorganism testing method and the microorganism testing kit of the present embodiment, for example, by performing PCR under the following reaction conditions, the target region of each microorganism 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

  Further, in the present embodiment and examples, the primers and probes are not limited to the respective base sequences themselves, and those in which one or several bases are deleted, substituted or added in each base sequence are used. be able to. Moreover, what consists of a nucleic acid fragment which can be hybridized on stringent conditions with respect to the nucleic acid fragment which consists of a base sequence complementary to each base sequence 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 a high homology (with a homology of 90% or more, preferably 95% or more) with a DNA comprising each base sequence is a DNA comprising a base sequence complementary to the DNA comprising the respective base sequence. And hybridization conditions. 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”.

  The primer and probe used in the microorganism testing method, microorganism testing kit, and microorganism testing microarray of this embodiment are all about 16 to 40 mer (base) in length and can be synthesized by a DNA synthesizer. .

The microarray for microbiological examination of this embodiment collects a sample from plant tissue or soil, water, or other environment, extracts DNA from a group of microorganisms contained in the sample, and performs PCR using the extracted DNA. , A microarray for microbiological examination for judging the presence or absence of microorganisms in a sample based on the obtained amplification product, and PCR for the DNA of Plasmodiophora and the β-tubulin gene in the DNA of Plasmodiophora A first primer set comprising a forward primer consisting of at least one of the base sequences shown in SEQ ID NOs: 1 to 3 and a reverse primer consisting of at least one of the base sequences shown in SEQ ID NOs: 4 and 5 SEQ ID NO: 12 which binds complementarily to the first amplification product obtained by PCR using To nucleotide sequences and wherein the immobilized first probe comprising at least one of the complementary sequences.
According to the microarray for microbiological examination of the present embodiment, it is possible to specifically detect plasmodifora spp. By using the microarray on which the first probe is immobilized.

In addition, the microarray for microbiological examination of the present embodiment is represented by SEQ ID NO: 13 or 14 that binds complementarily to the DNA of the genus Psium and the second amplification product obtained by PCR using the first primer set. A second probe comprising at least one of a base sequence and a complementary sequence thereof is immobilized.
According to the microarray for microbiological examination of this embodiment, it is possible to specifically detect the genus Psium by using the microarray on which the second probe is immobilized.
Further, according to the microarray for microbiological examination of the present embodiment, the plasmodiophora genus and the genus Pysium are specifically detected simultaneously by using the microarray in which the first probe and the second probe are immobilized. It is possible to do.

In addition, the microarray for microbiological examination of the present embodiment is represented by SEQ ID NO: 15 or 16 that binds complementarily to the DNA of Phytophthora sp. And the third amplification product obtained by PCR using the first primer set. A third probe comprising at least one of the base sequences is immobilized.
According to the microarray for microbiological examination of this embodiment, it is possible to specifically detect Phytophthora spp. By using the microarray on which the third probe is immobilized.
Further, according to the microarray for microbiological examination of the present embodiment, by using the microarray on which the first probe, the second probe, and the third probe are immobilized, Plasmodiophora spp., Psium spp. And Phytophthora spp. Can be specifically detected simultaneously.

Furthermore, the microarray for microbiological examination of this embodiment is obtained by PCR using at least one DNA of Plasmodiophora, Psium, Phytophthra, and other microorganisms and the first primer set. The base probe shown in SEQ ID NO: 17 that complementarily binds to the obtained fourth amplification product and the fourth probe comprising at least one of the complementary sequence are immobilized.
According to the microarray for microbiological examination of this embodiment, by using the microarray on which the fourth probe is immobilized, Plasmodiophora spp., Psium spp., Phytophthra spp., And, for example, Aspergillus barge color It is possible to detect other microorganisms.

Still further, the microarray for microbiological examination of the present embodiment includes a DNA sequence of Plasmodiophora and a base represented by SEQ ID NO: 6 which targets the rDNA gene ITS region in the DNA of Plasmodiophora by PCR amplification. As shown in SEQ ID NO: 18 that complementarily binds to the fifth amplification product obtained by PCR using the second primer set consisting of the forward primer consisting of the sequence and the reverse primer consisting of the base sequence shown in SEQ ID NO: 9. A fifth probe comprising at least one of a base sequence and a complementary sequence is immobilized.
According to the microarray for microbiological examination of this embodiment, it is possible to specifically detect Plasmodiophora spp. By using the microarray on which the fifth probe is immobilized.

Further, the microarray for microbiological examination of the present embodiment is at least one of the base sequence shown in SEQ ID NO: 7 or 8, which uses the DNA of Psium and the rDNA gene ITS region in the DNA of Psium as a target for amplification by PCR. A base shown in SEQ ID NO: 19 that complementarily binds to a sixth amplification product obtained by PCR using a third primer set consisting of a forward primer consisting of: and a reverse primer consisting of the base sequence shown in SEQ ID NO: 10 A sixth probe comprising at least one of the sequence and the complementary sequence is immobilized.
According to the microarray for microbiological examination of this embodiment, it is possible to specifically detect the genus Psium by using the microarray on which the sixth probe is immobilized.
Further, according to the microarray for microbiological examination of the present embodiment, the plasmodiophora genus and the genus Psium are specifically detected simultaneously by using the microarray in which the fifth probe and the sixth probe are immobilized. It is possible to do.

Furthermore, the microarray for microbiological examination of the present embodiment is represented by SEQ ID NO: 8 which targets PCR amplification with the DNA of Pisces or Phytophthra and the rDNA gene ITS region in the DNA of Pisium or Phytophthra. SEQ ID NO: 20 that complementarily binds to a seventh amplification product obtained by PCR using a fourth primer set consisting of a forward primer consisting of a base sequence and a reverse primer consisting of the base sequence shown in SEQ ID NO: 11 or A seventh probe comprising at least one of the base sequence shown in No. 21 and a complementary sequence thereof is immobilized.
According to the microarray for microbiological examination of this embodiment, it is possible to detect the genus Psium and the genus Phytophthra by using the microarray on which the seventh probe is immobilized.

The microarray for microbiological examination of the present embodiment is not particularly limited as long as at least one of the probes for detecting the microorganism to be detected is immobilized. For example, a spot type DNA microarray, a synthetic type A DNA microarray or the like can be used.
For example, in the case of creating a paste-type DNA chip as the microarray for microbiological examination of the present embodiment, the probe is immobilized on a glass substrate by a DNA spotter and formed by forming spots corresponding to each probe. 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. .

Next, the presence or absence of the amplification product is confirmed by dropping the amplification product onto the microarray for microbiological examination of the present embodiment and detecting the label of the amplification product hybridized to the probe immobilized on this microarray for microbiological examination. . As a result, the microorganism to be inspected can be specified.
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 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.

  As described above, according to the microorganism testing method, the microorganism testing kit, and the microarray for microorganism testing of the present embodiment, it is possible to suitably detect Plasmodiophora, Psium, and Phytophthra. It becomes possible.

(Test 1)
Using the primer set and probe targeting the β-tubulin gene by the microorganism testing method, microorganism testing kit, and microorganism testing microarray of this embodiment, Plasmodiophora spp., Psium spp., And A test was conducted to verify whether Phytophthora spp. Can be detected independently.

Specifically, using a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 targeting the β-tubulin gene, -The β-tubulin genes in DNAs of Brassicae, Psium aphanidermatum, Phytofutra infestans, and Aspergillus bargecolor were individually amplified by PCR.
Further, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5 targeting the β-tubulin gene, The β-tubulin gene in DNA of Pythium Ultimum, Phytophthora infestans and Aspergillus bargecolor was individually amplified by PCR.
Then, the amplification product by the above PCR is dropped onto a microarray on which probes having the nucleotide sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 are immobilized, and the amplified product is fluorescently labeled. Was detected.

Plasmodiophora brassicae, an absolute parasite, was used from the plant pathology laboratory of Kyoto Prefectural University. The genus Pythium and the genus Phytophthra were obtained from NBRC (NITE Biological Resource Center). The strains obtained from the center including the tests described below are as follows.
Pythium aphanidermatum NBRC7030
Pythium ultimum NBRC100125
Pythium zingiberis NBRC30818
Phytophthora infestans NBRC9174

As the PCR reaction solution, Ampdirect (R) (manufactured by Shimadzu Corporation) was used and the following composition was used. Each primer used was synthesized by Operon Technology Co., Ltd.
・ Ampdirect (G / Crich) 4.0μl
・ Ampdirect (addition-4) 4.0μl
・ DNTP Mixture (dATP, dCTP, dGTP, dTTP 2.5 mM each) 1.0 μl
・ Cy-5 dCTP 0.2μl
・ EX Taq Hot Start DNA polymerase (5U / μl) 0.2μl
・ Forward primer (10μM) 1.0μl
・ Reverse primer (10μM) 1.0μl
・ Sample DNA (100pg / μl) 1.0μl
・ Water (water is added until the total is 20.0 μl)

Amplification of the gene by the PCR method was performed by the nucleic acid amplification apparatus (TaKaRa PCR Thermal Cycler Dice (R) Gradient Takara Bio Inc.) under the same conditions as described in the embodiment.
(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, the PCR amplification product was mixed with a buffer solution (3 × SSC citrate-saline + 0.3% SDS), heated at 94 ° C. for 5 minutes, and dropped onto the microarray. The microarray was allowed to stand at 45 ° C. for 1 hour, and PCR products that were not hybridized using the buffer solution were washed away from the microarray.
Then, using a label detection device (BIOSHOT (R), manufactured by Toyo Kohan Co., Ltd.), the fluorescence intensity of each probe immobilized on the microarray (the fluorescence intensity of the amplification product bound to the probe) is measured, The S / N ratio value in each probe was acquired. The result is shown in FIG.

In FIG. 3, a β-tubulin gene in DNA of plasmodiophora brassicae using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 It is shown that the amplified product obtained by amplifying PCR was bound to the probe consisting of the nucleotide sequences shown in SEQ ID NOs: 12 and 17, and the label was detected.
Further, by using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, β- in the DNA of Pythium aphanidermatum and Phytophthora infestans The amplification products obtained by amplifying the tubulin gene by PCR bound to the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 13 and 17 and SEQ ID NOs: 15 and 17, respectively, indicating that the label was detected. Yes.
Furthermore, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, the β-tubulin gene in the Aspergillus barge color DNA is amplified by PCR The amplified product obtained in this manner was bound to the probe consisting of the base sequence shown in SEQ ID NO: 17, indicating that the label was detected.

Similarly, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, the β-tubulin gene in the DNA of plasmodiaphora brassicae It is shown that the amplified product obtained by PCR amplification was bound to the probe consisting of the nucleotide sequences shown in SEQ ID NOs: 12 and 17, and the label was detected.
Further, β-tubulin in DNA of Pythium Ultimum and Phytophthora infestans using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5 It is shown that the amplified products obtained by amplifying the gene by PCR were bound to probes consisting of the nucleotide sequences shown in SEQ ID NOs: 13 and 17 and SEQ ID NOs: 15 and 17, respectively, and the label was detected.
Furthermore, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, the β-tubulin gene in Aspergillus bargecolor DNA is amplified by PCR The amplified product obtained in this manner was bound to the probe consisting of the base sequence shown in SEQ ID NO: 17, indicating that the label was detected.

(Test 2)
Using the primer set and probe targeting the β-tubulin gene by the microorganism testing method, microorganism testing kit, and microorganism testing microarray of this embodiment, Plasmodiophora spp., Psium spp., And Further tests were conducted to verify whether phytofutra spp. Could be detected independently.

Specifically, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 targeting the β-tubulin gene, -The β-tubulin genes in DNAs of Brassicae, Psium aphanidermatum, Phytofutra infestans, and Aspergillus bargecolor were individually amplified by PCR.
In addition, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5 targeting the β-tubulin gene, The β-tubulin genes in DNA of P. gingivalis, Phytofutra infestans, and Aspergillus burgecolor were individually amplified by PCR.
Then, the amplification product by the above PCR is dropped onto the microarray on which the probes consisting of the base sequences shown in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17 are immobilized, and the amplified product is fluorescently labeled. Was detected.

PCR amplification and gene amplification by the PCR method were carried out under the same composition and the same conditions as in Test 1.
Then, the PCR amplification product and the probe were hybridized in the same manner as in Test 1, and the fluorescence intensity of each probe was measured with a label detection device to obtain the S / N ratio value. The result is shown in FIG.

In FIG. 4, a β-tubulin gene in DNA of Plasmodiophora brassicae using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 It is shown that the amplified product obtained by amplifying PCR was bound to the probe consisting of the nucleotide sequences shown in SEQ ID NOs: 12 and 17, and the label was detected.
Further, by using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, β-- in the DNA of Pythium aphanidermatum and Phytoftra infestans The amplification products obtained by amplifying the tubulin gene by PCR were bound to probes consisting of the nucleotide sequences shown in SEQ ID NOs: 14 and 17 and SEQ ID NOs: 16 and 17, respectively, indicating that the label was detected. Yes.
Furthermore, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, the β-tubulin gene in Aspergillus virgin color DNA is amplified by PCR The amplified product obtained in this manner was bound to the probe consisting of the base sequence shown in SEQ ID NO: 17, indicating that the label was detected.

Similarly, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, the β-tubulin gene in the DNA of Plasmodiophora brassicae It is shown that the amplified product obtained by PCR amplification was bound to the probe consisting of the nucleotide sequences shown in SEQ ID NOs: 12 and 17, and the label was detected.
In addition, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, the β-tubulin gene in DNA of P. gingivalis and phytofutra infestans It is shown that the amplification products obtained by PCR amplification were bound to probes consisting of the nucleotide sequences shown in SEQ ID NOs: 14 and 17 and SEQ ID NOs: 16 and 17, respectively, and the label was detected.
Furthermore, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, the β-tubulin gene in the Aspergillus virgin color DNA is amplified by PCR The amplified product obtained in this manner was bound to the probe consisting of the base sequence shown in SEQ ID NO: 17, indicating that the label was detected.

(Test 3)
Using the primer set and probe targeting the β-tubulin gene by the microorganism testing method, microorganism testing kit, and microorganism testing microarray of this embodiment, Plasmodiophora spp., Psium spp., And Further tests were conducted to verify whether phytofutra spp. Could be detected independently.

Specifically, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 targeting the β-tubulin gene and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, -The β-tubulin genes in the DNAs of Brassicae, Psium gingivalis, Phytofutra infestans, and Aspergillus bargecolor were individually amplified by PCR.
Further, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5 targeting the β-tubulin gene, DNA of these four types of microorganisms The β-tubulin genes in were individually amplified by PCR.
Then, the amplification product by the above PCR is dropped onto the microarray on which the probes consisting of the base sequences shown in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17 are immobilized, and the amplified product is fluorescently labeled. Was detected.

PCR amplification and gene amplification by the PCR method were carried out under the same composition and the same conditions as in Test 1.
Then, the PCR amplification product and the probe were hybridized in the same manner as in Test 1, and the fluorescence intensity of each probe was measured with a label detection device to obtain the S / N ratio value. The result is shown in FIG.

In FIG. 5, a β-tubulin gene in DNA of plasmodiaphora brassicae using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 It is shown that the amplified product obtained by amplifying PCR was bound to the probe consisting of the nucleotide sequences shown in SEQ ID NOs: 12 and 17, and the label was detected.
Further, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, a β-tubulin gene in DNA of P. gingivalis and phytofutra infestans It is shown that the amplification products obtained by PCR amplification were bound to probes consisting of the nucleotide sequences shown in SEQ ID NOs: 14 and 17 and SEQ ID NOs: 16 and 17, respectively, and the label was detected.
Furthermore, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, the β-tubulin gene in the Aspergillus virgin color DNA is amplified by PCR The amplified product obtained in this manner was bound to the probe consisting of the base sequence shown in SEQ ID NO: 17, indicating that the label was detected.

Similarly, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, the β-tubulin gene in the DNA of plasmodiophora brassicae It is shown that the amplified product obtained by PCR amplification was bound to the probe consisting of the nucleotide sequences shown in SEQ ID NOs: 12 and 17, and the label was detected.
Further, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, the β-tubulin gene in DNA of Pysium gingivalis and phytofutra infestans It is shown that the amplification products obtained by PCR amplification were bound to probes consisting of the nucleotide sequences shown in SEQ ID NOs: 14 and 17 and SEQ ID NOs: 16 and 17, respectively, and the label was detected.
Furthermore, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, the β-tubulin gene in the Aspergillus virgin color DNA is amplified by PCR The amplified product obtained in this manner was bound to the probe consisting of the base sequence shown in SEQ ID NO: 17, indicating that the label was detected.

(Test 4)
Using the primer set and probe targeting the β-tubulin gene by the microorganism testing method, microorganism testing kit, and microorganism testing microarray of this embodiment, Plasmodiophora spp., Psium spp., And A test was conducted to verify whether phytofutra spp. Could be detected at the same time.

Specifically, using a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 targeting the β-tubulin gene, -The β-tubulin gene in DNAs of Brassicae, P. aphanidermatum, Phytofutra infestans, and Aspergillus bargecolor was simultaneously amplified by PCR using one PCR reaction solution.
Further, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5 targeting the β-tubulin gene, The β-tubulin gene in DNA of Pythium Ultimum, Phytophthora infestans and Aspergillus bargecolor was simultaneously amplified by PCR using one PCR reaction solution.
Then, the amplification product by the above PCR is dropped onto a microarray on which probes having the nucleotide sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 are immobilized, and the amplified product is fluorescently labeled. Was detected.

PCR amplification and gene amplification by the PCR method were carried out under the same composition and the same conditions as in Test 1. However, the PCR reaction solution was used as a sample DNA containing 1.0 μl for each bacterium and a total of 4.0 μl.
Then, the PCR amplification product and the probe were hybridized in the same manner as in Test 1, and the fluorescence intensity of each probe was measured with a label detection device to obtain the S / N ratio value. The result is shown in FIG.

In FIG. 6, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, Plasmodiophora brassicae, Psium aphanidermatum, Amplification products obtained by PCR amplification of the β-tubulin gene in phytophthra infestans DNA were bound to probes consisting of the nucleotide sequences shown in SEQ ID NOs: 12, 13, and 15, respectively, and the label was detected. It has been shown.
In addition, the β-tubulin gene in Aspergillus bargecolor DNA was amplified by PCR using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4. The amplified product obtained in this manner was bound to the probe consisting of the nucleotide sequence shown in SEQ ID NO: 17, indicating that the label may have been detected.

Similarly, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, Plasmodiophora brassicae, Psium Ultimum, The amplification products obtained by amplifying the β-tubulin gene in the infestance DNA by PCR were bound to probes having the base sequences shown in SEQ ID NOs: 12, 13, and 15, respectively, and the label was detected. It is shown.
In addition, the β-tubulin gene in Aspergillus bargecolor DNA was amplified by PCR using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5. The amplified product obtained in this manner was bound to the probe consisting of the nucleotide sequence shown in SEQ ID NO: 17, indicating that the label may have been detected.

(Test 5)
Using the primer set and probe targeting the β-tubulin gene by the microorganism testing method, microorganism testing kit, and microorganism testing microarray of this embodiment, Plasmodiophora spp., Psium spp., And Further tests were conducted to verify whether phytofutra spp. Could be detected at the same time.

Specifically, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 targeting the β-tubulin gene, -The β-tubulin gene in DNAs of Brassicae, P. aphanidermatum, Phytofutra infestans, and Aspergillus bargecolor was simultaneously amplified by PCR using one PCR reaction solution.
In addition, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5 targeting the β-tubulin gene, The β-tubulin gene in DNA of P. gingivalis, Phytofutra infestans, and Aspergillus burgecolor was simultaneously amplified by PCR using one PCR reaction solution.

Further, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 targeting the β-tubulin gene, β -The tubulin gene was simultaneously amplified by PCR using one PCR reaction solution.
Further, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5 targeting the β-tubulin gene, DNA of these four types of microorganisms The β-tubulin gene in was simultaneously amplified by PCR using one PCR reaction solution.

  Then, the amplification product by the above PCR is dropped onto the microarray on which the probes consisting of the base sequences shown in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17 are immobilized, and the amplified product is fluorescently labeled. Was detected.

PCR amplification and gene amplification by the PCR method were carried out under the same composition and the same conditions as in Test 1. However, the PCR reaction solution was used as a sample DNA containing 1.0 μl for each bacterium and a total of 4.0 μl.
Then, the PCR amplification product and the probe were hybridized in the same manner as in Test 1, and the fluorescence intensity of each probe was measured with a label detection device to obtain the S / N ratio value. The result is shown in FIG.

In FIG. 7, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, Plasmodiophora brassicae, Psium aphanidermatum, Amplification products obtained by amplifying the β-tubulin gene in the DNA of Phytophthora infestans by PCR bind to the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 12, 14 and 16, respectively, and the label is detected. It has been shown.
In addition, the β-tubulin gene in Aspergillus bargecolor DNA was amplified by PCR using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4. The amplified product obtained in this manner was bound to the probe consisting of the nucleotide sequence shown in SEQ ID NO: 17, indicating that the label may have been detected.

Similarly, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, Plasmodiophora brassicae, Psium gingivalis, and Phytophthra inferior The amplification products obtained by amplifying the β-tubulin gene in the stance DNA by PCR were bound to probes consisting of the nucleotide sequences shown in SEQ ID NOs: 12, 14, and 16, respectively, indicating that the label was detected. Has been.
In addition, the β-tubulin gene in Aspergillus bargecolor DNA was amplified by PCR using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5. The amplified product obtained in this manner was bound to the probe consisting of the nucleotide sequence shown in SEQ ID NO: 17, indicating that the label may have been detected.

Furthermore, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4, Plasmodiophora brassicae, Pysium gingivalis, and Phytofutra infestance Amplification products obtained by amplifying the β-tubulin gene in the DNA by PCR bind to probes consisting of the nucleotide sequences shown in SEQ ID NOs: 12, 14, and 16, respectively, indicating that the label was detected. ing.
In addition, the β-tubulin gene in Aspergillus bargecolor DNA was amplified by PCR using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4. The amplified product obtained in this manner was bound to the probe consisting of the nucleotide sequence shown in SEQ ID NO: 17, indicating that the label may have been detected.

Similarly, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5, Plasmodiophora brassicae, Psium gingivalis, and Phytophthra inferior The amplification products obtained by amplifying the β-tubulin gene in the stance DNA by PCR were bound to probes consisting of the nucleotide sequences shown in SEQ ID NOs: 12, 14, and 16, respectively, indicating that the label was detected. Has been.
In addition, the β-tubulin gene in Aspergillus bargecolor DNA was amplified by PCR using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 5. The amplified product obtained in this manner was bound to the probe consisting of the nucleotide sequence shown in SEQ ID NO: 17, indicating that the label may have been detected.

(Test 6)
Using the primer set and probe targeting the rDNA gene ITS region by the microorganism testing method, the microorganism testing kit, and the microarray for microorganism testing of the present embodiment, Plasmodiophora spp., Psium spp. A test was conducted to verify whether each genus or the like can be detected alone.

Specifically, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 targeting the rDNA gene ITS region and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9, The rDNA gene ITS regions in the DNAs of Brassicae, Psium Ultimum, and Phytophylla infestans were each amplified by PCR.
In addition, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 7 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 10 targeting the rDNA gene ITS region, Each rDNA gene ITS region was individually amplified by PCR.

Furthermore, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 8 targeting the rDNA gene ITS region and a reverse primer consisting of the base sequence shown in SEQ ID NO: 10, in the DNA of these three types of microorganisms Each rDNA gene ITS region was individually amplified by PCR.
In addition, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 8 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 11 targeting the rDNA gene ITS region, Each rDNA gene ITS region was individually amplified by PCR.

  Then, the amplification product by the above PCR is dropped onto the microarray on which the probes consisting of the base sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 are immobilized, and the amplified product is fluorescently labeled. Was detected.

PCR amplification and gene amplification by the PCR method were carried out under the same composition and the same conditions as in Test 1.
Then, the PCR amplification product and the probe were hybridized in the same manner as in Test 1, and the fluorescence intensity of each probe was measured with a label detection device to obtain the S / N ratio value. The result is shown in FIG.

In FIG. 9, the rDNA gene ITS region in the DNA of Plasmodiophora brassicae was determined using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9. It is shown that the amplified product obtained by PCR amplification was bound to the probe having the base sequence shown in SEQ ID NO: 18 and the label was detected.
In addition, the rDNA gene ITS region in the DNA of Pythium ultimum was amplified by PCR using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 7 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 10. The amplified product obtained in this manner was bound to the probe consisting of the base sequence shown in SEQ ID NO: 19, indicating that the label was detected.
In addition, the rDNA gene ITS region in the DNA of Pythium ultimum was amplified by PCR using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 8 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 10. The amplified product obtained in this manner was also bound to the probe consisting of the base sequence shown in SEQ ID NO: 19, indicating that the label was detected.

  Furthermore, using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 8 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 11, the rDNA gene ITS region in the DNA of Pythium ultimum and Phytophthora infestans It is shown that the amplified product obtained by PCR amplification was bound to the probe consisting of the nucleotide sequences shown in SEQ ID NOs: 20 and 21, and the label was detected.

(Test 7)
Using the primer set and probe targeting the rDNA gene ITS region by the microorganism testing method, the microorganism testing kit, and the microarray for microorganism testing of the present embodiment, Plasmodiophora spp., Psium spp. A test was conducted to verify whether genus bacteria can be detected simultaneously.

  Specifically, a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9 targeting the rDNA gene ITS region, and a base shown in SEQ ID NO: 7 Using a primer set consisting of a forward primer consisting of the sequence and a reverse primer consisting of the base sequence shown in SEQ ID NO: 10, one PCR is performed on the rDNA gene ITS region in the DNA of Plasmodiophora brassicae and Psium Ultimum. The reaction solution was used for simultaneous amplification by PCR.

  In addition, a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9 targeting the rDNA gene ITS region, and a base sequence shown in SEQ ID NO: 8 Using a primer set consisting of a forward primer and a reverse primer consisting of the base sequence shown in SEQ ID NO: 11, a single PCR reaction solution containing the rDNA gene ITS region in the DNA of Plasmodiophora brassicae and Psium Ultimum And simultaneously amplified by PCR.

Furthermore, it consists of a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9 targeting the rDNA gene ITS region, and a base sequence shown in SEQ ID NO: 8. Using a primer set consisting of a forward primer and a reverse primer consisting of the base sequence shown in SEQ ID NO: 11, a single PCR reaction solution containing the rDNA gene ITS region in the DNA of Plasmodiophora brassicae and Phytophthora infestans And simultaneously amplified by PCR.
Then, the amplification product by the above PCR is dropped onto the microarray on which the probes consisting of the base sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 are immobilized, and the amplified product is fluorescently labeled. Was detected.

PCR amplification and gene amplification by the PCR method were carried out under the same composition and the same conditions as in Test 1. However, the PCR reaction solution was used as the sample DNA containing 1.0 μl for each bacterium and a total of 2.0 μl.
Then, the PCR amplification product and the probe were hybridized in the same manner as in Test 1, and the fluorescence intensity of each probe was measured with a label detection device to obtain the S / N ratio value. The result is shown in FIG.

  In FIG. 10, a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9, a forward primer consisting of the base sequence shown in SEQ ID NO: 7 and SEQ ID NO: 10 Amplification products obtained by amplifying the rDNA gene ITS region in the DNA of Plasmodiophora brassicae and Pythium ultimum by PCR using a primer set consisting of a reverse primer consisting of the base sequence shown, respectively, It is shown that the label was detected by binding to the probe having the nucleotide sequence shown in SEQ ID NOs: 18 and 19.

  In addition, a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9, a forward primer consisting of the base sequence shown in SEQ ID NO: 8 and a base shown in SEQ ID NO: 11 The amplification product obtained by amplifying the rDNA gene ITS region in the DNA of Plasmodiophora brassicae by PCR using a primer set consisting of a reverse primer consisting of a sequence consists of the base sequence shown in SEQ ID NO: 18 The amplified product obtained by amplifying the rDNA gene ITS region in the DNA of Pythium ultimum by PCR was shown that the label was detected by binding to the probe. The label is detected by binding to the probe consisting of It has been shown that was.

  Furthermore, a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9, a forward primer consisting of the base sequence shown in SEQ ID NO: 8 and a base shown in SEQ ID NO: 11 The amplification product obtained by amplifying the rDNA gene ITS region in the DNA of Plasmodiophora brassicae by PCR using a primer set consisting of a reverse primer consisting of a sequence consists of the base sequence shown in SEQ ID NO: 18 The amplified product obtained by amplifying the rDNA gene ITS region in the DNA of phytofutra infestans by PCR was shown that the label was detected by binding to the probe. To the probe consisting of There has been shown to have been detected.

  The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the present invention. For example, each of the above primer sets may be used alone in a PCR reaction solution, or a plurality of primer sets may be used simultaneously in one PCR reaction solution in all combinations. Further, in the microarray for microbiological examination, the above-mentioned probes can be appropriately changed, such as being used alone or in combination in all combinations.

  The present invention can be suitably used in plant tissue and soil tests, epidemiological environmental tests, environmental tests, clinical tests, livestock hygiene, and the like.

Claims (23)

A sample is taken from plant tissue or soil, water, or other environment, DNA is extracted from a microorganism group contained in the sample, PCR is performed using the extracted DNA, and the obtained amplification product is used based on the obtained amplification product. A method for testing microorganisms for determining the presence or absence of microorganisms in a sample,
A forward primer consisting of at least one of the nucleotide sequences shown in SEQ ID NOs: 1 to 3 targeting the β-tubulin gene in the DNA of Plasmodiophora spp. By PCR, and the nucleotide sequences shown in SEQ ID NOs: 4 and 5 PCR is performed using the first primer set consisting of the reverse primer consisting of at least one of the above and the extracted DNA, and based on the obtained amplification product, the presence or absence of Plasmodiophora spp. A method for inspecting a microorganism characterized by determining. From the base sequence shown in SEQ ID NO: 12 that binds complementarily to the first amplification product obtained by PCR using the DNA of Plasmodiophora and the first primer set, and at least one of the complementary sequences The first amplification product is contacted with a microarray having the first probe immobilized thereon,
The method for inspecting a microorganism according to claim 1, wherein the label of the first amplification product that is complementarily bound to the first probe is detected. PCR was performed using the first primer set and the extracted DNA with the β-tubulin gene in the DNA of Plasmodiophora, Psium, and Phytophthora as targets for amplification by PCR. The method for testing microorganisms according to claim 1 or 2, wherein the presence or absence of Plasmodiophora, Psium, and Phytophthra in the sample is simultaneously determined based on the obtained amplification product. From the base sequence shown in SEQ ID NO: 13 or 14 that binds complementarily to the second amplification product obtained by PCR using the DNA of Psium and the first primer set, and at least one of these complementary sequences The second amplification product is contacted with a microarray on which the second probe is immobilized,
The method for testing a microorganism according to claim 3, wherein the label of the second amplification product that is complementarily bound to the second probe is detected. A third probe comprising at least one of the nucleotide sequences shown in SEQ ID NO: 15 or 16, which binds complementarily to a third amplification product obtained by PCR using DNA of Phytophthora and the first primer set. The third amplification product is contacted with the microarray on which is immobilized,
The method for inspecting a microorganism according to claim 3 or 4, wherein the label of the third amplification product that is complementarily bound to the third probe is detected. Complementary binding to the fourth amplification product obtained by PCR using at least one of the DNAs of Plasmodiophora, Psium, Phytophthra, and other microorganisms and the first primer set Contacting the fourth amplification product with a microarray on which a fourth probe consisting of at least one of the base sequence shown in SEQ ID NO: 17 and its complementary sequence is immobilized,
The method for inspecting a microorganism according to any one of claims 3 to 5, wherein a label of the fourth amplification product that is complementarily bound to the fourth probe is detected. A second primer consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9 targeting the rDNA gene ITS region in the DNA of Plasmodiophora spp. PCR is performed using the primer set and the extracted DNA, and based on the obtained amplification product, the presence or absence of Plasmodiophora in the sample is determined. The inspection method of microorganisms in any one. From the nucleotide sequence shown in SEQ ID NO: 18 that complementarily binds to the fifth amplified product obtained by PCR using the DNA of Plasmodiophora and the second primer set, and at least one of the complementary sequences Contacting the fifth amplification product with a microarray on which the fifth probe is immobilized,
The method for inspecting a microorganism according to claim 7, wherein the label of the fifth amplification product that is complementarily bound to the fifth probe is detected. From a forward primer consisting of at least one of the base sequences shown in SEQ ID NO: 7 or 8, which targets the rDNA gene ITS region in the DNA of the genus Psium, by PCR, and a reverse primer consisting of the base sequence shown in SEQ ID NO: 10 PCR is performed using the extracted third DNA set and the extracted DNA, and the presence or absence of Pythium spp. In the sample is determined based on the obtained amplification product. The inspection method of microorganisms in any one. A sixth sequence comprising at least one of the base sequence shown in SEQ ID NO: 19 and the complementary sequence that binds complementarily to the sixth amplification product obtained by PCR using the DNA of the genus Psium and the third primer set Contacting the sixth amplification product with the microarray on which the probe of
The method for inspecting a microorganism according to claim 9, wherein the label of the sixth amplification product that is complementarily bound to the sixth probe is detected. A first primer consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 8 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 11 targeting the rDNA gene ITS region in the DNA of Psium or Phytophthra sp. PCR is performed using the four primer sets and the extracted DNA, and the presence or absence of Pythium spp. Or Phytophthora spp. In the sample is determined based on the obtained amplification product. The method for examining a microorganism according to any one of 10. The base sequence shown in SEQ ID NO: 20 or 21 that complementarily binds to the seventh amplified product obtained by PCR using the DNA of Psium or Phytophthra and the fourth primer set, and the complementary sequence thereof Contacting the seventh amplification product with a microarray on which a seventh probe comprising at least one is immobilized;
The microorganism testing method according to claim 11, wherein the label of the seventh amplification product that is complementarily bound to the seventh probe is detected. A sample is taken from plant tissue or soil, water, or other environment, DNA is extracted from a microorganism group contained in the sample, PCR is performed using the extracted DNA, and the obtained amplification product is used based on the obtained amplification product. A test kit for microorganisms used to determine the presence or absence of microorganisms in a sample,
A forward primer consisting of at least one of the base sequences shown in SEQ ID NOs: 1 to 3 targeting the β-tubulin gene in the DNA of Plasmodiophora spp., Psium spp., And Phytophthra spp. A microorganism testing kit comprising a primer set consisting of a reverse primer consisting of at least one of the base sequences shown in SEQ ID NOs: 4 and 5. Primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 6 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9 that targets the rDNA gene ITS region in the DNA of Plasmodiophora spp. The microorganism testing kit according to claim 13, further comprising: From a forward primer consisting of at least one of the base sequences shown in SEQ ID NO: 7 or 8, which targets the rDNA gene ITS region in the DNA of the genus Psium, by PCR, and a reverse primer consisting of the base sequence shown in SEQ ID NO: 10 The microorganism testing kit according to claim 13 or 14, further comprising a primer set. Primer consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 8 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 11 that targets the rDNA gene ITS region in the DNA of Psium or Phytophthora sp. The microorganism testing kit according to any one of claims 13 to 15, further comprising a set. A sample is taken from plant tissue or soil, water, or other environment, DNA is extracted from a microorganism group contained in the sample, PCR is performed using the extracted DNA, and the obtained amplification product is used based on the obtained amplification product. A microarray for microbiological examination for judging the presence or absence of microorganisms in a sample,
A forward primer consisting of at least one of the nucleotide sequences shown in SEQ ID NOs: 1 to 3, which uses the β-tubulin gene in the DNA of Plasmodiophora sp. As shown in SEQ ID NO: 12, which binds complementarily to a first amplification product obtained by PCR using a first primer set consisting of a reverse primer consisting of at least one of the base sequences shown in SEQ ID NOs: 4 and 5 A microarray for microbiological examination, wherein a first probe comprising at least one of a base sequence and a complementary sequence is immobilized. At least one of the base sequence shown in SEQ ID NO: 13 or 14, which binds complementarily to the DNA of the genus Psium and the second amplification product obtained by PCR using the first primer set, and their complementary sequences The microarray for microbiological examination according to claim 17, further comprising a second probe comprising: A third DNA comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 15 and 16 that binds complementarily to the third amplification product obtained by PCR using the first primer set and the DNA of Phytophthora spp. The microarray for microbiological examination according to claim 17 or 18, wherein a probe is further immobilized. Complementary to DNA of at least one of Plasmodiophora, Psium, Phytophthra, and other microorganisms, and a fourth amplification product obtained by PCR using the first primer set The microarray for microbiological examination according to any one of claims 17 to 19, wherein a fourth probe comprising at least one of the base sequence shown in SEQ ID NO: 17 and the complementary sequence is further immobilized. A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 6 and the DNA shown in SEQ ID NO: 9 that targets the rDNA gene ITS region in the DNA of Plasmodiophora sp. A base sequence shown in SEQ ID NO: 18 that complementarily binds to a fifth amplification product obtained by PCR using a second primer set consisting of a reverse primer consisting of a sequence and a first sequence consisting of at least one of the complementary sequences The microarray for microbiological examination according to any one of claims 17 to 20, wherein five probes are further immobilized. A forward primer consisting of at least one of the base sequence shown in SEQ ID NO: 7 or 8 that targets the rDNA gene ITS region in the DNA of Psium sp. A base sequence shown in SEQ ID NO: 19 that complementarily binds to a sixth amplification product obtained by PCR using a third primer set consisting of a reverse primer consisting of a sequence and a first sequence consisting of at least one of the complementary sequences The microarray for microbiological examination according to any one of claims 17 to 21, wherein six probes are further immobilized. A forward primer consisting of the base sequence shown in SEQ ID NO: 8 with the DNA of Pisium spp. Or Phytophthora sp., The rDNA gene ITS region in the DNA of Psium spp. A base sequence shown in SEQ ID NO: 20 or 21 that complementarily binds to a seventh amplification product obtained by PCR using a fourth primer set consisting of a reverse primer consisting of the base sequence shown, and at least one of these complementary sequences The microarray for microbiological examination according to any one of claims 17 to 22, wherein the seventh probe made of any of these is further immobilized.

Images