WO2019244860A1

WO2019244860A1 - Method for detection and discrimination of phytopathogenic bacteria

Info

Publication number: WO2019244860A1
Application number: PCT/JP2019/024005
Authority: WO
Inventors: 税増田; 達児畑谷; 和義古田
Original assignee: 国立大学法人北海道大学; ホクサン株式会社
Priority date: 2018-06-20
Filing date: 2019-06-18
Publication date: 2019-12-26
Also published as: JP7445924B2; JPWO2019244860A1

Abstract

[Problem] To provide a method whereby bacteria belonging to the genus Pectobacterium can be detected while discriminating bacterial species thereof even in a single reaction system. [Solution] A nucleic acid probe for detecting bacteria belonging to the genus Pectobacterium that comprises all or part of the following base sequence (i) or (ii): (i) a base sequence corresponding to the 1st to 40th positions, from the amino terminus, of the amino acid sequence of cellulase B of a bacterium belonging to the genus Pectobacterium; and (ii) a base sequence complementary to base sequence (i). According to the present invention, bacteria belonging to the genus Pectobacterium can be quickly and easily detected on a species level, which can contribute to the prevention of the onset of plant diseases caused by the bacteria and suppression of damages caused thereby.

Description

Methods for detecting and identifying plant pathogenic bacteria

The present invention relates to a method for detecting and identifying a plant pathogenic bacterium, and more particularly to a method for detecting a bacterium belonging to the genus Pectobacterium while identifying its species.

Pectobacterium bacteria are known as bacteria that cause disease in plants. For example, Pectobacterium wasabie (hereinafter sometimes abbreviated as “Pw”), Pectobacterium atroseptica (hereinafter sometimes abbreviated as “Pa”), and Pectobacterium carotobolum Subspecies Pectobacterium carotovorum subspecies brasiliense (hereinafter sometimes abbreviated as "Pcb") causes black spot disease of potatoes. Potatoes that have developed black rust show symptoms such as decay of the rhizome and withering of the aerial part. Pectobacterium carotovorum subspecies carotovorum (Pcc) (hereinafter sometimes abbreviated as “Pcc”) includes potato black rust, radish, Chinese cabbage, cabbage, cauliflower and the like. , Lettuce, ginger, celery, parsley, scallions, onions, carrots, potatoes, tomatoes, peppers, bok choy, komatsuna, leek, turnips, garlic, etc., which cause soft rot in many agricultural crops. In soft-rot crops, fresh soft tissues (fruits, flowers, leaves, stems, roots, etc.) above and below the ground are invaded, softening and decaying, causing odors.

In order to prevent or minimize the occurrence of such diseases, it is necessary to quickly detect Pectobacterium spp. And take measures such as effective control, investigation of the causes, and prevention of outbreaks based on the results. is there. Conventionally, an enrichment PCR method has been used as a method for detecting Pectobacterium bacteria. This is a method in which microorganisms contained in a sample such as soil or plant tissue are first cultured and increased, and a polymerase chain reaction (PCR) is performed using the DNA extracted therefrom as a template to detect the microorganisms ( Non-Patent Documents 1 and 2).

However, the conventional methods described in Non-Patent Document 1 and Non-Patent Document 2 have a problem that it is necessary to perform PCR separately for each detection target bacterial species, which lacks quickness and simplicity. The reason for this is that, as shown in Example 7 below, in the conventional PCR, the gene to be amplified differs for each bacterial species, so that when PCR is performed in a single reaction system, specific amplification is remarkable. And the detection sensitivity is not sufficient for the determination. The present invention has been made in order to solve the above-mentioned problem, and an object of the present invention is to provide a method for detecting a bacterium belonging to the genus Pectobacterium, even in a single reaction system, while identifying its species. And

The present inventors have conducted intensive studies and found that the 1st to 40th amino acids from the amino terminus of cellulase B of a bacterium belonging to the genus Pectobacterium are sequences unique to the bacterial species. Hereinafter, in the present invention, the amino acid sequence at positions 1 to 40 from the amino terminus of cellulase B of the genus Pectobacterium is referred to as “Pec cell B non-conserved region”, and the amino acid sequence after position 41 is referred to as “Pec cell B conserved region”. Area ".

Then, by detecting all or a part of the base sequence corresponding to the Pec cell B non-conserved region, even in a single reaction system, it is possible to detect a bacterium belonging to the genus Pectobacterium while identifying its species, In addition, it has been found that Pcc can be detected while identifying it. Then, based on this knowledge, the following inventions were completed.

(1) The nucleic acid probe according to the present invention is a nucleic acid probe for detecting a bacterium belonging to the genus Pectobacterium, and includes all or a part of the following nucleotide sequence (i) or (ii); (i) Pec A base sequence corresponding to the cell B non-conserved region, (ii) a base sequence complementary to (i).

(2) In the nucleic acid probe according to the present invention, the Pec cell B non-conserved region may be the one shown in SEQ ID NO: 1.

(3) In the nucleic acid probe according to the present invention, the bacterium belonging to the genus Pectobacterium may be one or more selected from the group consisting of Pw, Pcc, Pa, and Pcb.

(4) In the nucleic acid probe according to the present invention, the bacterium belonging to the genus Pectobacterium may be a pathogenic bacterium of black spot or soft rot of potato.

(5) The method for detecting a bacterium belonging to the genus Pectobacterium according to the present invention includes a step of detecting the nucleic acid probe according to the present invention.

(6) The method for detecting a bacterium belonging to the genus Pectobacterium according to the present invention may further include a step of amplifying the nucleic acid probe according to the present invention by PCR.

(7) The kit for detecting a bacterium belonging to the genus Pectobacterium according to the present invention includes the nucleic acid probe according to the present invention.

(8) The carrier for detecting a bacterium belonging to the genus Pectobacterium according to the present invention has the nucleic acid probe according to the present invention immobilized thereon.

(9) The PCR primer according to the present invention is a PCR primer for detecting a bacterium belonging to the genus Pectobacterium, and contains all or a part of the base sequence corresponding to the Pec cell B non-conserved region.

(10) In the PCR primer according to the present invention, the Pec cell B non-conserved region may be the one shown in SEQ ID NO: 1.

(11) In the PCR primer according to the present invention, the bacterium belonging to the genus Pectobacterium may be one or more selected from the group consisting of Pw, Pcc, Pa, and Pcb.

(12) In the PCR primer according to the present invention, the bacterium belonging to the genus Pectobacterium may be a pathogenic bacterium of black spot or soft rot of potato.

(13) The PCR primer set according to the present invention includes the following first PCR primer and the following second PCR primer used as a pair with the first PCR primer; PCR primer according to the present invention, second primer: a PCR primer containing a part of a base sequence complementary to a base sequence corresponding to the Pec cell B conserved region.

(14) In the PCR primer set according to the present invention, the Pec cell B conserved region may be the one shown in SEQ ID NO: 2.

According to the present invention, even if it is a single reaction system, bacteria of the genus Pectobacterium can be detected while distinguishing the species. Therefore, according to the present invention, a bacterium belonging to the genus Pectobacterium can be detected quickly and easily at the bacterial species level, thereby contributing to prevention of plant disease caused by the bacterium and suppression of the damage.

It is a figure which shows the amino acid sequence multiple alignment of cellulase B of bacteria of the genus Pectobacterium. In the figure, the enclosed portion is the Pec cell B non-storage area. The notation “.” In the amino acid sequence indicates that the amino acid is the same as the amino acid at the top. FIG. 4 is a view showing a designed region (underlined portion) of a forward primer at 1 to 120 mer from the 5 ′ end of a cellulase B gene (celB) of a bacterium belonging to the genus Pectobacterium. Using genomic DNA extracted from various strains of the genus Dikey bacterium and Pectobacterium as a template, the pectate lyase gene (pelADE) of the genus Dikey bacterium or the cellulase B gene (celB) of the bacterium of the genus Pectobacterium as the gene to be amplified FIG. 3 is an agarose gel electrophoresis diagram of a product of PCR performed. The left side is a photograph showing the spot position of each DNA on the four-species detection membrane. The right side is a photograph showing the spot position of each DNA on the 6-species detection membrane. On the upper side, genomic DNA extracted from each species or strain of the genus Dikea and the bacterium of the genus Pectobacterium was used as a template, and seven types of primers were mixed. It is a photograph which shows the result detected with the membrane for species detection. The lower side is an agarose gel electrophoresis diagram of the PCR product. On the upper side, genomic DNA extracted from each species or strain of bacteria belonging to the genus Dikeya and bacterium belonging to the genus Pectobacterium was used as a template, and eight types of primers were mixed. It is a photograph which shows the result detected with the membrane for species detection. The lower side is an agarose gel electrophoresis diagram of the PCR product. It is a photograph which shows the spot position of each DNA in the conventional type 4 membrane for detection of bacterial species. 4 is a photograph showing a position where a section is collected in a potato seedling exhibiting a symptom that a seed potato rots by inoculation of a Pw @ Ecc-2 strain. On the upper side, a genomic DNA extracted from a diseased potato tissue was used as a template, and eight types of conventionally used primers were mixed to perform PCR in a single reaction system. It is a photograph which shows the result of detection. The lower side is an agarose gel electrophoresis diagram of the PCR product. The upper part is a photograph showing the results of detection of a PCR product obtained in a single reaction system using genomic DNA extracted from a diseased potato tissue as a template, mixing seven types of primers, and using a four-species detection membrane. is there. The lower side is an agarose gel electrophoresis diagram of the PCR product.

Hereinafter, the present invention will be described in detail. In the present invention, the “single reaction system” refers to a reaction performed under one reaction condition in one phase (often a liquid phase). Further, “detecting a microorganism while identifying it” refers to detecting a microorganism in a manner that can identify the species to which the microorganism belongs.

において In the present invention, “Bacteria belonging to the genus Pectobacterium” refers to bacteria belonging to the genus Pectobacterium. As described above, such bacteria are known to be pathogenic bacteria that cause black rust of potato and soft rot of various crops. Examples of such species include Pw, Pa, Pcb, and Pcc described above.

Since the non-conserved region of Pec cell B is a sequence specific to the strain, by detecting a base sequence corresponding thereto or a base sequence complementary thereto, Pectobacterium bacteria can be detected while distinguishing the strain. can do. That is, the nucleic acid probe according to the present invention is a nucleic acid probe for detecting a bacterium belonging to the genus Pectobacterium, and includes all or a part of the following nucleotide sequence (i) or (ii);
(I) a base sequence corresponding to the Pec cell B non-conserved region,
(Ii) a base sequence complementary to (i).

A probe generally refers to a substance used to detect a certain substance. That is, in the present invention, the “nucleic acid probe” refers to a nucleic acid used for detecting a bacterium belonging to the genus Pectobacterium. Note that a nucleic acid refers to a biopolymer in which nucleotides consisting of a base, a sugar, and a phosphate are linked by a phosphodiester bond.

セル Cellulase B of a bacterium belonging to the genus Pectobacterium refers to cellulase B inherent to a bacterium belonging to the genus Pectobacterium. The gene encoding cellulase B of the bacterium belonging to the genus Pectobacterium is, in addition to the cellulase B gene (celB), in the present invention, a cellulase S gene (celS) which is highly homologous to celB and is considered to be present in the bacterium SCC3193. ) Are also included in the same gene (Genbank accession number M32399 and Koskinen et al. (2012) vol. 194, no. 21 p6004, Genbank accession number CP003415).

Examples of the amino acid sequence of cellulase B of the genus Pectobacterium include those of Pcc, Pa, Pcb and Pw (total length 264 aa) shown in FIG. 1 and SEQ ID NOS: 12 to 20. Based on the sequence illustrated in FIG. 1 and SEQ ID NOS: 12 to 20, the Pec cell B non-conserved region can be represented as SEQ ID NO: 1, and the Pec cellulase conserved region can be represented as SEQ ID NO: 2. In addition, amino acid sequence information of cellulase, Pec cellulase non-conserved region and Pec cellulase conserved region of bacteria belonging to the genus Pectobacterium can be found in PIR (http://pir.georgetown.edu/) and UniProtKB (http: //www.uniprot. org / uniprot /), Entrez @ Protein (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein).

において In the present invention, the term "base sequence corresponding to an amino acid sequence" refers to a base sequence specified by converting the amino acid sequence according to codons (gene codes). That is, the base sequence (i) can be specified by converting the amino acid sequence information of the Pec cell B non-conserved region according to codons.

In addition, the predetermined base sequence and the “complementary base sequence” are in the opposite direction to the predetermined base sequence according to Watson-Crick base pair, and are adenine (A), guanine and guanine of the predetermined base sequence. (G) refers to a base sequence in which bases are arranged to be T, C, A and G with respect to thymine (T) and cytosine (C), respectively. That is, the base sequence (ii) can be specified by converting the base sequence information (i) according to Watson-Crick base pairing.

The length of the base sequence (i) or (ii) contained in the nucleic acid probe according to the present invention is not particularly limited, but from the viewpoint of detection sensitivity, at least 18 mer of the base sequence (i) or (ii). It is preferable to include Further, the nucleic acid probe may be labeled or modified with various substances such as radioactive phosphate, biotin, a fluorescent dye molecule, and an enzyme for detection and purification.

Next, examples of how to use nucleic acid probes will be given. The nucleic acid probe can be used, for example, for detecting a bacterium belonging to the genus Pectobacterium contained in a sample collected from nature such as soil or plant tissue.

Specifically, DNA is extracted from a sample and its base sequence is decoded. The presence or absence of the nucleic acid probe according to the present invention in the sequence obtained by decoding is detected. In some cases, it can be determined that the sample contains Pectobacterium bacteria, and when there is no sample, it can be determined that the sample does not contain Pectobacterium bacteria.

When a nucleic acid probe is present in the DNA base sequence extracted from the sample, the base sequence of the nucleic acid probe is higher than the base sequence of a known Pw nucleic acid probe (for example, the 1st to 120th positions of SEQ ID NOS: 3 to 5). When the sample exhibits identity, it can be determined that the sample contains Pw, and when the sample exhibits high identity with the base sequence of a known Pcc nucleic acid probe (eg, positions 1 to 120 of SEQ ID NOs: 6 and 7). Can be determined to contain Pcc in the sample, and if it shows a high identity with the base sequence of a known nucleic acid probe of Pa (eg, positions 1 to 120 of SEQ ID NOs: 8 and 9), Pa can be determined to be included, and if the sample exhibits high identity with the base sequence of a known Pcb nucleic acid probe (eg, positions 1 to 120 of SEQ ID NOS: 10 and 11), the sample contains P It can be determined that b is included.

Alternatively, a method of performing PCR for amplifying the nucleic acid probe according to the present invention using DNA extracted from the above sample as a template can be mentioned. In this PCR, a nucleic acid probe consisting of about 20 to 30 mer of the base sequence (i) is used as a forward primer. If the nucleic acid probe is detected in the PCR product, it can be determined that the sample contains Pectobacterium sp., And if the nucleic acid probe is not detected in the PCR product, the sample contains Pectobacterium sp. Can be determined not to be present.

Then, when a nucleic acid probe is detected in the PCR product, if the nucleotide sequence shows a high identity with the nucleotide sequence of a known Pw nucleic acid probe (eg, positions 1 to 120 of SEQ ID NOS: 3 to 5) If it can be determined that the sample contains Pw, and the sample exhibits high identity with the base sequence of a known nucleic acid probe of Pcc (for example, positions 1 to 120 of SEQ ID NOs: 6 and 7), the sample contains Pcc If the sample exhibits high identity with the base sequence of a known nucleic acid probe of Pa (eg, positions 1 to 120 of SEQ ID NOS: 8 and 9), it is determined that the sample contains Pa. If the sample exhibits high identity with the base sequence of a known Pcb nucleic acid probe (eg, positions 1 to 120 of SEQ ID NOS: 10 and 11), it is determined that the sample contains Pcb. It can be.

As described above, the present invention also provides a method for detecting a bacterium belonging to the genus Pectobacterium, comprising the step of detecting the nucleic acid probe according to the present invention. The present detection method may further include a step of amplifying the nucleic acid probe by PCR. The present invention also provides a PCR primer for detecting a bacterium belonging to the genus Pectobacterium, which comprises all or a part of the nucleotide sequence corresponding to the Pec cell B non-conserved region.

By the way, detection of a nucleic acid probe in DNA extracted from a sample or a PCR product using the DNA as a template can be performed, for example, by decoding a base sequence (sequencing) or by hybridization.

In the case of detection by hybridization, a carrier in which a single-stranded nucleic acid probe of each known bacterial species is fixed in a different position for each bacterial species is prepared in advance. A nucleic acid contained in a sample such as a DNA or a PCR product extracted from the sample is labeled with a labeling substance such as biotin, and then hybridized to a carrier. A detection reaction is performed according to the labeling substance, and the position where the signal is detected on the carrier is confirmed. If a signal is detected at the position where the Pw nucleic acid probe is fixed, it can be determined that Pw is contained in the sample. If a signal is detected at the position where the Pa nucleic acid probe is fixed, Pa Is contained, and if a signal is detected at the position where the Pcb nucleic acid probe is fixed, it can be determined that the sample contains Pcb, and the signal is detected at the position where the Pcc nucleic acid probe is fixed. Is detected, it can be determined that the sample contains Pcc.

As described above, the present invention also provides a carrier for detecting a bacterium belonging to the genus Pectobacterium, on which the nucleic acid probe according to the present invention is immobilized. The support of the nucleic acid probe used in the present carrier may be any support capable of immobilizing nucleic acid, and examples thereof include a resin thin film (membrane) and a resin or glass base (chip). The present invention also provides a kit for detecting a bacterium belonging to the genus Pectobacterium, comprising the nucleic acid probe according to the present invention. The kit includes a nucleic acid probe of each species of Pectobacterium bacteria or a carrier for detection of Pectobacterium bacteria, and other reagents (medium, DNA extraction buffer, PCR enzyme, PCR enzyme, etc.) depending on the detection method. Reaction buffers, washing solutions, purification columns, etc.), containers, instruments, instruction manuals and the like.

細菌 Representative bacteria causing the black spot disease of potato include Pectobacterium spp., As well as Dickeya dianthicola (hereinafter sometimes abbreviated as “Ddi”). Conventionally, Ddi has been detected by an enrichment PCR method using the pectate lyase gene as an amplification target. The PCR primers include ADE1 (forward primer: SEQ ID NO: 27) and ADE2 (reverse primer: SEQ ID NO: 29). (Nassar et al. (1996), Applied and Enviromental Microbiology 62 p2228-2235).

As shown in Example 6 described below, the nucleic acid probe according to the present invention can be used in a single reaction system with the ADE1 primer and the ADE2 primer, or with the partial sequence of the pectate lyase gene of Ddi. Does not increase or the specific reaction decreases. Therefore, in addition to the nucleic acid probe, a partial sequence of the pectate lyase gene of Ddi may be immobilized on the carrier for detecting Pectobacterium bacteria. Similarly, the kit for detecting a bacterium belonging to the genus Pectobacterium may include, in addition to the nucleic acid probe, all or part of the base sequence of the ADE1 primer, the ADE2 primer, and the pectate lyase gene of Ddi. According to such a kit or carrier, a pathogenic bacterium of black spot of potato can be comprehensively detected or identified by a single reaction system.

Finally, the present invention provides a PCR primer set. The present PCR primer set includes the following first PCR primer and the following second PCR primer used as a pair with the first PCR primer;
First primer: PCR primer according to the present invention,
Second primer: a PCR primer containing a part of the base sequence complementary to the base sequence corresponding to the Pec cell B conserved region.

２The second primer is common among species of bacteria belonging to the genus Pectobacterium. Therefore, in PCR for amplifying nucleic acid probes of a plurality of species in a single reaction system, the number of types of primers can be suppressed by using the above-mentioned primer set. That is, according to this primer set, even in a single reaction system, it is possible to more effectively amplify the nucleic acid probe of each bacterial species without causing non-specific amplification or reduction of specific amplification. it can.

Hereinafter, the present invention will be described based on each embodiment. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Test method>
In this example, the following test methods were used unless otherwise specified. In this example, "%" represents% by mass unless otherwise specified.

(1) Strains The strains shown in Table 1 were used. The PccSR HS-SR strain was isolated from Hokusan Co., Ltd., and the others were obtained from the Tokachi Agricultural Research Station, Agricultural Research Division, Hokkaido Prefectural Research Organization.

(2) Extraction of Nucleic Acid from Strain A single colony of each strain on King's B agar medium (* Note 1) was inoculated into King's B liquid medium (Note 1), and 1-2 in a dark place at 25 ° C. Culture was performed for a day to obtain a culture solution. The culture was subjected to centrifugation to remove the supernatant, and the cell pellet was collected. DNA was extracted from the cell pellet by the CTAB method (* Note 2), and a 100 ng / uL DNA solution was prepared in sterile water.

* Note 1: King's B medium King's B liquid medium (1L composition; peptone 20 g, K ₂ HPO ₄ 1.5 g, MgSO ₄ .7H ₂ O 1.5 g, glycerin 10 mL, distilled water 990 mL, pH 7.2), 1.5% (w / v) of agar was added, and the mixture was autoclaved at 121 ° C. for 30 minutes to obtain King's B agar medium.

* Note 2: CTAB method At least 5 times the amount of nucleic acid extraction buffer (composition: 2% hexadecyltrimethylammonium bromide (CTAB), 100 mM Tris-HCl (pH 8.0), 1.4 M NaCl) , 50 mM ethylenediaminetetraacetic acid (EDTA), 0.1% 2-mercaptoethanol) and heated at 65 ° C. After allowing the mixture to stand at room temperature, an equal amount of the added nucleic acid extraction buffer and chloroform: isoamyl alcohol (24: 1 by volume) was added, and the mixture was mixed and stirred for 1 minute or more. Thereafter, the supernatant (aqueous layer) was collected by centrifugation, and purified using a commercially available silica column (Qiagen, etc.). When RNA was selected as the extracted nucleic acid, the RNA was fractionated by performing a DNase treatment and a lithium chloride precipitation treatment before purifying the silica column.

(3) PCR
The PCR enzyme used was AmpliTaq Gold360 (Thermo Fisher Scientific). The reaction solution volume was 25 μL or 50 μL, and the composition of the reaction solution was 0.02 U / μL of PCR enzyme, 0.2 to 0.4 μM of primer, and 100 mg of template DNA. The first cycle of PCR was performed at 95 ° C. for 10 minutes, the second to 35th cycles were performed at 95 ° C. for 30 seconds, 56 ° C. for 30 seconds and 72 ° C. for 30 seconds, and 34 cycles were completed. C. for 10 minutes. When using RNA as a template nucleic acid, M-MLV reverse transcriptase (Invirtogen) treatment was performed prior to PCR, and a kit such as PrimeScript One Step RT-PCR kit ver. 2 (TAKARA) was used.

(4) Preparation of Membrane for Macro Array The PCR product was purified to obtain a DNA fragment. To this, sterile water and a dye solution containing xylene cyanol (XC) were added to prepare a spot solution having a DNA concentration of 50 ng / μL and an XC concentration of 0.01 mg / mL. Using a HYDR96 DNA spotter, 0.2 μL of the spot solution was spotted on a nylon membrane (Biodyne Pall). The nylon membrane after the spot was heat-denatured by placing it at 120 ° C. for 30 minutes while sandwiching it between filter papers to form a single strand. Subsequently, the DNA was immobilized on the membrane by setting it on a UV crosslinker (CL-1000; UVP) with the spot surface facing upward and irradiating with 120 mJ / cm ² of ultraviolet light. This membrane was cut and used according to the arrangement of spotted DNA.

(5) Detection of Signal in Macroarray Membrane The operation of subjecting the test sample to the macroarray membrane to detect signals was performed in the following steps 1 to 4.
1. Prehybridization 1.7 mL of PerfectHyb solution (TOYOBO) was placed in a 2-mL microtube, and heated by placing it in a heat block set at 69 ° C. for 20 minutes or more. A macroarray membrane fixed with a holder was placed in the microtube, and incubated at 69 ° C. for at least 40 minutes.

2. Hybridization The sample to be tested (biotin-labeled DNA fragment) was thermally denatured by placing it at 99 ° C. for 5 minutes, and then rapidly cooled by placing it on ice for 3 minutes. 15 μL of the sample to be tested was added to the microtube from which the pre-hybridized membrane had been removed, and then the membrane was again put therein. Thereafter, incubation was performed at 69 ° C. for 2 to 16 hours.

3. Washing 2 mL microtube containing 1.7 mL of Wash Solution 1 (2 × SSC, 0.1% SDS) and 2 mL microtube containing 1.7 mL of Wash Solution 2 (0.1 × SSC, 0.1% SDS) Were prepared for each sample, and were placed on a 69 ° C. heat block and heated for 20 minutes. The membrane after hybridization was added to the first washing solution 1, incubated for 1 minute, and then mixed by inversion about 5 times. The membrane was transferred to the second washing solution 1, incubated for 1 minute, and then mixed by inversion about 5 times. The membrane was transferred to the third washing solution 1, incubated for 10 minutes, and then mixed by inversion about 5 times. The membrane was transferred to the first washing solution 2 and incubated for 1 minute, and then mixed by inversion about 5 times. The membrane was transferred to the second washing solution 2, incubated for 1 minute, and then mixed by inversion about 5 times. The membrane was transferred to the third washing solution 2 and incubated for 10 minutes.

4. Coloring reaction i) The membrane after washing was put into a vessel containing 15 mL of BLOCK solution (5% SDS, 125 mM NaCl, 25 mM sodium phosphate, pH 7.2) and lightly rinsed. The BLOCK solution was discarded, 15 mL of a new BLOCK solution was added, and the mixture was shaken for 5 minutes.
ii) The BLOCK solution was discarded, and 15 mL of alkaline phosphatase-labeled streptavidin solution (SAP solution; prepared by adding 10 μL of Streptavidin-Alkaline Phosphataase (BioRad) to 15 mL of BLOCK solution) was added and shaken for 5 minutes.
iii) The SAP solution was discarded, and rinsing with 15 mL of Wash Solution 3 (0.5% SDS, 12.5 mM NaCl, 2.5 mM sodium phosphate, pH 7.2) was repeated twice. After adding the washing solution 3 for the third time, the mixture was shaken for 5 minutes.
iv) Washing solution 3 was discarded, and rinsing with washing solution 4 (10 mM Tris-HCl (pH 9.5), 10 mM NaCl, 1 mM MgCl ₂ ) was repeated twice. After the washing liquid 4 was added for the third time, the mixture was shaken for 5 minutes.
v) NBT / BCIP ready-to-use tablet (Roche) One tablet (0.34 g) dissolved in 10 mL of distilled water is put in a new container, and the membrane taken out from the washing solution 4 is immersed in the container, and the container is lightened. Leave for 1 minute while shaking.
vi) The membrane was taken out and immediately wrapped in a wrap, and the presence or absence of a color spot or the color intensity within 15 minutes was visually checked.

<Example 1> Multiple alignment (1) Sequencing of cellulase B gene Primers were designed based on published reports on cellulase B of a bacterium belonging to the genus Pectobacterium and nucleotide sequence information registered in GenBank. PCR was performed using the genomic DNA of the strain shown in Table 1 as a template and these primers to obtain a DNA fragment. By direct sequencing of this DNA fragment, the base sequence of the cellulase B gene (celB) of each strain was determined, and the amino acid sequence of cellulase B was determined based on the base sequence. The results are shown in Table 2 and SEQ ID NOS: 3 to 20. Regarding Pw, from the high homology of the base sequence, it is estimated that what is reported as the cellulase S gene corresponds to the cellulase B gene (Koskinen et al. (2012) vol. 194, no. 21). p6004, Genbank accession number CP003415).

Subsequently, it was obtained based on the base sequences of the cellulase B gene of Pectobacterium bacterium Pcc @ LY34 (Genbank @ accession number AF025769) and the cellulase S gene of Psp @ SCC3193 (Genbank @ accession number @ M32399) registered in Genbank. The amino acid sequence and the amino acid sequence of cellulase B determined as described above were compared with Genetyx @ ver. 12, multiple alignment was performed. The result is shown in FIG.

{Circle around (1)} As shown in FIG. 1, it was revealed that diversity exists between the bacterial species at positions 1 to 40 from the amino terminal of cellulase B (SEQ ID NO: 1). From these results, the base sequence corresponding to the amino acid sequence at positions 1 to 40 from the amino terminus of cellulase B of the genus Pectobacterium (Pec cell B non-conserved region), or all or part of the base sequence complementary thereto It has been clarified that Pectobacterium genus bacteria can be detected by distinguishing the species of the bacterium.

<Example 2> Design of PCR primer In the 1st to 120th region from the 5 'end of SEQ ID NOS: 3 to 11 (base sequence corresponding to the non-conserved region of Pec cell B), a forward primer having a length of 21 to 31 mer ( Fw primer). This Fw primer had a sequence unique to each strain. In addition, Fw primer and reverse primer (Rv primer) were designed so as to have a common sequence among the bacterial species in the region from the 5 'end to the 121st region (base sequence corresponding to the Pec cell B conserved region). The sequences of these primers are shown in Table 3. Table 3 also shows ADE1 (SEQ ID NO: 27) and ADE2 (SEQ ID NO: 29) which are PCR primers for Ddi detection. FIG. 2 shows the design region of the Fw primer.

<Example 3> Confirmation of specific DNA amplification Genomic DNA of the strains shown in Table 1 ([1] Ddi Echr93431, [2] Ddi Echr8263, [3] Pw EccNR-2, [4] Pw BNS2-2, [5] Pw 4-1-1, [6] Pcc HS-SR, [7] Pa Eca2, [8] Pa P-14, [9] Pcb Kbs-1) as a template and PCR using the primers in Table 3 The presence or absence and size of the PCR product were confirmed by agarose gel electrophoresis. The primer sets used are shown in the following [i] to [vi]. In addition, PCR was similarly performed on a control sample containing no template DNA. The result is shown in FIG.
《Primer set》
[I] Fw; SEQ ID NO: 27, Rv; SEQ ID NO: 29 (amplified fragment length: 420 bp, gene to be amplified: pelADE, bacterial species to be detected: Ddi),
[Ii] Fw; SEQ ID NO: 26, Rv; SEQ ID NO: 28 (amplified fragment length: 525 bp, gene to be amplified: celB, species to be detected: bacteria belonging to the genus Pectobacterium),
[Iii] Fw; SEQ ID NO: 21, Rv; SEQ ID NO: 28 (amplified fragment length: 755 bp, gene to be amplified: celB, bacterial species to be detected: Pw),
[Iv] Fw; SEQ ID NO: 22, Rv; SEQ ID NO: 28 (amplified fragment length: 753 bp, gene to be amplified: celB, species to be detected: Pcc)
[V] Fw; SEQ ID NO: 23, Rv; SEQ ID NO: 28 (amplified fragment length: 716 bp, gene to be amplified: celB, strain to be detected: Pa)
[Vi] Fw; SEQ ID NO: 24, Rv; SEQ ID NO: 28 (amplified fragment length: 718 bp, gene to be amplified: celB, bacterial species to be detected: Pcb Kbs-1)

As shown in FIG. 3, in the primer set [i], a band having a size corresponding to 420 bp was confirmed in the lanes [1] and [2], and the band was not confirmed in the lanes [3] to [10]. Was. In the primer set [ii], a band having a size of 525 bp was confirmed in the lanes [3] to [9], and the band was not confirmed in the lanes [1], [2] and [10]. In the primer set [iii], a band of a size equivalent to 755 bp was confirmed in the lanes [3] to [5], and the band was not confirmed in the lanes [1] [2] [6] to [10]. Was. In the primer set [iv], a band having a size equivalent to 753 bp was confirmed in the lane [6], and the band was not confirmed in the lanes [1] to [5] and [7] to [10]. In the primer set [v], a band corresponding to a size of 716 bp was confirmed in the lanes [7] and [8], and the band was confirmed in the lanes [1] to [6], [9] and [10]. Did not. In the primer set of [vi], a band of a size equivalent to 718 bp was confirmed in the lane of [9], and the band was not confirmed in the lanes of [1] to [8] and [10].

That is, in PCR using the primer sets [i] to [vi], the DNA of each bacterial species was specifically amplified, and there was no non-specific amplification. From this result, it was revealed that by performing PCR using a base sequence corresponding to the Pec cell B non-conserved region as an amplification target, bacteria belonging to the genus Pectobacterium can be detected while discriminating the species.

Example 4 Preparation of Membrane for Macroarray In the cellulase B gene of each of three strains of Pw, Pcc and Pa, and two strains of Pcb Kbs-1 and Pcb SUPP2564, the 1st to 120th region (Pec) from the 5 ′ end A primer was designed using the 80-mer at the 5 'end of the base sequence corresponding to the non-conserved region of cell B) as the Fw primer and the Rv primer as the base sequence complementary to the 80-mer at the 3' end. In addition, primers for amplifying a partial sequence of the Ddi pectate lyase gene were designed (Nassar et al. (1996), Applied and Environmental Microbiology 62 p2228-2235). The sequences of these primers are shown in Table 4.

Genomic DNA of each strain of Ddi was mixed to prepare genomic DNA of the bacterial species. Using this Ddi genomic DNA as a template, PCR was performed using primer sets of SEQ ID NOS: 30 and 31, and a PCR product was obtained. For Pw, Pcc, Pa, Pcb @ Kbs-1 and Pcb @ SUPP2564, the Fw primer and the RV primer were mixed for each bacterial type because the primer set in Table 4 was also a template, and PCR was performed to obtain a PCR product. Subsequently, a membrane for macroarray (membrane for detecting four bacterial species) was prepared by spotting the amplified fragments of the cellulase B gene of Pw, Pa, Pcb @ Kbs-1 and Pcc by the test method (4). Similarly, a membrane for macroarray (membrane for detecting 6 bacterial species) spotted with an amplified fragment of the pectate lyase gene of Ddi and an amplified fragment of the cellulase B gene of Pw, Pa, Pcb @ Kbs-1, Pcb @ SUPP2564 and Pcc was prepared. . The spot position on each membrane was as shown in FIG.

<Example 5> Detection in a single reaction system: Membrane for detection of 4 strains Genomic DNA of each strain Ddi, Pw and Pa was mixed for each strain to prepare strain genomic DNA. Genomic DNA of this strain and genomic DNA of Pcb Kbs-1, Pcc HS-SR and Pcc EccS-1B ([1] Ddi, [2] Pw, [3] Pa, [4] Pcb Kbs-1, [5] PCR was performed using Pcc HS-SR and [6] Pcc EccS-1B) as templates. [7] PCR was similarly performed on a control sample containing no template DNA. The primer used was a mixture of the primer sets [i] to [vi] of Example 3 (seven types of primers of SEQ ID NOS: 21 to 24 and 27 to 29). The primers of SEQ ID NO: 27 and SEQ ID NO: 28 used had been labeled with biotin in advance. Thereafter, the presence or absence and size of the PCR product were confirmed by agarose gel electrophoresis. Further, the presence / absence and position of the signal were confirmed by applying to the membrane for detecting four bacterial species in Example 4. The result is shown in FIG.

As shown in FIG. 5, when the template was Ddi genomic DNA ([1]), a band corresponding to 420 bp was confirmed by electrophoresis, but no signal was detected with the membrane for detecting four strains.
On the other hand, when the template is Pw genomic DNA ([2]), a band corresponding to 755 bp was confirmed by electrophoresis, and a signal was detected at a position where the amplified fragment of cellulase B gene of Pw was spotted on the membrane for detection of four strains. No signal was detected at other positions.
When the template was Pa genomic DNA ([3]), a band corresponding to 753 bp was confirmed by electrophoresis, and a signal was detected at the spot where the amplified fragment of the cellulase B gene of Pa was spotted even on the membrane for detecting four bacterial species. No signal was detected at the position.
In the case where the template was Pcb Kbs-1 genomic DNA ([4]), a band corresponding to 716 bp was confirmed by electrophoresis, and even in the membrane for detection of four strains, the amplified fragment of the cellulase B gene of Pcb Kbs-1 was spotted. A signal was detected, and no signal was detected at other positions.
When the template was Pcc HS-SR genomic DNA ([5]), a band equivalent to 753 bp was confirmed by electrophoresis, and a signal was detected at the position where the amplified fragment of Pcc cellulase B gene was spotted even on the membrane for detecting four strains. No signal was detected at other positions.
When the template was Pcc EccS-1B genomic DNA ([6]), a band equivalent to 753 bp was confirmed by electrophoresis, and a signal was detected at the position where the amplified fragment of Pcc cellulase B gene was spotted on the membrane for detection of 4 strains. No signal was detected at other positions.
In the case of the control sample without the template DNA ([7]), no band was confirmed by electrophoresis, and no signal was detected even with the membrane for detecting four bacterial species.

That is, even in PCR performed in a single reaction system by mixing seven types of primers, a sufficient amount of DNA to be detected is amplified in a specific manner for each species of Ddi, Pw, Pa, Pcb and Pcc, No non-specific amplification or reduction in specific amplification was observed. From this result, by detecting all or a part of the base sequence corresponding to the Pec cell B non-conserved region, or the base sequence complementary thereto, Pectobacterium sp. It became clear that it could be detected.

<Example 6> Detection in a single reaction system: Membrane for detecting 6 strains Genomic DNA of each strain Ddi, Pw and Pa was mixed for each strain to prepare genomic DNA of each strain. Genomic DNA of this strain and genomic DNAs of Pcb Kbs-1, Pcb SUPP2564, Pcc HS-SR and Pcc EccS-1B ([1] Ddi, [2] Pw, [3] Pa, [4] Pcb Kbs-1, PCR was performed using [5] Pcb SUPP2564, [6] Pcc HS-SR, and [7] Pcc EccS-1B) as templates. [8] A control sample containing no template DNA was similarly subjected to PCR. As the primer, in addition to the primer sets [i] to [vi] of Example 3, a mixture of the following primer sets [vii] (eight kinds of primers of SEQ ID NOS: 21 to 25 and 27 to 29) was mixed. What was used was used. The primers of SEQ ID NO: 27 and SEQ ID NO: 28 used had been labeled with biotin in advance. Thereafter, the presence or absence and size of the PCR product were confirmed by agarose gel electrophoresis. In addition, the presence or absence and position of the signal were confirmed by applying to the membrane for detecting 6 bacterial species of Example 4. FIG. 6 shows the result.
[Vii] Fw; SEQ ID NO: 25, Rv; SEQ ID NO: 28 (amplified fragment length: 748 bp, gene to be amplified: celB, strain to be detected: Pcb SUPP2564)

As shown in FIG. 6, when the template is Ddi genomic DNA ([1]), a band corresponding to 420 bp was confirmed by electrophoresis, and the position where the amplified fragment of pectic acid lyase gene of Ddi was spotted even on the membrane for detection of 6 strains. And no signal was detected at other positions.
When the template was Pw genomic DNA ([2]), a band corresponding to 755 bp was confirmed by electrophoresis, and a signal was detected at the position where the amplified fragment of the cellulase B gene of Pw was spotted even on the membrane for detecting 6 bacterial species. No signal was detected at other positions.
When the template was Pa genomic DNA ([3]), a band corresponding to 753 bp was confirmed by electrophoresis, and a signal was detected at the position where the amplified fragment of the cellulase B gene of Pa was spotted even on the membrane for detecting 6 bacterial species. No signal was detected at the position.
When the template was Pcb Kbs-1 genomic DNA ([4]), a band corresponding to 716 bp was confirmed by electrophoresis, and the membrane for detection of 6 strains was located at the position where the amplified fragment of the cellulase B gene of Pcb Kbs-1 was spotted. A signal was detected, and no signal was detected at other positions.
Also when the template was Pcb SUPP2564 genomic DNA ([5]), a band corresponding to 748 bp was confirmed by electrophoresis, and a signal was detected at the position where the amplified fragment of the cellulase B gene of Pcb SUPP2564 was spotted even on the 6-species detection membrane. No signal was detected at other positions.
When the template was Pcc HS-SR genomic DNA ([6]), a band equivalent to 753 bp was confirmed by electrophoresis, and a signal was detected at the position where the amplified fragment of Pcc cellulase B gene was spotted even on the membrane for detecting 6 strains. No signal was detected at other positions.
When the template was Pcc EccS-1B genomic DNA ([7]), a band corresponding to 753 bp was confirmed by electrophoresis, and a signal was detected at the position where the amplified fragment of Pcc cellulase B gene was spotted even on the membrane for detecting 6 strains. No signal was detected at other positions.
In the case of the control sample without the template DNA ([8]), no band was confirmed by electrophoresis, and no signal was detected even with the 6-species detection membrane.

That is, even in PCR performed in a single reaction system by mixing eight kinds of primers, a sufficient amount of DNA to be detected is amplified in a specific manner for each species of Ddi, Pw, Pa, Pcb and Pcc, No non-specific amplification or reduction in specific amplification was observed. From this result, by detecting all or a part of the base sequence corresponding to the Pec cell B non-conserved region, or the base sequence complementary thereto, Pectobacterium sp. It became clear that it could be detected. In addition, a PCR primer for amplifying a gene fragment of the Pec cell B non-conserved region or a gene fragment of the Pec cell B non-conserved region, a PCR primer primer for amplifying a Ddi pectate lyase gene fragment or a pectic acid of Ddi It was revealed that the pathogenic bacteria of the black spot disease of potato can be comprehensively detected or identified by using the lyase gene fragment in a single reaction system.

<Comparative Example 1> Preparation of Macroarray Membrane Based on Conventional Detection System As a primer conventionally used for detecting Ddi, a target for amplification of the pectate lyase gene of Ddi (Fw; ADE1 (SEQ ID NO: 27), Rv; ADE2 (SEQ ID NO: 29)) were prepared. Further, as a conventionally used primer for detecting Pw, a Pw universal rice primer (URP) to be amplified (Fw; contig1F, Rv; contig1R, de Haan et al. (2008), European Journal) of Plant Pathology 122 p561-569). In addition, as a primer conventionally used for detecting Pa, a Pa-specific DNA probe is used as an amplification target (Fw; ECA1f, Rv; ECA2r, de Bore & Ward (1995), Phytopathology 85 p854-858). Was prepared. In addition, as primers conventionally used for detecting Pcb, those which target the intergenic spacer region of 16S rRNA and 23S rRNA (16S-23S IGS) (Fw; BR1f, Rv; L1r, Duarte et al.) . (2004), Jounal of Applied Microbiology 96 p535-545). The sequence is shown in Table 5.

In addition, primers were designed to amplify a partial sequence of a gene to be amplified with the primers shown in Table 5 so as to be fixed to a macroarray membrane. The sequences of these primers are shown in Table 6.

The genomic DNA of each strain of Ddi, Pw, Pa and Pcb was mixed for each strain to prepare the genomic DNA of the strain. Using this genomic DNA as a template, PCR was performed using the primer set in Table 6 to obtain a PCR product. Subsequently, according to the test method (4), a membrane for a macroarray on which a Ddi pectate lyase gene amplified fragment, a Pw URP amplified fragment, a Pa-specific DNA probe amplified fragment, and a Pcb 16S-23S @ IGS amplified fragment were spotted (conventional method). A membrane for detecting type 4 strains) was prepared. The spot position on each membrane was as shown in FIG.

<Example 7> Detection in diseased potato tissue (1) Extraction of DNA from potato tissue Potato seedlings inoculated with Pw Ecc-2 strain and showing symptoms of spoilage of potatoes (Hokkaido Prefectural Research Organization) Agricultural Research Division Tokachi Agricultural Experiment Station). As shown in FIG. 8, sections were collected from three different tissues of the potato, DNA was extracted by the CTAB method, a 100 ng / uL DNA solution was prepared in sterile water, and this was used as the DNA of the diseased tissue DNA1. ～ 3. In addition, a section was collected from a healthy potato seedling and a DNA solution was prepared in the same manner, and this was used as a healthy tissue DNA.

(2) Detection by Macroarray Membrane [2-1] Macroarray Membrane of Comparative Example 1 The DNA solution of Example 7 (1) ([1] diseased tissue DNA1, [2] diseased tissue DNA2, [3] PCR was performed using the diseased tissue DNA 3 and [4] healthy tissue DNA) as templates. [5] PCR was similarly performed on a control sample containing no template DNA. As the primer, a mixture of eight kinds of primers shown in Table 6 (SEQ ID NOS: 30, 31, and 48 to 53) was used. In addition, each Fw primer used was previously labeled with biotin. Thereafter, the presence or absence and size of the PCR product were confirmed by agarose gel electrophoresis. In addition, the presence / absence and position of a signal were confirmed by using the membrane of Comparative Example 1 for detection of four conventional bacterial species. The result is shown in FIG.

As shown in FIG. 9, when the template was diseased tissue DNAs 1 to 3 ([1] to [3]), no band was confirmed by electrophoresis, and the URP amplified fragment of Pw was purified using the conventional type 4 membrane detection membrane. Very little signal was detected at the spotted position, but the intensity was not sufficient to determine whether it was positive or negative. In both cases where the template was healthy tissue DNA ([4]) and the control sample without template DNA ([5]), no band was confirmed by electrophoresis, and the conventional four-species detection membrane was not used. No signal was detected. In other words, it has been found that it is difficult to detect or discriminate Ddi, Pw, Pa and Pcb by a single reaction system using conventional amplification target genes and PCR primers.

[2-2] Membrane for macroarray of the present invention DNA solution of Example 7 (1) ([1] diseased tissue DNA 1, [2] diseased tissue DNA 2, [3] diseased tissue DNA 3, [4] healthy tissue DNA ) Was used as a template to perform PCR. [5] PCR was similarly performed on a control sample containing no template DNA. The primer used was a mixture of the primer sets [i] to [vi] of Example 3 (seven types of primers of SEQ ID NOS: 21 to 24 and 27 to 29). The primers of SEQ ID NO: 27 and SEQ ID NO: 28 used had been labeled with biotin in advance. Thereafter, the presence or absence and size of the PCR product were confirmed by agarose gel electrophoresis. Further, the presence / absence and position of the signal were confirmed by applying to the membrane for detecting four bacterial species in Example 4. The result is shown in FIG.

As shown in FIG. 10, when the template was diseased tissue DNAs 1 to 3 ([1] to [3]), a band corresponding to 755 bp was confirmed by electrophoresis, and Pw cellulase B gene amplification was performed even on the membrane for detecting four bacterial species. A signal was detected at the position where the fragment was spotted, and no signal was detected at other positions. In addition, in both cases where the template was healthy tissue DNA ([4]) and the control sample without the template DNA ([5]), no band was confirmed by electrophoresis, and the conventional four-species detection membrane was not used. No signal was detected. That is, even when the DNA extracted from the diseased tissue was used as a template, a sufficient amount of DNA for detecting pathogenic bacteria could be specifically amplified, and non-specific amplification and a decrease in specific amplification were not observed. From this result, by detecting all or a part of the base sequence corresponding to the Pec cell B non-conserved region, or the base sequence complementary thereto, Pectobacterium sp. It became clear that it could be detected.

Claims

A nucleic acid probe for detecting a bacterium belonging to the genus Pectobacterium, comprising a whole or a part of the base sequence of (i) or (ii) below;
(I) a nucleotide sequence corresponding to the 1st to 40th amino acid sequence from the amino terminal of cellulase B of a bacterium belonging to the genus Pectobacterium;
(Ii) a base sequence complementary to (i).
核酸 The nucleic acid probe according to claim 1, wherein the amino acid sequence at positions 1 to 40 from the amino terminus of the cellulase B of the genus Pectobacterium is the sequence shown in SEQ ID NO: 1.
The bacterium of the genus Pectobacterium is Pectobacterium wasabier (Pw), Pectobacterium carotobolam subspecies carotobolum (Pectobacterium carotovorum subspecies carotovorum (Pcc)), Pectobacterium atroseptica (Pectobacterium atroseptica and Pectobacterium atroseptica). The nucleic acid probe according to claim 1, wherein the nucleic acid probe is at least one selected from the group consisting of bacteria, carotovorum, subspecies, and brassienense (Pcb).
(4) The nucleic acid probe according to any one of (1) to (3), wherein the bacterium belonging to the genus Pectobacterium is a pathogenic bacterium of black spot or soft rot of potato.
A method for detecting a bacterium belonging to the genus Pectobacterium, comprising the step of detecting the nucleic acid probe according to any one of claims 1 to 4.
The detection method according to claim 5, further comprising a step of amplifying the nucleic acid probe by a polymerase chain reaction (PCR).
(4) A kit for detecting a bacterium belonging to the genus Pectobacterium, comprising the nucleic acid probe according to any one of (1) to (4).
(4) A carrier for detecting a bacterium belonging to the genus Pectobacterium, on which the nucleic acid probe according to any one of (1) to (4) is immobilized.
(4) A PCR primer for detecting a bacterium belonging to the genus Pectobacterium, comprising all or a part of the nucleotide sequence corresponding to the amino acid sequence of amino acids 1 to 40 from the amino terminus of cellulase B of the bacterium of the genus Pectobacterium.
(10) The PCR primer according to (9), wherein the amino acid sequence at positions 1 to 40 from the amino terminus of the cellulase B of the genus Pectobacterium is the sequence shown in SEQ ID NO: 1.
The bacterium of the genus Pectobacterium is Pectobacterium wasabier (Pw), Pectobacterium carotobolam subspecies carotobolum (Pectobacterium carotovorum subspecies carotovorum (Pcc)), Pectobacterium atroseptica (Pectobacterium atroseptica and Pectobacterium atroseptica). The PCR primer according to claim 9 or 10, wherein the PCR primer is one or more selected from the group consisting of Bacterium {Carotovorum} subspecies {Pectobacterium {carotovorum} subspecies {brasiliense} (Pcb)).
The PCR primer according to any one of claims 9 to 11, wherein the bacterium belonging to the genus Pectobacterium is a pathogenic microorganism of black spot or soft rot of potato.
A PCR primer set comprising the following first PCR primer and the following second PCR primer used in pairs with the first PCR primer;
A first primer: the PCR primer according to any one of claims 9 to 12,
Second primer: a PCR primer containing a part of a base sequence complementary to a base sequence corresponding to the amino acid sequence of amino acids 41 and on from the amino terminus of cellulase B of a bacterium belonging to the genus Pectobacterium.
(14) The PCR primer set according to (13), wherein the amino acid sequence from the 41st amino acid of the cellulase B of the bacterium belonging to the genus Pectobacterium is the sequence shown in SEQ ID NO: 2.