KR100514431B1 - Primer Sets for Specifid Identification of a Pathogen Xanthomonas campestris pv. campestris, and Specific-Identification Methods of the Pathogen using the Primers - Google Patents

Abstract

The present invention relates to a rapid, quick heukbu pathogen detection method using a PCR primer set, and the primer set that can accurately detect the specific DNA fragment of heukbu pathogen (Xanthomonas campestris pv. Campestris) of cruciferous acids. The primer set provided in the present invention specifically acts on the N-terminal part of the hrpF gene of the black pathogen.

Since the primer set according to the present invention forms an amplification band only for the black pathogen, the presence of the black pathogen can be detected at a DNA level within a short time, thereby enabling the rapid response to the prevention, treatment and post-treatment of the black disease. .

Description

Primer sets for specific detection of cruciferous pathogens and methods for detecting them using the same {Primer Sets for Specifid Identification of a Pathogen Xanthomonas campestris pv. campestris, and Specific-Identification Methods of the Pathogen using the Primers}

The present invention relates to a specific DNA fragment of heukbu pathogen (Xanthomonas campestris pv. Campestris) of cruciferous flow quickly, the PCR primers (primer) that can be detected accurately set. The primer of the present invention is a pair of primers made of 20mer which detects only cruciferous black pathogen by using polymerase chain reaction (PCR) method.

Heukbu pathogen (Xanthomonas campestris pv. Campestris) has penetrated the radish, cabbage, cauliflower, turnips, bok choy and many cruciferous leaf, stem, flower stalk and roots of plants to cause the black bubyeong. Among them, cabbage, broccoli and cauliflower are the most damaging. The pathogen survives in the soil in which the diseased plant grew and then penetrates into new plants or spreads through the seeds of the diseased plant. Invasion is done by hand in the leaves and through the wound in the roots.

In seedlings immediately after germination, the whole cotyledons wither and die after blackening from the apical tip of cotyledons. In the case of cauliflower, the stem of the branch is infected and the catheter is blackened, and when the symptoms become severe, the cortex rots and then falls off and is exposed to the duct, and the ground part may wither and die. In the case of leaves and roots, the conduit becomes black, which leads to a significant drop in commodity value. Radishes begin with yellowing at the edges of the leaves, then the leaves or veins turn black and eventually the entire leaf becomes black.

To date, there is no bactericide specific to this bacterium, and currently, an alternative drug called benomil is used. However, it does not give much help to seed QC (Quality Control).

At present, a method of using a selective medium or detecting the physiological and biochemical characteristics of pathogens has been made to detect the black pathogens.

However, these methods can be diagnosed only when the pathogen is isolated and cultured for a long time and the concentration of the isolate is significantly higher (above 103 CFU), and because of similar physiological characteristics, the same genus ( Xanthomonas ) or the same species ( Xanthomonas campestris This is a problem that can only be achieved by a professional taxonomy because it is very difficult to accurately identify in relation to the various non-pathogenic subspecies in.

On the other hand, ELISA (Enzyme-Linked ImmunoSorbent Assay) diagnostic method using the antibody reaction, although the detection sensitivity is high, there is a problem that the procedure is complicated, expensive analysis cost, and time-consuming identification method.

Therefore, the present inventors have attempted to diagnose the black pathogen by using a method of identifying a species at the gene (DNA) level by the recent development of molecular biological techniques.

In general, the most widely used method for identifying species at the genetic level is DNA fingerprinting. This method is further divided into a restriction fragment length polymorphism (RFLP) assay, a method using a difference in ribosome constituent nucleic acid sequences, and a polymerase chain reaction (PCR) diagnosis using a specific primer. Among them, the RFLP method is a method of detecting DNA polymorphism by using a probe labeled with a radioisotope after cutting DNA by restriction enzymes, transferring the fragments to a nylon membrane. When the PCR method is the genome gene of a bacterium specific DNA fragments (primers) consisting 10~20bp after contact (annealing) to (genomic DNA) was added a thermostable DNA polymerase (Taq polymerase), and the synthesis reaction performed repeatedly, primer The amplified region rapidly amplifies, and the PCR amplification product is electrophoresed on an agarose gel or an acrylamide gel, and the DNA is ethidium bromide and silver stained. It is a method of detecting and diagnosing DNA polymorphism of bacterial species such as the presence or absence of DNA bands or differences in morphology after staining. Currently, such a method for detecting bacteria by PCR is used to diagnose bacterial diseases of humans or animals, and PCR diagnostic kits for detecting food poisoning-related pathogenic bacteria, such as Escherichia coli and cholera bacteria, have been developed. In addition, in foreign countries, PCR diagnostic methods for various pathogens, including Erwinia amylovora, have been developed for plant pathogens, which are used for the diagnosis of diseases and the import and export of agricultural products.

Actually, the time required for PCR diagnosis is very short compared to other diagnostic methods, and is completed within 8 hours. The cost of diagnosis is also low, and many samples can be tested simultaneously. We attempted to diagnose the black pathogen of Cruciferaceae.

The present invention is a specific DNA fragment of heukbu pathogens of cruciferous acids (Xanthomonas campestris pv. Campestris) in a quick, object of the present invention to provide a PCR primer set that can be correctly detected.

In addition, another object of the present invention is to provide a detection method capable of quickly and accurately determining the authenticity of infection of the black pathogen at the DNA level using the primer set.

The present invention for achieving the above object, the cruciferous black disease disease pathogen Xanthomonas campestris pv. of campestris hrpF gene (SEQ ID NO: 1) relates to a primer set capable of specifically amplifying all or part of 1 to 650 bp. Furthermore, the present invention relates to a primer set capable of specifically amplifying all or part of the bp 90-625 gene. In the present invention, the primer set is a DNA fragment prepared based on SEQ ID NO: 1, and includes a primer set having a nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3.

In another aspect, the present invention, cruciferous black disease pathogen Xanthomonas campestris pv. The present invention relates to a method for detecting campestris . That is, the present invention, (A) isolating plant samples to extract the total DNA, (B) PCR amplification of the extracted DNA using the primer set according to any one of claims 1 to 3. (C) PCR amplification result Xanthomonas campestris pv. of campestris hrpF gene (SEQ ID NO: 1) provides a method for detecting cruciferous black disease, including the step of confirming whether all or part of the 1 to 750 times bp amplified.

The invention ① heukbu pathogenic bacteria (Xanthomonas campestris pv. Campestris) with this in that the (Xanthomonas) microorganisms in there with a protein gene hrpF involved in host recognition in common and ② sequencing a substantial homology of these hrpF gene but Only the nucleotide sequence corresponding to the N-terminal region of the hrpF protein has a high difference. ③ In particular, the nucleotide sequence corresponding to the N-terminal region of the hrpF protein of the black pathogen is systematically and biochemically related to the black pathogen. It is based on the fact that it has a very different specificity from other microorganisms, that is, different strain microorganisms belonging to the same species, microorganisms of the recent genus of the same genus ( Xanthomonas ) and microorganisms belonging to other genera.

The primer prepared in the present invention is a primer set of 20mer size that detects only the black pathogen by using polymerase chain reaction (PCR) method. The primer set is a single band of approximately 540bp is formed only for cruciferous heukbu pathogen (Xanthomonas campestris pv. Campestris) (i.e., amplified) and does not form a band at all, the similar species and other pathogenic strains in other Xanthomonas genus. This amplification is confirmed whether it is possible to determine the authenticity of infection quickly, accurately heukbu pathogen (Xanthomonas campestris pv. Campestris) at the DNA level it is possible to sufficiently in about 6 hours.

Hereinafter, the present invention will be described in more detail with reference to Examples and Application Examples. The following examples and application examples are merely illustrative of the present invention, and thus, the nature and content of the technical spirit of the present invention are not changed or reduced. It will be apparent to those skilled in the art that various modifications can be made with reference to the technical spirit of the present invention and the following examples and applications.

Preparation: related microorganisms and their culture conditions

Xanthomonas campestris pv. Other microorganisms, including campestris, were distributed by the Korean Agricultural Culture Collection (KACC) with Suwon Institute of Agricultural Science and Technology Genetic Resources and the Belgian Co-ordinated Collections of Micro-organisms (BCCMTM) in Belgium. The microorganisms, fungi and their sources used are summarized in Table 1. The meaning of each symbol in the table is as follows: a: KACC, Korean Agricultural Culture Collection, Korea (http://mgd.niast.go.kr); ATCC, American Type Culture Collection, USA; LMG, The Belgian Co-ordinated Collections of Micro-organisms (BCCMTM), Belgium. b: Other Collection No.

Xanthomonas strains were YGC medium [D-(+)-glucose 2.0%, CaCO3 2.0%, Yeast extract 1.0%: 1L], Fusarium spp. Was potato dextrose agar (PDA, Difco), Escherichia coli was LB agar (Sambrook and Maniatis). In 1989), other bacteria were cultured in nutrient agar (NA, Difco).

Example 1 by microorganism hrpF  Confirmation of Gene Existence

The DNA dot blot was used to determine the presence of the hrpF gene in several strains of the same genus, Xanthomonas , and other microorganisms of the same genus, which are thought to be biochemically similar to the pathogens and the microorganisms. It was confirmed through.

Total DNA of Xanthomonas strain was extracted by CTAB method, and total DNA of other microorganisms was extracted using genomic DNA extraction kit (Genomic-tips) of Qiagen (Hilden, Germany). 20 ng of genomic DNA extracted from each microorganism was dispensed on Hybond-N + nylon membrane (Amersham pharmacia biotech, UK), respectively and fixed using UV cross-link. Xanthomonas campestris pv. Gene fragments amplified from campestris were labeled with radioisotope [32P] dCTP using Ladderman ™ Labeling kit (Takara, Japan) according to the random prime method. Hybridization of radioisotope labeled probes with each microbial genomic DNA immobilized on a Hybond-N + nylon membrane was followed by hybridization buffer (NaCl 0.75M, sodium citrate 75mM, 0.5% SDS, 0.1% BSA, 0.1) at 65 ° C for 18 hours. % Ficoll, 0.1% polyvinylpyrrolidone and denatured salmon sperm DNA (50µg / ml). The hybridized nylon membrane was washed twice (at 10 min each) at room temperature with 2 × SSC buffer containing 0.1% sodium dodecyl sulfate (SDS) and 65 × 0.1 × SSC containing 0.1% sodium dodecyl sulfate (SDS). Wash twice again at 15 ° C. (15 min each). After hybridization and washing, the nylon membrane was developed after exposure to X-ray film (AGFA, Belgium) at -70 ° C. for one day (FIG. 1).

As a result, as shown in the figure, it was confirmed that the hrpF gene is present in the total DNA of all strains belonging to Xanthomonas . On the other hand, it was not found at all in the microorganisms of the other genus.

Judging from these results, hrpF gene was identified specific genes that exist in Xanthomonas only microorganism belonging to Xanthomonas.

Table 2 shows the microorganisms indicated by each number in the figure.

Example 2: hrpF  Gene Sequencing

The degree of mutation of the hrpF gene identified as specific to the genus Xanthomonas was confirmed.

Xanthomonas strains using the DNASTAR software package (DNASTAR Inc.) ( X. c. Pv. Campestris, X. a. Pv. Citri, X. o. Pv. Oryzae, X. c. Pv. Vesicatoria) (Da Silva etc. Amino acid or base sequence of each hrpF derived from Ochiai et al. (2001, Noel et al. 2002) and the amino acid or base sequence of nolX derived from Mesorhizobium loti (Kaneko et al. 2000) known to have similar sequence and biochemical properties to hrpF- Analyzed. The nucleotide sequence and amino acid sequences referred to in the present invention was used to investigate the GeneBank database accession number that is AAM35285.1 (X. c. Pv. Citri ), BAB07869.1 (X. o. Pv. Oryzae), AAM40515 0.1 a (X. c. pv. campestris) , AAB86527.1 (X. c. pv. vesicatoria), NP_106860.1 (Mesorhizobium loti).

A comparative analysis of the amino acid sequence of the Hrp F gene of the Xanthomonas strain and the nolX gene of M. loti is shown in FIGS. 2 and 3, and the base sequence of the Hrp F gene of the Xanthomonas strain is shown in SEQ ID NO: 1.

As can be seen in the figure , the hrpF gene and nolX gene showed genetic similarity in the range of 3.5 to 48.7% in total, while the strains belonging to the same genus ( Xanthomonas ) were X. a. pv. citri , X. o. pv. oryzae , X. c. pv. HrpF amino acid sequence of vesicatoria is X. c. pv. The HrpF amino acid sequence of campestris showed a high degree of homology (over 60%). It can be seen. Specifically, the black disease pathogen ( X. c. Pv. Campestris ) is X. o. pv. 63% in oryzae , X. a. pv. citri to 63.1%, X. c. pv. 64.5% similarity with vesicatoria . In particular X. o. pv. oryzae and X. c. pv. vesicatoria showed a very high similarity of 92.5%.

However, as a whole, a considerable degree of dissimilarity was confirmed in the nucleotide sequence corresponding to the N-terminal part (in the range of 1 to about 650 bp, and thus 1 to about 217aa for the protein). Hereinafter, in the present invention, the sequence of the above portion is referred to as "specific sequence".

Therefore, even in spite of the gene of hrpF hrpF gene and the closely related microorganisms heukbu pathogens is very high similarity in Xanthomonas, and specifically to the nucleotide sequence on the N- end with a non-like sequence is concentrated ( "sequence-specific") compared to the - Analysis shows that the black pathogen can be detected specifically for other related microorganisms.

2 and 3, the microorganism corresponding to each code is shown in the following table.

Example 3 Preparation and Specificity of Primer for Amplification of "Specific Sequence"

Since the "sequence" part mentioned above may be a sequence which can distinguish the hrpF of the black pathogen from among the microorganism hrpF of Xanthomonas genus, only the examination of the "sequence sequence" part detects the black disease pathogen among microorganisms of Xanthomonas genus Check if it can be done.

(1) Preparation of primer set for amplification of "special sequence"

Among the microorganisms of the genus Xanthomonas, a primer set capable of amplifying the "sequence" identified as having only the black disease pathogen was produced.

X. c. pv. A HFF primer having the same sequence as the nucleotide sequence of Nos. 90 to 109 of the N-terminal region of the hrpF gene of campestris and an antisequence of the nucleotide sequences of 605 to 624 were prepared (see FIG. 4). The sequence of the primer is as follows.

① HFF Primer: 5'CGATTCGGCCATGAATGACT-3 '20mer (SEQ ID NO: 2)

② HFR primer: 5'CTGTTGATGGTGGTCTGCAA-3 '20mer (SEQ ID NO: 3)

These primer sets are expected to amplify gene fragments of 535 bp in length.

(2) Confirm the specificity of the primer set

Xanthomonas campestris pv. Of HFF and HFR primers . In order to confirm the specificity of campestris , PCR was performed on Xanthomonas strains and other microorganisms.

PCR amplification reaction was carried out using PTC-200TM thermocycler (MJ research, Watertown, mass), each reaction of Tris-HCl 10mM, KCl 50mM, MgCl2 1.5mM, gelatin 0.01%, dNTP 0.2mM, primer respectively PCR mixture containing 10pM, Taq polymerase 2unit (Promega, Madison, Wis.) Was carried out in a total amount of 50ul. The amount of genomic DNA of some microorganisms added to the PCR mixture was about 50ng.

The reaction is denatured at 94 ° C. for 10 minutes, repeated 25 times at 94 ° C. 15 seconds, 60 ° C. 30 seconds, and 72 ° C. 30 seconds, followed by reaction at 72 ° C. for 7 minutes. After the amplification reaction, 8 μl of the PCR product was electrophoresed on agarose gel at 1% concentration and then stained with ethidium bromide, and confirmed by UV transilluminator.

As a result of the reaction X. c. pv. Only campestris was amplified and other Xanthomonas strains and microorganisms were not amplified by PCR products (FIG. 5). HFF and HFR primers were mixed under 50ng DNA concentration extracted from each microorganism and appropriate conditions (50μl PCR reaction containing 1.5mMgCl2, 0.2mM each of dNTP, 10pM of each primer, 2units of Taq polymerase, and 5μl of 10X PCR buffer). When PCR was performed X. c. pv. 535bp long gene fragment was amplified from campestris . In addition, PCR results were obtained using Perkin Elmer 9600 thermal cyclers (Perkin Elmer International, Rotkreuz). In addition, PCR amplification using Taq polymerase enzymes sold by other manufacturers (Promega, Takara, Toyobo) was also possible.

Therefore, by performing PCR amplification experiments using primer sets (eg, HFF and HFR primer sets) capable of amplifying all or part of the aforementioned "sequence sequences", it is possible to specifically detect the black pathogen. Clearly confirmed.

Arrows in the figure are shown in Xanthomonas campestris pv. Specific PCR fragments commonly present in campestris strains are shown. Table 4 shows the microorganisms indicated by each number in the figure.

Application example: field application experiment

When the PCR amplification using the primer set according to the present invention from the cabbage leaves infected with the actual black rot pathogen Xanthomonas campestris pv. It was confirmed whether the campestris can be detected.

In order to confirm the specificity of the HFF and HFR primers prepared by the examples for Xanthomonas campestris pv. Cabbage leaves were collected and subjected to PCR amplification. Infected and uninfected cabbage tissues were cut to size using sterile scissors and total genomic DNA was extracted using CTAB method. 20 ng of genomic DNA extracted from cabbage tissue was taken and PCR amplification was carried out in the same manner as described in Example 2 (2) using HFF and HFR.

Blot photographs of the PCR amplification results are shown in FIG. 6. As can be seen in the figure, only the cabbage tissues suspected of black-brown disease were found to be PCR-amplified gene fragments in the same way as positive-control ( Xanthomonas campestris pv. Campestris ). As a result, pathogens that infected cabbage were Xanthomonas campestris pv . It was confirmed that the campestris .

Therefore, PCR amplification experiments using primer sets (eg, HFF and HFR primer sets) capable of amplifying all or a portion of the aforementioned "sequence sequences" are specific for whether the cruciferous plants are infected with the black pathogen. It has been clearly confirmed that the detection can be done quickly and quickly.

Table 5 shows the microorganisms indicated by each number in the figure.

The present invention by performing a PCR amplification method using the specific DNA fragment PCR primer sets that can be quickly and accurately detected (the "specific sequences") of the pathogenic bacteria (Xanthomonas campestris pv. Campestris), which causes the black bubyeong cruciferous acids In addition, it enables rapid, precise and easy diagnosis of black disease infection.

In addition, the present invention can be usefully used to track the path of infection of the black pathogens, or to diagnose the infection of the black pathogens on seeds or seeds, which contributes to preventing and controlling the disease.

1 is a DNA dot-blot results showing the presence of hrpF gene in various microorganisms.

2 is an amino acid sequence comparison chart of the hrpF protein of the Xanthomonas strains and the nolX protein of Mesorhizobium loti .

Figure 3 is a diagram analyzing the genetic flexibility of the hrpF gene of the Xanthomonas strains and the nolX gene of Mesorhizobium loti . a is the genetic similarity analysis in percent and b is the genetic relationship analysis.

Figure 4 is a diagram showing the binding site of the primer corresponding to a specific site of the hrpF gene of the black pathogen.

5 is a blot photograph showing the results of specific PCR amplification of various microorganisms using the primer set according to the present invention.

6 is from the cabbage infected with the black pathogen using the primer set according to the present invention X. campestris pv . Blot picture showing the detection results of campestris .

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Primer Sets for Specifid Identification of a Pathogen Xanthomonas campestris pv. campestris, and Specific-Identification Methods of the Pathogen using the Primers <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 535 <212> DNA <213> Xanthomonas campestris <220> <221> gene (222) (1) .. (535) <400> 1 cgattcggcc atgaatgact ccgatctgtt gctggcgatg gacaacctgt ttctgcagca 60 gatttaccgt ttgattgccg ccacctacgg caatacgtcg ctcaatgggc caggcagtgg 120 tattccgggg cttgacacgc cttcagcaga cgaccttcaa gccagccagc cgattgagaa 180 acggacctcc tggccgacgc tatcggctcc gttcaatgtc aaggacatca agggatccag 240 gttgccgcct gcggtggatg ggtcatcggt gacctgggag ggcggcacgc tcaccccatc 300 ggagcttcag attgtttcga cactcaatca gcacaaagac aagacgccgc tggagttcgc 360 aaaactcgac gacaagatca atgatccatc tacacctccg gatctgaaga gtgcgctgca 420 aggcttgcag aaagatccgc gtcttttctt cgccatcggt tcgcagggcg acggcaaatg 480 cggcggcaag atcaaggcgg gtgatttgtg ggattttgca gaccaccatc aacag 535 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for identifying hrpF sequence <400> 2 cgattcggcc atgaatgact 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for identifying hrpF sequence <400> 3 ctgttgatgg tggtctgcaa 20

Claims (4)

delete delete Xanthomonas campestris pv. of campestris A primer set for detecting a cruciferous black disease pathogen having a nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3 capable of specifically amplifying the hrpF gene (SEQ ID NO: 1). (A) isolating plant specimens to extract total DNA; (B) PCR amplifying the extracted DNA using the primer set according to claim 3; (C) PCR amplification result Xanthomonas campestris pv. of campestris Checking whether or not the hrpF gene (SEQ ID NO: 1); Cruciferous black disease disease detection method comprising a.