KR101424103B1 - Primer sets for detecting Xanthomonas campestris pv. campestris and detection method using the same - Google Patents

Abstract

The present invention relates to a method for treating Xanthomonas campestris pv. campestris) using the primer set and the primer set for the specific detection of infected seeds and cruciferous crops heukbu pathogen (Xanthomonas campestris pv. campestris ) detection method.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer set for detecting cruciferous black rot fungi, and a method for detecting the same. campestris and detection method using the same}

The present invention relates to a primer set for detection of cruciferous and fungal pathogens and a detection method using the primer set. More particularly, the present invention relates to a method for detecting cruciferous black pathogens including the primer set, a composition for detection, and a kit for detection.

Black pathogens ( Xanthomonas campestris pv. campestris ) infiltrate leaf, stem, flower bud, and root of many cruciferous plants such as radish, cabbage, carly flower, turnip, In particular, blacks have the greatest damage from cabbage, broccoli and carlyflower. Black pandemic is a serious disease that occurs after a low temperature and high rainfall in May and around September to October, and when it occurs in a young seedling immediately after germination, it begins to darken from the cotyledon and eventually it will die. It grows mainly from the lower leaf in the main field and makes a thin V - shaped yellow lesion around the vein around the leaf. In the case of severe damage, the bacteria invade the stem, causing the inside of the bulb to darken and occur in the entire field.

Currently, there is no germicide specific to this bacterium, and currently an alternative drug called benomyl is used. However, it does not provide much help for seed quality control (QC) and the bacterial growth rate is slow during cultivation, For the detection of black pathogens, a selection medium is used, or the physiological and biochemical characteristics of the pathogens are tested.

However, these methods can be diagnosed only when the isolation and cultivation of the pathogenic bacteria takes a long time and the concentration of the isolated bacteria is high (above 10 ³ CFU), and because of similar physiological characteristics, the same genus ( Xanthomonas ) Xanthomonas campestris ), it is very difficult to pinpoint the exact identity.

On the other hand, although the ELISA (Enzyme-Linked Immunosorbent Assay) method using the antibody reaction has a high detection sensitivity, it requires complicated procedures, requires expensive analysis cost, and has a problem that it takes a long time to identify.

Korean Patent Registration No. 10-0514481

In order to solve the above-mentioned problem, the present invention provides a method for treating Xanthomonas campestris pv. campestris) infected seeds and specific primers and samples in cruciferous heukbu pathogens nano-concentration using the primer set for the detection of plant (Xanthomonas campestris pv. campestris .

The present invention relates to the use of the cruciferous black pathogens ( Xanthomonas < RTI ID = 0.0 > campestris pv. campestris ) detection primer set.

The present invention also relates to the use of said primer set for the isolation of cruciferous black pathogens ( Xanthomonas campestris pv. campestris ).

The present invention also relates to a method of treating or preventing Xanthomonas < RTI ID = 0.0 > campestris pv. campestris ).

The present invention also relates to the use of said primer set for the isolation of cruciferous black pathogens ( Xanthomonas campestris pv. campestris ) detection kit.

According to the present invention, the cruciferous black pathogens ( Xanthomonas campestris pv. Campestris detection primer set and the detection method using the primer set, it is possible to check the disease and health of the seeds and the crops, and it is possible to confirm the infection quickly within 1 hour through the above test.

FIG. 1 is a schematic view of an embodiment of the present invention. ≪ RTI ID = 0.0 > Xanthomonas campestris pv. campestris ) lipoprotein (blastn search).
Figure 2 shows electrophoretic images after PCR using XCC202F and XCC202R primer sets.
FIG. ≪ RTI ID = 0.0 > Xanthomonas campestris pv. campestris ) was detected by real-time PCR.

The present invention relates to a medicament comprising a cruciferous black pathogen ( Xanthomonas campestris pv. campestris ) detection primer set.

As used herein, the term "primer " refers to a short nucleic acid sequence that can form a base pair with a complementary template and serves as a starting point for template strand copying.

The present invention relates to the use of the cruciferous black pathogens ( Xanthomonas < RTI ID = 0.0 > campestris pv. campestris ) detection primer set, wherein the primer set can have the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3. The primer set includes the cruciferous black pathogens ( Xanthomonas campestris pv. a campestris) may be a primer that can be detected specifically. The primer set according to the present invention is a set of Xanthomonas campestris pv. (e.g., addition, deletion, substitution) within a range that does not affect the specific detection of the test compound (e.g., campestris ).

The primer set according to the present invention is a set of Xanthomonas campestris pv. campestris ). < / RTI > More specifically, the primer set includes a gene coding for lipoprotein (registered trademark: GenBank accession No. CP000050.1, region: 1839433-1840443, protein ID: AAY48596.1) represented by SEQ ID NO: 1 of cruciferous black rot fungus A 202 bp fragment can be specifically amplified.

The primers of the present invention can be used as a primer of restriction fragment length polymorphism (RFLP), Randomly Amplified Polymorphic DNA (RAPD), DNA Amplification Fingerprinting (DAF), Arbitrarily Primed PCR, Sequence Tagged Site (EST) ), Sequence Characterized Amplified Regions (SCAR), Inter-Simple Sequence Repeat (ISSR), Amplified Fragment Length Polymorphism (AFLP), Cleaved Amplified Polymorphic Sequences (CAPS), and Single-Strand Conformation Polymorphism Can be designed to be suitable for various known DNA analyzes.

The present invention also relates to the use of a primer set of SEQ ID NO: 2 and SEQ ID NO: 3 for the production of Xanthomonas campestris pv. campestris ).

The primer set of the present invention was used to isolate Xanthomonas campestris pv. a method capable of detecting a campestris) is, PCR (Polymerase Chain Reaction) analysis, DNA sequencing (DNA sequencing), the hybridization of the microarray (microarray), PCR-RFLP ( Restriction Fragment Length Polymorphism) analysis, RAPD (Randomly Amplified Polymorphic DNA, DNA Amplification Fingerprinting (DAF), Arbitrarily Primed PCR, Sequence Tagged Site (STS), Expressed Sequence Tag (EST), Sequence Characterized Amplified Regions (SCAR) , Amplified Fragment Length Polymorphism (AFLP), Cleaved Amplified Polymorphic Sequences (CAPS), Single-Strand Conformation Polymorphism (PCR-SSCP), Northern hybridization Southern hybridization and Western hybridization Western hybridization, and the like, but are not limited thereto. In one embodiment of the present invention, the PCR reaction using the primer set of the present invention showed that Xanthomonas campestris pv. campestris ) can be detected specifically. In one embodiment of the present invention, the PCR reaction was carried out by denaturing at 95 ° C for 5 minutes, repeating 35 times at 95 ° C for 60 seconds, 64 ° C for 30 seconds, 72 ° C for 60 seconds, and 72 ° C for 10 minutes .

The present invention also relates to the use of said primer set for the isolation of cruciferous black pathogens ( Xanthomonas campestris pv. campestris ).

The present invention also relates to the use of said primer set for the isolation of cruciferous black pathogens ( Xanthomonas campestris pv. campestris ) in the presence of a recombinant DNA polymerase chain reaction (real-time PCR). The PCR method may be a conventional real-time PCR, and in one embodiment of the present invention, a real-time PCR amplification reaction from the plant is performed at 95 ° C Denatured for 30 seconds, and repeated 45 times at 95 ° C for 5 seconds and 64 ° C for 30 seconds, followed by reaction at 65 ° C for 0.5 seconds at intervals of 10 seconds, followed by reaction at 95 ° C while increasing the reaction time.

In one embodiment, the sensitivity of the pathogen analysis according to the present invention is 50 fg, which is the lowest detectable DNA concentration in the case of DNA isolation assay, and corresponds to about 10 of the crucifer and black rot fungi, and the concentration of the pathogen is directly analyzed using a spectrophotometer If it may be about 0.1 x 10 ^-5 OD 600 nm.

The present invention also relates to a method for screening for Xanthomonas strains comprising the primers set forth in SEQ ID NO: 2 and SEQ ID NO: 3 campestris pv. campestris ).

In the composition of the present invention, the above-described primer set is used to treat Xanthomonas campestris pv. campestris) the course of the experiment (for example, required to detect: the PCR, Southern blot, etc.) and the resulting number of reagents necessary to determine, for example, PCR reaction mixture, restriction enzyme, agarose, hybridization, and / or buffer or the like required for the electrophoretic May be further included.

The composition for detection may be a composition for PCR. More specifically, the composition for detection may be a composition for real-time PCR.

In the PCR composition of the present invention, the above primer set is used to transform Xanthomonas campestris pv. campestris) the course of the experiment (for example, required to detect: the PCR, Southern blot, etc.) and the resulting number of reagents necessary to determine, for example, PCR reaction mixture, restriction enzyme, agarose, hybridization, and / or buffer or the like required for the electrophoretic May be further included.

The present invention also relates to the use of a primer set of SEQ ID NO: 2 and SEQ ID NO: 3 for the production of Xanthomonas campestris pv. campestris ) detection kit.

The present invention also relates to the use of said primer set for the isolation of cruciferous black pathogens ( Xanthomonas campestris pv. campestris ) of the present invention.

The kit for PCR includes a primer set of the present invention, and a PCR reaction was carried out using the Xanthomonas campestris pv. campestris ) can be detected. More specifically, the PCR kit may be a kit for real-time PCR.

The kit includes a set of primers according to the present invention and reagents necessary for PCR, and includes Xanthomonas campestris pv. The present invention relates to a kit for diagnosing the presence or absence of a cancerous cell .

The present invention includes a step of detecting a PCR amplification product. The detection of the amplification product can be performed by DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one method of detecting the amplification product, gel electrophoresis can be performed. Gel electrophoresis can utilize agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is carried out, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as P or S is added to the PCR reaction solution to mark the amplified product, and then the radioactivity is measured using a radioactive measuring device such as a Geiger counter or a liquid scintillation counter A liquid scintillation counter can be used to measure radioactivity.

The reagents required for the PCR kit of the present invention may be, for example, but are not limited to, primer sets, ribonuclease inhibitors, heat-resistant polymerase and reaction buffers, etc., and the reaction buffer includes both Taq polymerase buffer and PCR buffer And EF Taq polymerase can be used as the heat-resistant polymerase. PCR buffer is a compound added to the amplification reaction that alters the stability, activity, and / or longevity of one or more components of the amplification reaction by modulating the pH of the amplification reaction. The buffering agent is compatible with PCR amplification and site-specific RNase H cleavage activity. Some buffering agents are well known in the art and include tris, tricine, MOPS (3- (N-morpholino) propanesulfonic acid), and HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid ), But are not limited thereto. In addition, the PCR buffer may generally comprise KCl, MgCl ₂ , and the respective nucleotide dATP, dCTP, dGTP and dTTP. The buffers of the present invention can contain efficient reverse transcriptase-PCR or additive materials to optimize the PCR reaction.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<Examples>

Example  One. primer  Production of sets

(1) Bacterial strain and culture conditions;

The Zetamon &apos; s monoclonal antibody used in the present Example. Chem Fest lease other pathogens, including (Xanthomonas campestris pv. Campestris) is The Belgian Co-ordinated Collections of Micro -organisms (BCCM TM) and the Korean Agricultural Culture Collectio received pre-sale at (Korea Agriculture Microbial Resource Center, KACC), their source Are shown in Table 1. The mutant strains were cultured on PSA medium (Peptone: 1%, Sucrose: 1%, Sodium glutamate: 0.1%, Agar: 1.5%).

number Name NO. Geographic origin One Xanthomonas campestris pv. campestris ^T KACC 10913 U.K 2 Xanthomonas campestris pv. campestris LMG 559 U.K 3 Xanthomonas campestris pv. campestris LMG 565 Mauritius 4 Xanthomonas campestris pv. campestris LMG 567 Malawi 5 Xanthomonas campestris pv. campestris LMG 569 Uganda 6 Xanthomonas campestris pv. campestris LMG 7460 New Zealand 7 Xanthomonas campestris pv. campestris LMG 7515 USA 8 Xanthomonas campestris pv. campestris LMG 7662 Belgium 9 Xanthomonas campestris pv. campestris LMG 8005 Kenya 10 Xanthomonas campestris pv. campestris LMG 8030 France 11 Xanthomonas campestris pv. malvasecrum KACC 11127 Unknown 12 Xanthomonas campestris pv. carotae KACC 10164 USA 13 Xanthomonas campestris pv . poinsettiicola KACC 12894 New Zealand 14 Xanthomonas campestris pv. armoraciae LMG 7383 USA 15 Xanthomonas campestris pv. vesicatoria LMG 921 USA 16 Xanthomonas cucurbitae LMG 8662 New Zealand 17 Xanthomonas oryzae pv. oryzae KACC 10331 Unknown 18 Xanthomonas axonopodis pv. aurantifolii KACC 10161 Unknown 19 Xanthomonas axonopodis pv. citri KACC 10443 Korea 20 Xanthomonas axonopodis pv. begoniae LMG 551 U.K 21 Xanthomonas axonopodis pv. glycines KACC 10445 Unknown 22 Xanthomonas axonopodis pv. dieffenbachiae LMG 695 Brazil 23 Xanthomonas axonopodis pv. physeoli LMG 7455 Unknown 24 Xanthomonas axonopodis pv. phyllanthi LMG 844 Sudan 25 Xanthomonas axonopodis pv. vesculorum LMG 901 Mauritius 26 Xanthomonas axonopodis pv. vesicatoria LMG 905 Unknown 27 Xanthomonas theicola ^T LMG 8684 Japan 28 Xanthomonas translucis pv. c erelis LMG 679 USA 29 Xanthomonas translucis pv. hordei LMG 882 Canada 30 Xanthomonas translucis pv. phleipratensis LMG 843 USA 31 Xanthomonas fragariae ^T KACC 11115 USA 32 Xanthomonas cassavae ^T KACC 12914 Malawi 33 Xanthomonas kitty ^T KACC 12964 Japan 34 Pseudomonas libanesis ^T KACC 10809 Unknown 35 Pseudomonas fuscovaginae ^T LMG 2158 Japan 36 Pectobacterium atroseptocum ^T KACC 10477 USA 37 Ralstonia solanacearum ^T KACC 10814 USA 38 Escherichia coli ^T LMG 2092 Denmark

(2) Extraction of total DNA

The total DNA of the microorganisms (Table 1) was extracted using Qiagen (Hilden, Germany) genomic-DNA extraction kit (Genomic-tips).

(3) Analysis of use of Blastn program for searching specific nucleic acid sequence information

Cruciferous black pathogens ( Xanthomonas campestris pv. For the production of a primer set for specifically detecting the genomic DNA of a mammal, Campestris, for example, Zanthomonas campestris pv. registered at Genbank (http://www.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information (NCBI) . Xanthomonas campestris pv. campestris) genome information of lipoprotein (GenBank accession CP000050.1, region: through blastn and blastx program 1839433-1840443, protein ID AAY48596.1) National Center for Biotechnology Information (NCBI) Gene confirm the specificity and produce primer Respectively.

(4) Production of specific primer

Zanthomonas Campfirst v. V. Xanthomonas campestris pv. The XCC202F primer and the XCC202R primer set amplifying a fragment of 202 bp in size were prepared. The sequence of the primer is as follows.

XCC202F primer: 5'-ACCG CGTA CAGC AGCT CTTC GTC-3 '23mer

XCC202R primer: 5'-CCGC CGGG GCCT TTTT GA-3 '18mer

The primers were prepared by confirming the specificity of the sequence information of lipoprotein genes registered in GenBank through blastn program.

Example  2. PCR  Amplification reaction

(SEQ ID NO: 2: 23mer) and XCC202R (5'-CCGC CGGG GCCT TTTT GA-3 ') (SEQ ID NO: 3) amplifying a 202 bp fragment (5'- ACCG CGTA CAGC AGCT CTTC GTC- (SEQ ID NO: 1) sequence information of NCBI registered in the GenBank of NCBI (registration number: CP000050.1, region: 1839433-1840443, protein ID: AAY48596.1) was added to NCBI's blastn and blastx programs After confirming the specificity, it was produced.

PCR amplification reaction was carried out using a ^{PTC-200 TM thermocycler (MJ research} , Watertown, mass), each reaction is Tris-HCl 20 mM, KCl 100 mM, 50% Glycerol, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% Nonidet P-40, 1 mM PMSF and 0.2 mM dNTP, 20 pM each of primers and 2 units of Taq polymerase (SolGent, Korea). The amount of genomic DNA of the microorganism added to the PCR mixture was about 50 ng. The reaction was denatured at 95 ° C for 5 minutes, repeated 35 times at 95 ° C for 60 seconds, 64 ° C for 30 seconds, and 72 ° C for 60 seconds, followed by reaction at 72 ° C for 10 minutes. After amplification reaction, 10 μl of the PCR product was electrophoresed on 1.5% agarose gel, stained with ethidium bromide, and confirmed on a UV transilluminator (FIG. 2). Zanthomonas Campfirst v. V. Xanthomonas campestris pv. it was confirmed that a nucleic acid fragment having a size of 202 bp was specifically amplified only in the case of the wild type .

Example  3. Diseased Plants real - time PCR  Amplification reaction

Real-time PCR from Real Plants To measure the sensitivity of the real-time PCR, a part of the cabbage leaf (about 1 cm x 1 cm) was soaked in 1.0 ml sterilized water for 30 to 40 minutes and then 2 μl of the sample immersion liquid And then real-time PCR was performed. Real-time PCR amplification was performed using a CFX96 real-time PCR system (Bio-Rad laboratories, Inc., USA) ^. Premix The Ex Taq ™ primer was performed in a total volume of 20 μl with SYBR Green PCR mixture containing 2 pM each. The microorganisms ( Xanthomonas campestris pv. campestris KACC 10913) was about 5 ng. The reaction was carried out at 95 ° C for 30 sec, followed by 5 cycles of 95 ° C for 5 sec and 64 ° C for 30 sec. The reaction was repeated 45 times at 65 ° C for 0.5 sec and then for 10 sec. Melting peak was carried out.

As a result, as shown in Fig. 3, specific curves and peaks of each of the individual leaves were confirmed.

Fig. 3 is a graph showing the results obtained by using the primers XCC202F and XCC202R of the present invention. Xanthomonas campestris pv. campestris ), and 1 lane represents the results of real-time PCR. Xanthomonas campestris pv. campestris ) genomic DNA, and 2 lanes were obtained from Mantus monocytoph. Xanthomonas campestris pv. campestris ) cell suspension, and lane 3 to 6 were treated with Zanthomonas campestris pv. Chem plague is the infected sample in the lease (Xanthomonas campestris pv. Campestris), 7 ~ 10 lanes janto Pseudomonas Chem paste blood-less V. Xanthomonas campestris pv. campestris ), and the lane 11 is a negative control containing only distilled water. The results show that there is amplification corresponding to each of the cruciferous and black pathogens in lanes 1 to 6, and that amplification does not occur in lanes 7 to 11. In addition, Mantas Campbellis Pvt. &Lt; RTI ID = 0.0 &gt; Xanthomonas campestris pv. campestris) it was confirmed that the nucleic acid fragments only leaves infected with genomic DNA, cell suspension, cruciferous heukbu pathogen-specific amplification.

Thus, the XCC202F and XCC202R primers were obtained from Xanthomonas campestris pv. it can amplify only the specific DNA fragment of campestris), it can be seen that the use is possible as PCR primers for infection diagnosis of cruciferous heukbu pathogenic bacteria (Xanthomonas campestris pv. campestris), also produced from a specific DNA fragment of the lipoprotein gene Was found to be able to specifically detect crucifer and black rot fungi.

<110> The Foundation of Agri. Tech. Commercialization & Transfer <120> Primer sets for detecting Xanthomonas campestris pv. campestris          and detection method using the same <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 1011 <212> DNA <213> Xanthomonas campestris pv. campestris <400> 1 tcatggccgt gtgagcagca caaaggtcgt gccgtgagtg accagtccgc cgttgtcgct 60 tgcgagcaga acatgccgcg catccagcaa cgtcatgccc tcaatattgt ccggcccgac 120 cggcagcacg ataatcgtct ggctcgcaca ttgttggtct ctcgtgcagt tggaaaggtc 180 cacgcggcgc acgctcgccg acagcgcgtc cttcgctcct tgtcggcttc gctcaagaac 240 aagcagcgag ccatcgggca ggacgtccat tcccttaagc cggctgtctg gcgcttggcg 300 cacgaaggac caatgcccgc caattgcgta aagtgcgtgt tgatcctgcg gttgctgcaa 360 cagtggagac tcgggcgcag tgatcaggcc atgcgctgga tggatagcca acgattccag 420 gccgcgtccc tttttgcgat agttgttgag gtcgtcggct ggtggtggca cgggcagatt 480 gcccagcacc gtgccatctg gactgaagcg gacaatcttg ggcagagtgc cttccagcga 540 cgcaatgagc tctgtatcgc cagggatgcc gtttgtggca ttcaccagcg tcaatccctc 600 tgcgttgaat gcctcgggcg tgccacctgc aaggtgcggg tccgccaggg gcgcagcgcg 660 aattggctgg atgtcggcga tcgcatcgcc atcgagcgtg agacggaagt gaaacacgta 720 gcccgtgtcg gagaccgcgt acagcagctc ttcgtcggca tcccaggcca ggccggaaag 780 ctcggtcaga tgaatgtcgc caaccatcgc tgtgggagga aatgtgaaac gggtggatgc 840 ggcttgcatc gggccgggtt gcggatgtac tcgctcagca tgaccggtcg ccacatctgc 900 gtcctgattg cacgcagtca aaaaggcccc ggcggccagg atgatggcca gcagtgtttt 960 aggatgcatg cgcaaaacct ttcgaagagt agaggcttgt tgggcaggca c 1011 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> XCC202F <400> 2 accgcgtaca gcagctcttc gtc 23 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> XCC202R <400> 3 ccgccggggc ctttttga 18

Claims (8)

A primer set for the detection of cruciferous melanoma comprising nucleic acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 amplifying a lipoprotein gene consisting of SEQ ID NO: 1 of cruciferous black pathogens (Xanthomonas campestris pv. Campestris).
1) immersing the plant specimen in sterilized water; And
2) performing a real-time PCR by adding a primer set consisting of the nucleic acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 to the sterile water containing the sample of the step 1); And
3) detecting a black pathogen (Xanthomonas campestris pv. Campestris) by confirming the presence of a lipoprotein gene comprising SEQ ID NO: 1 amplified by the primer set.
delete delete delete delete A kit for the detection of cruciferous black disease, comprising the primer set of claim 1 which amplifies a lipoprotein gene consisting of SEQ ID NO: 1 of a cruciferous black pathogen (Xanthomonas campestris pv. Campestris). delete

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