KR20120053792A - Primer set of polymerase chain reaction for detecting the genus xanthomonas and method for detecting the genus xanthomonas using the same - Google Patents

Abstract

PURPOSE: A PCR primer pair for detecting Xanthomonas sp. microbes is provided to usefully detecting the microbes. CONSTITUTION: A PCR primer pair for detecting Xanthomonas sp. microbes is denoted by sequence numbers 1 and 2 or 3 and 4. A composition for detecting the microbes contains the primer pair. A kit for detecting the microbes contains the primer pair. A method for detecting the microbes comprises: a step of isolating DNA from a sample; a step of amplifying PCR using the primer pair; and a step of detecting electrophoresis of the PCR products.

Description

Primitive set of polymerase chain reaction for detecting the genus Xanthomonas and method for detecting the genus Xanthomonas using the same}

The present invention relates to a PCR primer pair for detecting Xanthomonas, a PCR composition and kit for detecting Xanthomonas, and more specifically, specifically binding to a sequence of a part of 23S rRNA gene of a genus microorganism of Xanthomonas. Characterized by the forward primer (Xan23SF5) of SEQ ID NO: 1 and the reverse primer (Xan23SR6) pair of SEQ ID NO: 2 or a primer pair consisting of a base sequence complementary thereto, a composition and kit for detecting xanthomonas comprising the same, and xantho using the same A method for detecting a monas.

Dot-blot, polymerase chain reaction (PCR), etc. are used as a method for diagnosing plant pathogens. In particular, PCR is a widely used method because time, effort, and manpower are more economical than the dot-blot method. PCR (polymerase chain reaction) diagnostic method is to anneal a specific DNA fragment (primer) consisting of 10 ~ 20bp to the genomic DNA of the bacteria and then heat-resistant DNA polymerase (Taq polymerase) After repeated addition and repeated synthesis, the primer-bound region rapidly amplifies, and the PCR amplification product is electrophoresed on an agarose gel or acrylamide gel and ethidium bromide It is a method for detecting and diagnosing DNA polymorphisms of bacterial species such as the presence or absence of DNA bands and staining after DNA staining with bromide and silver stain. At present, the PCR method is mainly used for diagnosing bacterial diseases of humans or animals, but recently, PCR diagnostic methods have been developed to diagnose plant pathogens and are used for quarantine of imported and imported agricultural products. . In addition, the PCR diagnostic method is shorter in time than other methods, is inexpensive to diagnose, and is economical because many samples can be simultaneously tested. Recently, a PCR diagnostic method for amplifying a nucleic acid, which is a genetic material, is most preferred among Xanthomonas detection methods.

Microorganisms in the genus Xanthomonas are Gram-positive rod-like bacteria that are involved in the denitrification of soil nitrogen and cause various plant diseases such as peach tree bacterial pore disease, radish black rot, citrus ulcer disease, rice white leaf blight and Gangnam bean blight. do. Among the existing Xanthomonas microorganisms, rice leaf blight pathogen ( Xanthomonas oryzae pv. oryzae ), bacterial spot pattern pathogen ( Xanthomonas campestris pv. vesicatoria ), cruciferous black pathogen ( Xanthomonas campestris pv. campestris ), and citrus ulcer pathogen ( Xanthomonas axonopodis pv. citri) and kongbul diamond pathogen (Xanthomonas axonopodis pv. glycines), such as research on the individual PCR primers used for the detection of various microorganisms has been made, the specific potential wide range of applications in janto Pseudomonas genus microorganism belonging to janto Pseudomonas species and species sub enemy No primers have been reported yet. In the case of plants infected with Xanthomonas microorganisms, it takes more than 2 days to cultivate on the diseased partial surface sterilization and solid incubator for the diagnosis and identification of bacteria in diseased plants. It takes more than 3 days for the pure separation process, and it takes more than 2 days to analyze the purely separated microorganisms by Biolog, a commercialized program, or by fatty acid analysis. Therefore, in order to quickly and easily diagnose the genus Xanthomonas, the development of primers specific for the genus Xanthomonas that can be used in the PCR diagnostic method is required.

The inventors of the present invention, while searching for species-specific sequences in the pathogens of xanthomonas to design PCR primers that can be used to detect pathogenic bacteria belonging to the genus Xanthomonas, confirmed that the base sequence of 23S rRNA is species-conserved. In addition, a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4 specific to some sequences of 23S rRNA was invented.

Therefore, an object of the present invention is to provide a pair of PCR primers for detecting the genus Xanthomonas ( Xanthomonas ) represented by SEQ ID NO: 1 and SEQ ID NO: 2.

Another object of the present invention is to provide a PCR primer pair for detecting Xanthomonas genus microorganisms represented by SEQ ID NO: 3 and SEQ ID NO: 4.

Still another object of the present invention is to provide a composition for detecting microorganisms of the genus Xanthomonas comprising a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4.

Another object of the present invention is to provide a kit for detecting xanthomonas bacteria comprising a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4.

Another object of the present invention is the step of (a) separating the DNA from the sample; (b) amplifying the DNA extracted in step (a) by PCR using a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4; And (c) it provides a method for detecting Xanthomonas genus microorganism comprising the step of confirming the amplified PCR product of step (b) by electrophoresis.

In order to achieve the object of the present invention as described above, the present invention provides a PCR primer pair for detecting Xanthomonas genus microorganisms represented by SEQ ID NO: 1 and SEQ ID NO: 2.

In order to achieve another object of the present invention, the present invention provides a PCR primer pair for detecting Xanthomonas genus microorganisms represented by SEQ ID NO: 3 and SEQ ID NO: 4.

In order to achieve another object of the present invention, the present invention provides a composition for detecting a microorganism of the genus Xanthomonas comprising a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4. .

In order to achieve another object of the present invention, the present invention provides a kit for detecting a microorganism of the genus Xanthomonas comprising a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4. .

In order to achieve another object of the present invention, the present invention comprises the steps of (a) separating the DNA from the sample; (b) amplifying the DNA extracted in step (a) by PCR using the primer pair of claim 1 or 2; And (c) provides a method for detecting Xanthomonas genus microorganism comprising the step of confirming the amplified PCR product of step (b) by electrophoresis.

Hereinafter, the present invention will be described in detail.

In the present invention, the term "primer" refers to a short nucleic acid sequence which can form a base pair with a complementary template, and serves as a starting point for template strand copying.

The present invention relates to a pair of PCR primers for detecting the genus Xanthomonas represented by SEQ ID NO: 1 and SEQ ID NO: 2.

PCR primer pair for detecting the genus Xanthomonas of the present invention is a microorganism belonging to the genus Xanthomonas Xanthomonas Campestris ( X. campestris pv. campestris ; GenBank Accession No .: AE008922) and the complementary nucleotide sequence of 2 to 28 bp portion and 2,832 to 2,805 bp portion of the 23S rRNA gene. The primer pair of the present invention can specifically amplify the 2 to 2805bp portion of the 23S rRNA gene of Xanthomonas microorganism through polymerase chain reaction (PCR).

Various genes such as rRNA and house keeping genes are used for the production of polymerase chain reaction primers specific to microbial genes. Especially, 16S rRNA and 23S rRNA among the rRNA genes are stable for evolution and are in the same species or species subclass. Due to the rare genetic variation, it is ideal as a polymerase chain reaction primer for diagnosing pathogens. In the present invention, the polymerase chain reaction (PCR) primer pair targets a species conserved 23S rRNA region in the gene sequence of the Xanthomonas genome and thus does not specifically bind to microbial genes other than the genus Xanthomonas. It is characterized by being able to specifically detect only Genus microorganisms.

Although not limited thereto, it is included in the microorganism of the genus Xanthomonas of the present invention, Xanthomonas ( Xanthomonas albilineans ) , Xanthomonas subsp. Alfalfa ( X. alfalfae subsp.alfalfae ), xanthomonas alfalfa subsp. X. alfalfae subsp. Citromelonis xanthomonas arboricola pv. X. arboricola pv.celebennsis , Xanthomonas arboricola pv. X. arboricola pv. Corylina , Xanthomonas arboricola pv. Plastic trying to point (X. arboricola pv. Fragariae) janto Monastir ahreubo Ricola pv. X. arboricola pv.juglandis , Xanthomonas arboricola pv. Shetty point cola (X. arboricola pv. Poinsettiicola), Pseudomonas janto ahreubo Ricola pv. Popley ( X. arboricola pv.populi), Xanthomonas arboricola pv. X. arboricola pv. Pruni Xanthomonas axotopodis pv. Axonopodis ( X. axonopodis pv. axonopodis ), xanthomonas axonopodis pv. Bauer Hebrews niae (X. axonopodis pv. Bauhiniae), Pseudomonas janto solution Sono Pho display pv. X. axonopodis pv. Begoniae , Xanthomonas axonopodis pv. X. axonopodis pv.cajani , Xanthomonas axonopodis pv. Casabae (X. axonopodis pv. cassavae ), xanthomonas axonopodis pv. X. axonopodis pv.cassiae , Xanthomonas axotopodis pv. X. axonopodis pv.clitoriae , Xanthomonas axonopodis pv. Coracane ( X. axonopodis pv. Coracanae), Xanthomonas axonopodis pv. X. axonopodis pv.cyamopsidis , Xanthomonas axonopodis pv. X. axonopodis pv.desmodii , Xanthomonas axonopodis pv. X. axonopodis pv.desmodiigangetici , Xanthomonas axonopodis pv. X. axonopodis pv.desmodiilaxiflori , Xanthomonas axonopodis pv. Teondi Rhodes Modified poly (X. axonopodis pv. Desmodiirotundifolii), Pseudomonas janto liquid discharge port Sono dedicate pen in her die (X. axonopodis pv. Dieffenbachiae), Pseudomonas janto liquid discharge port Sono erythritol Rhine (X. axonopodis pv. Erythrinae) , Xanthomonas axonopodis glycine (X. axonopodis pv. glycines ), Xanthomonas axonopodis resfede (X. axonopodis pv. lespedezae), Pseudomonas janto liquid discharge port Sono Mani arc tooth (X. axonopodis pv. manihotis), Pseudomonas janto liquid discharge port Sono Naka state cor chori (X. axonopodis pv. nakataecorchori), Pseudomonas janto liquid discharge port Sono wave Terry (X . axonopodis pv. patelii), Pseudomonas janto solution Sonoran port dispatcher frost (X. axonopodis pv. phaseoli), Pseudomonas janto solution Sono Pho display pilran Tea (X. axonopodis pv. phyllanthi), Pseudomonas janto solution Sonora Four killed Liddy cola display (X. axonopodis pv. physalidicola), Pseudomonas janto solution Sonora Four point display Shetty cola (X. axonopodis pv. poinsettiicola), Pseudomonas janto solution Sono Pho display Linkoping City kids (X. axonopodis pv. rhynchosiae), Pseudomonas janto solution Sono Pho Discover your Rich (X. axonopodis pv. ricini), Pseudomonas janto solution Sonora Po Ke Bani disabled access (X. axonopodis pv. sesbaniae), Pseudomonas janto solution Sono Pho Tama display Lindy (X. axonopodis pv. tamarindi), Pseudomonas solution janto Sono Pho disk Bath particulate rarum (X. axonopodis pv. vascularum ), Pseudomonas janto liquid discharge port Sono TOV radio AP (X. axonopodis pv. Vignaeradiatae), Pseudomonas janto liquid discharge port non sono Nicolas (X. axonopodis pv. Vignicola), Pseudomonas janto liquid Sono Four discharge Gravity Alliance (X. axonopodis pv . vitians , X. bromi , X. campestris pv. aberrans , X. campestris pv. armoracieae , Xanthomonas campyris Fast-less bar fade (X. campestris pv. barbarae), Pseudomonas janto Kam Kam paste less paste-less (X. campestris pv. campestris), Pseudomonas janto Kam paste less pv. Fish ( X. campestris pv. Fici ), Xanthomonas populi ), xanthomonas campestris pv. Incaae ( X. campestris pv. Incanae ), Xanthomonas campestris pv. Mori ( X. campestris pv.mori ), Xanthomonas campestris pv. Negro makyul lance (X. campestris pv. Nigromaculans), Pseudomonas janto Kam paste less pv. Paulineia ( X. campestris pv. Paulliniae ), Xanthomonas campestris pv. X. campestris pv.raphani , Xanthomonas campestris pv. X. campestris pv.sesami , Xanthomonas campestris pv. Bt coke (X. campestris pv. Viticola), Pseudomonas janto Casa times (X. cassavae, LMG 673), Pseudomonas janto sheet Lee pv. Citri ( X. citr i subsp. Citri ), xanthomonas citri pv. X. citri subsp. Malvaceaerum , X. codiae i, X. cucurbitae , X. cynarae , X. cynarae , Xantonas ubesicataria X. euvesicatoria ), Xanthomonas pragariae ( X. fragariae ), Xanthomonas fuscanns pv. X. fuscans pv. Fuscans , Xanthomonas Fuscans pv. Orantipoli ( X. fuscans subsp. Aurantifolii ), Xantho gardneri ( X. gardneri ), Xanthomonas hortorum pv. X. hortorum pv. Carotae , Xanthomonas hortorum pv. X. hortorum pv.hederae , Xanthomonas hortorum pv. Pelargoni ( X. hortorum pv. Pelargonii ), Xanthomonas hortorum pv. X. hortorum pv.taraxaci , Xanthomonas hortorum pv. X. hortorum pv. Vitians , X. hyacinthi , X. melonis , Xanthomonas duckwood pv. Duck ( X. oryzae pv. Oryzae ), Xanthomonas duckwood pv. Originality Cola (X. oryzae pv. Oryzicola), Pseudomonas janto perforated lance (X. perforans), Pseudomonas janto fish (X. pisi), Pseudomonas janto campus tris pv. Sesame ( X. campestris pv . sesami ), Xanthomonas sakari ( X. sacchari ), Xanthomonas teicola ( X. theicola ), Xanthomonas trance lusen pv. Arhenateri ( X. translucens pv. Cerealis ( X. translucens pv. Cerealis ), Xanthomonas Translucens pv. Graminis ( X. translucens pv. Graminis ), Xanthomonas translucence pv. X. translucens pv. Phlei , xanthomonas translucens pv. X. translucens pv. Phleipratensis , xanthomonas translucence pv. X. translucens pv. Poae , xanthomonas translucence pv. X. translucens pv. Secalis , xanthomonas translucence pv. Translucens ( X. translucens pv.translucens ), xanthomonas translucens pv. X. translucens pv.undulosa , Xanthomonas Bashicola pv. Holsy cola ( X. vasicola pv. There is such holcicola), Pseudomonas janto Betsy Oh Katori (X. vesicatoria).

In one embodiment of the present invention by comparing the phytopathogenic microorganisms analyzed the entire nucleotide sequence registered in the GenBank (GenBank) to search for a sequence that is specifically present only in xanthomonas (see Example 1-1) specific for the microorganism of the genus Pseudomonas janto shown in SEQ ID NO: 1 and SEQ ID NO: 2, from 23S rRNA genes:; (GenBank registration No. AE008922 X. campestris pv campestris.) based on this, the microorganism of the genus Pseudomonas janto the janto Pseudomonas Kam paste-less Polymerase chain reaction forward and reverse primer pairs were prepared. In addition, the present invention provides a PCR primer pair for detecting the genus Xanthomonas represented by SEQ ID NO: 3 and SEQ ID NO: 4 having a sequence complementary to SEQ ID NO: 1 and SEQ ID NO: 2. The primer pair of the present invention may be modified (eg, added, deleted, substituted) within a range that does not affect specific detection of xanthomonas.

The present invention also relates to a composition for detecting the genus Xanthomonas comprising a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4. The composition for detecting the genus Xanthomonas microorganism of the present invention comprises the primer pair of the present invention, and may include cofactors used for DNA polymerase, dNTP and other PCR. Detecting composition in one embodiment of the present invention means a PCR composition, and included Tris HCl, KCl, dNTP, Taq DNA polymerase, MgCl ₂ , primers and template DNA.

The present invention also relates to a kit for detecting microorganisms of the genus Xanthomonas comprising a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4.

The kit of the present invention includes a variety of reagents, such as PCR compositions, restriction enzymes, agarose, hybridization, and electricity, for experiments (eg, PCR) and confirmation of results required for detecting microorganisms of Xanthomonas using the primer pair. A buffer solution necessary for the electrophoresis may be additionally included. More specifically, the PCR composition may contain an appropriate amount of DNA polymerase (eg, Thermus) in addition to the complementary DNA synthesized by reverse transcription and the PCR primer pair provided in the present invention. aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or Pyrococcus heat stable DNA polymerase obtained from furiosus (Pfu), dNTP, PCR buffer and water (dH ₂ O). The PCR buffer solution includes Tris-HCl, MgCl 2, KCl, and the like. At this time, the concentration of MgCl ₂ greatly affects the specificity and quantity of amplification. Generally, if an excess of the Mg ^{2 +} increase the non-specific PCR amplification products, and reduce the yield of a PCR product if the Mg ^{2 +} insufficient. The PCR buffer contains an appropriate amount of Triton X-100, dimethylsulfoxide (DMSO), Tween20, nonidet P40, PEG6000, formamide or bovine serum albumine (BSA). May be further included. In one embodiment of the present invention Tris HCl 50 mM, KCl 20 mM, nucleic acid polymerase 2 units, dNTP 2.0 mM, MgCl ₂ 1.5 mM, using a PCR composition comprising 20 pmol primer.

In addition, the kit may further include a DNA polymerase and a PCR reaction buffer solution of the composition in order to easily perform the PCR reaction, and the components necessary for performing electrophoresis to confirm whether or not amplification of the PCR product It may be further included in the kit of the present invention.

In addition, the present invention comprises the steps of (a) separating the DNA from the sample; (b) amplifying the DNA extracted in step (a) by PCR using primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4; And (c) relates to a method for detecting microorganism of the genus Xanthomonas comprising the step of confirming the amplified PCR product of step (b) by electrophoresis.

Polymerase chain reaction (PCR) is used to isolate small amounts of DNA in vivo and use it as a template, and by using primer pairs consisting of oligonucleotides complementary to both ends of the target DNA sequence, only the target DNA fragment can be hundreds of thousands of times. It is a method to amplify abnormally. Such PCR can be used to quickly and accurately detect plant-infected microorganisms. In the present invention, the detection method including the PCR was used to detect the microorganism of the genus Xanthomonas.

Looking at this step by step, the separation of DNA in the step (a) can be performed according to the method commonly used in the art from the microorganism belonging to the genus Xanthomonas, it can be carried out using a commercially available DNA extraction kit have.

In step (b), the DNA isolated in step (a) may be used as a template, and the primer pair of the present invention may be used to amplify the 23S rRNA gene of the genus Xanthomonas. This step is repeated several times, including the steps of (i) denaturation step, ii) annealing the target of the primer, and iii) elongation step. As used herein, the term "cycle" refers to the process of making one copy of a target nucleic acid. The denaturation, temperature and time conditions in the target nucleic acid binding step and extension step of the primer can be selected by a person skilled in the art suitable conditions and are not limited to a specific range, but preferably (i) 55 to 65 seconds at 90 to 98 ℃ 30 cycles of one cycle consisting of denaturation process of (ii) annealing for 55 to 60 seconds at 55 to 63 ° C. and (e) 180 to 190 seconds elongation at 65 to 75 ° C. And (iv) an additional stretching step at 72 ° C. for 10 minutes. The time and number of cycles in each step can be determined according to the conditions generally made in the art.

Step (c) may separate the PCR product by DNA size according to methods well known in the art. Preferably, it can be confirmed by an agarose gel or a polyacrylamide gel electrophoresis or fluorescence spectrometer. In this case, in order to use the fluorescence analyzer, PCR should be performed in step (b) using a primer pair attached with a fluorescent die known in the art. PCR amplification results can be preferably confirmed by agarose gel electrophoresis. After electrophoresis, the result of electrophoresis can be analyzed by ethidium bromide staining. For methods of general reverse transcription, PCR performance and analysis of the results, methods well known in the art are used.

Meanwhile, standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and described in the following literature (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984); and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

The present invention relates to a pair of PCR primers for detecting microorganisms belonging to the genus Xanthomonas, more specifically, PCR primer pairs represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4, Xanthomonas comprising the primer pair The present invention relates to a composition for detecting genus microorganisms, a kit for detection, and a method for detecting microorganisms of genus Xanthomonas using the same. Since the primer pair of the present invention has a sequence specific to a gene specific to the genus Xanthomonas, it may be used to detect the microorganism included in the xanthomonas without excessive time and effort.

Figure 1 shows a process for detecting the genus Xanthomonas by PCR analysis using a primer pair specific to the genus Xanthomonas microorganism of the present invention.
Figure 2 shows the comparison of the similarity of the 23S rRNA sequences of the genus Xanthomonas using ClastaW to design the forward and reverse primers for nucleic acid amplification of the genus Xanthomonas microorganism of the present invention.
3 is a position diagram of 23S rRNA which is a target region of a pair of PCR primers for detecting genus Xanthomonas of the present invention.
Figure 4 shows the specificity of the genus Xanthomonas of the PCR primer pair of the present invention, the microorganism of each lane is as follows. 1: Xanthomonas albilineans , ICMP 196), 2: xanthomonas campestris pv. Inca ( X. campestris pv . incanae , CFBP 2527), 3: xanthomonas alfalfa subsp. Alpal L (X. alfalfae subsp. Alfalfae, ICMP 5718), 4: Xanthomonas alfalfa subsp. Citromelonis ( X. alfalfae subsp. Citromelonis , ICMP15808), 5: xanthomonas arboricola pv. Selevensis ( X. arboricola pv.celebennsis , CFBP 3523), 6: Xanthomonas arboricola pv. Corylina ( X. arboricola pv. Corylina , CFBP 1159), 7: Xanthomonas arboricola pv. Plastic point Ke (X. arboricola pv fragariae, CFBP 6771 .), 8: Pseudomonas janto ahreubo Ricola pv. Jugglandis ( X. arboricola pv. Juglandis , LMG 747), 9: Xanthomonas arboricola pv. Shetty point cola (X. arboricola pv poinsettiicola, ICMP 6274 .), 10: janto Pseudomonas ahreubo Ricola pv. Four pyulri (X. arboricola pv populi, CFBP 3123 .), 11: janto Pseudomonas ahreubo Ricola pv. Pruni ( X. arboricola pv. Pruni, ICMP 51), 12: Xanthomonas axonopodis pv. Axonopodis ( X. axonopodis pv. axonopodis , LMG 982), 13: xanthomonas axonopodis pv. Bauer Hebrews niae (X. axonopodis pv. Bauhiniae ICMP 5720), 14: Xanthomonas axonopodis pv. Begonia ( X. axonopodis pv. Begoniae CFBP 2524), 15: Xanthomonas axonopodis pv. Kazani ( Xanthomonas axonopodis pv. cajani , LMG 558), 16: Xanthomonas axonopodis pv. Casabae (X anthomonas axonopodis pv. cassavae , LMG 8049), 17: Xanthomonas axonopodis pv. Cassia ( Xanthomonas axonopodis pv. cassiae , LMG 675), 18: Xanthomonas Xanthomonas axonopodis pv. clitoriae , LMG 9045 ), 19: Xanthomonas axonopodis pv. Coracao ( Xanthomonas axonopodis pv. coracanae LMG 686), 20: Xanthomonas Xanthomonas axonopodis pv. cyamopsidis , LMG 686), 21: Xanthomonas axonopodis pv. Des Modi ( Xanthomonas axonopodis pv. desmodii , LMG 692), 22: Xanthomonas axonopodis pv. Des Modi Gange City ( Xanthomonas axonopodis pv. desmodiigangetici , LMG 693), 23: Xanthomonas axonopodis pv. Des mododilacciflori ( Xanthomonas axonopodis pv. desmodiilaxiflor , LMG 9046), 24: Xanthomonas axonopodis pv. Desmododitondipoli ( Xanthomonas axonopodis pv. desmodiirotundifolii, LMG 694), 25: Xanthomonas axonopodis pv. Dieter pen dedicate her (Xanthomonas axonopodis pv dieffenbachiae, LMG 695 .), 26: janto Monastir solution Sono Pho display pv. Erie tree within (. Xanthomonas axonopodis pv erythrinae, LMG 698), 27: janto Pseudomonas liquid discharge port Sono pv. Sines glycidyl (Xanthomonas axonopodis pv glycines, ICMP 5732 .), 28: janto Pseudomonas liquid discharge port Sono pv. Lespedesae ( Xanthomonas axonopodis pv.lespedezae , LMG 757), 29: Xanthomonas axonopodis pv. Maniotis ( Xanthomonas axonopodis pv. Manihotis , ICMP 5741), 30: Xanthomonas axonopodis pv. Nakata- Cocor ( Xanthomonas axonopodis pv. nakataecorchori , LMG 786), 31: Xanthomonas axonopodis pv. Parteri ( Xanthomonas axonopodis pv. patelii , LMG 811), 32: Xanthomonas axonopodis pv. Passery ( Xanthomonas axonopodis pv. phaseoli , ICMP 5834), 33: Xanthomonas axonopodis pv. Pilanti ( Xanthomonas axonopodis pv. Phyllanthi , LMG 844), 34: Xanthomonas axonopodis pv. Fisalidicola ( Xanthomonas axonopodis pv. physalidicola , LMG 845), 35: xanthomonas axonopodis pv. Poinseticola ( Xanthomonas axonopodis pv. poinsettiicola , LMG 849), 36: Xanthomonas axonopodis pv. Lincosea ( Xanthomonas axonopodis pv. rhynchosiae , LMG 8021), 37: Xanthomonas axonopodis pv. Richie ( Xanhomonas axonopodis pv. ricini , LMG 861), 38: Xanthomonas Xanthomonas axonopodis pv. sesbaniae, ICMP 367), 39: Xanthomonas axonopodis pv. Tamarindi ( Xanthomonas axonopodis pv. tamarindi , LMG 955), 40: Xanthomonas axonopodis pv. Bascula Room ( Xanthomonas axonopodis pv. vascularum , CFBP 5823), 41: xanthomonas pv. Xanthomonas axonopodis pv.vignaeradiatae , LMG 936, 42: Xanthomonas axonopodis pv. Vinico , ( Xanthomonas axonopodis pv. vignicola , LMG 8752), 43: Xanthomonas axonopodis pv. Vitians ( Xanthomonas axonopodis pv . vitians , CFBP 2538), 44: Xanthomonas bromi X. bromi , LMG 947), 45: Xanthomonas campestris pv. Everance ( Xanthomonas campestris pv . aberrans , CFBP 6865), 46: Xanthomonas campestris pv. Amadorasiea ( X. campestris pv . armoracieae , CFBP 3838), 47: Xanthomonas Campestris pv. Creeper ( Xanthomonas campestris pv . barbarae , CFBP 5825), 48: Xanthomonas campestris pv. Campestris ( X. campestris pv . campestris , ICMP 13), 49: Xanthomonas Campestris pv. Fish ( X. campestris pv . fici , LMG 701), 50: Xanthomonas populi , CFBP 1817), 51: Xanthomonas campestris pv. Mori (Xanthomonas campestris pv mori, ICMP 13199 .), 52: janto Pseudomonas Kam paste less pv. Nigromaculans ( Xanthomonas campestris pv . nigromaculans , LMG 787), 53: Xanthomonas campestris pv. Paulineia ( Xanthomonas campestris pv . paulliniae , CFBP 5862), 54: Xanthomonas Campestris pv. Raphani ( Xanthomonas campestris pv . raphani , ICMP 1404), 55: Xanthomonas Campestris pv. Sesame ( Xanthomonas campestris pv . sesami , ICMP 621), 56: Xanthomonas Campestris pv. Viticola ( X. campestris pv . Viticola , ICMP 3867), 57: Xanthomonas cassavae, LMG 673, 58: Xanthomonas citri supsp. Citri ( Xanthomonas citri subsp . citri , ICMP 15804), 59: xanthomonas citri subsp. Malvaserum ( Xanthomonas citri subsp . malvaceaerum , ICMP 217), 60: Xanthomonas codiaei , CFBP 4690), 61: Xanthomonas cucurbitae ( ICMP 2299), 62: Xanthomonas cynarae , CFBP 4188), 63: Xanthomonas euvesicatoria , ICMP 109), 64: Xanthomonas fragariae ( CFBP 2157), 65: Xanthomonas fuscanns pv. Fucans ( Xanthomonas fuscans pv . Fuscans , ICMP 239), 66: Xanthomonas Fuscans subsp. Orantipoli ( Xanthomonas fuscans subsp . Aurantifolii , ICMP15806), 67: Xanthomonas gardnery ( Xanthomonas gardneri , NCPPB 881), 68: Xanthomonas Hortorum pv. Carotene ( Xanthomonas hortorum pv . carotae , CFBP 4997), 69: Xanthomonas Hortorum pv. Heiderae ( Xanthomonas hortorum pv . hederae , CFBP 4925), 70: xanthomonas hortorum pv. Pelargoni ( Xanthomonas hortorum pv . pelargonii , CFBP 2533), 71: xanthomonas hortorum pv. Fallen sashimi ( Xanthomonas hortorum pv . taraxaci , CFBP 410,), 72: xanthomonas hortorum pv. Vitians ( Xanthomonas hortorum pv . vitians LMG 938), 73: Xanthomonas hyacinthi ( LMG 739), 74: Xanthomonas Melonis melonis , CFBP 4644), 75: xanthomonas duck pv. Duck ( Xanthomonas oryzae pv . oryzae , ICMP 3125), 76: Xanthomonas duckwood pv. Original Cola ( Xanthomonas oryzae pv . oryzicola , LMG 797), 77: Xanthomonas perforans ( NCPPB 4321), 78: Xanthomonas pisi , ICMP 570, 79: Xanthomonas sacchari , CFBP 4641), 80: Xanthomonas theicola ( CFBP 4691), 81: Xanthomonas translucense pv. Arhenateri ( Xanthomonas translucens pv. arrhenatheri , CFBP 2056), 82: Xanthomonas translucens pv . cerealis , CFBP 2541), 83: Xanthomonas translucens pv. Graminis ( Xanthomonas translucens pv . graminis , CFBP 2053), 84: xanthomonas translucense pv. Frey ( Xanthomonas translucens pv . phlei , CFBP 2062), 85: Xanthomonas translucense pv. Playpratensis ( Xanthomonas translucens pv . phleipratensis , ICMP 5744 ), 86: Xanthomonas translucens pv. Pore ( Xanthomonas translucens pv . poae , CFBP 2057), 87: xanthomonas translucense pv. Sepalis ( Xanthomonas translucens pv . secalis , CFBP 2539), 88: xanthomonas translucence pv. Xanthhomonas translucens pv . translucens , CFBP 2054) 89: Xanthomonas translucens pv. Underrosa ( Xanthomonas translucens pv . undulosa , ICMP 5755), 90: Xanthomonas Bashicola pv. Whole Cola ( Xanthomonas vasicola) pv . holcicola , CFBP 2543), 91: Xanthomonas vesicatoria, CFBP 2537, 92: Erwinia amylovora , ATCC 15580 ) , 93: Erwinia psidii , ATCC 49406 ) , 94: Erwinia rhapondici, ATCC 29283), 95: Oh, I know the panto-Tees (Pantoea ananatis , ATCC 33244) , 96: Pectobacterium betavascolorum ( ATCC 43762) , 97: Pectobacterium cipripedi ( Pectobacterium cypripedii , LMG 1268) , 98: Pectobacterium chrysanthemi , ATCC 11663) , 99: Brenneria nigrifluens , LMG 2696 , 100: Brenneria salissis salicis , LMG2698) , 101: Enterobacter dissolves ( Enterobacter dissolvens , LMG 2683) , 102: Enterobacter tracheiphila ( ATCC 33245) , 103: Pantoea Dispulsar dispersa , LMG 2603) , 104: Pantoea stewarti subsp. Stewarti ( Pantoea stewartii subsp. sewartii , ATCC 8199), 105: Pseudomonas syringae pv. syringae , ICMP 3319) , 106: Pseudomonas savastonnoy pv. Pseudomonas savastonoi pv. savastonoi , CFBP 1670) , 107: Pseudomonas tolaasii , CFBP 2068), 108: Pseudomonas cichorii, LMG 2162), 109: Pseudomonas irregularities deployment rubber (Pseudomonas viridiflava, CFBP 2107), 110: Pseudomonas koreo Gata (Pseudomonas corrugata , CFBP 2431), 111: Dickeya dianthicola , CFBP 1200), 112: Acidovorax konjaci, LMG 5691 , 113: Ralstonia solanasearum, LMG2297), 114: Agrobacterium non-Tees (Agrobacterium vitis, BC3096), 115: ruby Agrobacterium (Agrobacterium rubi, BC3097), 116: Agrobacterium jeneseu separation tank (Agrobacterium rhizogenes , BC3098), 117: Agrobacterium tumefaciens ( BC3099)

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

Xanthomonas Xanthomonas ) Design of Primer Pairs for Detection

<1-1> Information search of specific sequencing

Genopathogenic genus pathogenic rRNA and housekeeping genes that are frequently used in PCR primer development to select nucleic acids with bacterial specific bases, are stable in evolution in the xanthomonas genomic sequence, and variations among the same species or subclasses Rarely, the target 23S rRNA region was targeted with species-conserved and pathogenic PCR primers. In the present invention, 23S rRNA genes of phytopathogens and some bacteria whose total sequences are registered in the GenBank are analyzed by BioEdit Sequence Alignment Editor Version 7.0.9.0 (Hall, TA 1999. BioEdit: a user-friendly biological sequence). alignment editor and analysis program for Windows 95/98 / NT.Nucl.Acids.Symp.Ser. 41: 95-98.

As a result, as shown in Figure 2, 2 to 28 bp and 2,832 to 2,805 bp of the base sequence of the 23S rRNA of Xanthomonas campestris ( X. campestris pv. Campestris; GenBank Accession No .: AE008922) Was confirmed to be a nucleotide sequence specifically present in 23S rRNA genes of other microorganisms belonging to the genus Xanthomonas. The position of the base sequence is shown in FIG. 3.

<1-2> for specific sequence amplification primer  Pair making

Using the 23S rRNA gene confirmed in the specificity in 1-1 to prepare a forward and reverse primer pair of the polymerase chain reaction specific to xanthomonas as follows. This janto Pseudomonas 23S rRNA gene is designed from the 23S rRNA gene of the AE008922 (X. campestris pv. Campestris) registered in GenBank (http://www.ncbi.nlm.nih.gov/) of the (National Center for Biotechnology Information) NCBI Nucleotide sequences complementary to bases 2 to 28 (5'-TGGTCAAGCCGCACGGATCATTAGTAT-3 ', 27 mer, Xan23SF5, (SEQ ID NO: 1)) and base sequences complementary to bases 2,832 to 2,805 (' 5-ACGTGGATAGCCTGCGAAAAGTGTC -'3, 25 mer, Xan23SR6, (SEQ ID NO: 2)) can be represented by the base sequence shown in Table 1 below. The positions and base sequences of the PCR primers at 23S rRNA of Xanthomonas campestris are shown in FIGS. 2 and 2. In addition, the position in Table 2 is GenBank Acession NO. The 23S rDNA sequence of AE008922 was prepared.

SEQ ID NO: primer Sequence location One Xan23SF5 5-TGGTCAAGCCGCACGGATCATTAGTAT-3 2 ~ 28bp 2 Xan23SR6 5-ACGTGGATAGCCTGCGAAAAGTGTC-3 2,832-2,805bp

< Example  2>

Preparation of Sample

Using a primer prepared in Example 1 was prepared a sample to be used in the polymerase chain reaction for detecting xanthomonas. Bacteria used in the experiment were pathogens of the genus Xanthomonas of 91 species subclasses and 26 bacteria were used as controls. Specifically, the bacteria used in the experiment are 1: Xanthomonas ( Xanthomonas) albilineans , ICMP 196), 2: xanthomonas campestris pv. Inca ( X. campestris pv . incanae, CFBP 2527), 3: xanthomonas alfalfa subsp. Alpal L (X. alfalfae subsp. Alfalfae, ICMP 5718), 4: Xanthomonas alfalfa subsp. Citromelonis ( X. alfalfae subsp. Citromelonis, ICMP15808), 5: Xanthomonas arboricola pv. Selevensis ( X. arboricola pv.celebennsis, CFBP 3523), 6: Xanthomonas arboricola pv. Corylina ( X. arboricola pv. Corylina, CFBP 1159), 7: Xanthomonas arboricola pv. Pragaria ( X. arboricola pv.fragariae , CFBP 6771), 8: xanthomonas arboricola pv. Jugglandis ( X. arboricola pv. Juglandis, LMG 747), 9: Xanthomonas arboricola pv. Poinseticola ( X. arboricola pv.poinsettiicola , ICMP 6274), 10: xanthomonas arboricola pv. Populi ( X. arboricola pv.populi , CFBP 3123), 11: xanthomonas arboricola pv. Pruni ( X. arboricola pv. Pruni, ICMP 51), 12: Xanthomonas axonopodis pv. Axonopodis ( X. axonopodis pv. Axonopodis, LMG 982), 13: Xanthomonas axonopodis pv. Bauer Hebrews niae (X. axonopodis pv bauhiniae ICMP 5720. ), 14: janto Monastir solution Sono Pho display pv. Begonia ( X. axonopodis pv. Begoniae CFBP 2524), 15: Xanthomonas axonopodis pv. Kazani ( Xanthomonas axonopodis pv.cajani, LMG 558), 16: Xanthomonas axonopodis pv. Casabae (X anthomonas axonopodis pv.cassavae, LMG 8049), 17: Xanthomonas axonopodis pv. Xanthomonas axonopodis pv.cassiae, LMG 675, 18: Xanthomonas axonopodis pv.clitoriae, LMG 9045 , 19: Xanthomonas axonopodis pv. Xanthomonas axonopodis pv.coracanae LMG 686, 20: Xanthomonas axonopodis pv. Cyamopsidis, LMG 686, 21: Xanthomonas axonopodis pv.coracanae LMG 686. Desmodii ( Xanthomonas axonopodis pv. Desmodii, LMG 692), 22: Xanthomonas axonopodis pv. Xanthomonas axonopodis pv. Desmodiigangetici, LMG 693, 23: Xanthomonas axonopodis pv. Desmodiilaxiflor ( LMG 9046), 24: Xanthomonas axonopodis pv. Teondi Rhodes Modified poly (Xanthomonas axonopodis pv desmodiirotundifolii, LMG 694 .), 25: janto Pseudomonas liquid discharge port Sono pv. Diefenbachia ( Xanthomonas axonopodis pv. Dieffenbachiae , LMG 695), 26: Xanthomonas axonopodis pv. Erythrida ( Xanthomonas axonopodis pv.erythrinae , LMG 698), 27: Xanthomonas axonopodis pv. Glycines ( Xanthomonas axonopodis pv. Glycines , ICMP 5732), 28: Xanthomonas axonopodis pv. Xanthomonas axonopodis pv.lespedezae, LMG 757, 29: Xanthomonas axonopodis pv. Maniotis ( Xanthomonas axonopodis pv. Manihotis , ICMP 5741), 30: Xanthomonas axonopodis pv. Nakata- Cocor ( Xanthomonas axonopodis pv. nakataecorchori , LMG 786), 31: Xanthomonas axonopodis pv. Parteri ( Xanthomonas axonopodis pv. patelii , LMG 811), 32: Xanthomonas axonopodis pv. Passery ( Xanthomonas axonopodis pv. phaseoli , ICMP 5834), 33: Xanthomonas axonopodis pv. Pilanti ( Xanthomonas axonopodis pv. Phyllanthi , LMG 844), 34: Xanthomonas axonopodis pv. Fisalidicola ( Xanthomonas axonopodis pv. physalidicola , LMG 845), 35: xanthomonas axonopodis pv. Poinseticola ( Xanthomonas axonopodis pv. poinsettiicola , LMG 849), 36: Xanthomonas axonopodis pv. Lincosea ( Xanthomonas axonopodis pv. rhynchosiae , LMG 8021), 37: Xanthomonas axonopodis pv. Richie ( Xanhomonas axonopodis pv. ricini , LMG 861), 38: Xanthomonas Xanthomonas axonopodis pv. sesbaniae , ICMP 367), 39: Xanthomonas axonopodis pv. Tamarindi ( Xanthomonas axonopodis pv. tamarindi , LMG 955), 40: Xanthomonas axonopodis pv. Bascula Room ( Xanthomonas axonopodis pv. vascularum , CFBP 5823), 41: xanthomonas pv. Xanthomonas axonopodis pv.vignaeradiatae , LMG 936, 42: Xanthomonas axonopodis pv. Vinico , ( Xanthomonas axonopodis pv. vignicola , LMG 8752), 43: Xanthomonas axonopodis pv. Vitians ( Xanthomonas axonopodis pv . vitians , CFBP 2538), 44: Xanthomonas bromi X. bromi , LMG 947), 45: Xanthomonas campestris pv. Everance ( Xanthomonas campestris pv . aberrans , CFBP 6865), 46: Xanthomonas campestris pv. Amadorasiea ( X. campestris pv . armoracieae , CFBP 3838), 47: Xanthomonas Campestris pv. Creeper ( Xanthomonas campestris pv . barbarae , CFBP 5825), 48: Xanthomonas campestris pv. Campestris ( X. campestris pv . campestris , ICMP 13), 49: Xanthomonas Campestris pv. Fish ( X. campestris pv . fici , LMG 701), 50: Xanthomonas populi , CFBP 1817), 51: Xanthomonas campestris pv. Mori (Xanthomonas campestris pv mori, ICMP 13199 .), 52: janto Pseudomonas Kam paste less pv. Nigromaculans ( Xanthomonas campestris pv . nigromaculans , LMG 787), 53: Xanthomonas campestris pv. Paulineia ( Xanthomonas campestris pv . paulliniae , CFBP 5862), 54: Xanthomonas Campestris pv. Raphani ( Xanthomonas campestris pv . raphani , ICMP 1404), 55: Xanthomonas Campestris pv. Sesame ( Xanthomonas campestris pv . sesami , ICMP 621), 56: Xanthomonas Campestris pv. Viticola ( X. campestris pv . viticola , ICMP 3867), 57: Xanthomonas cassavae , LMG 673 ) , 58: Xanthomonas citri supsp. Citri ( Xanthomonas citri subsp . citri , ICMP 15804), 59: xanthomonas citri subsp. Malvaserum ( Xanthomonas citri subsp . malvaceaerum , ICMP 217), 60: Xanthomonas codiaei , CFBP 4690), 61: Xanthomonas cucurbitae ( ICMP 2299), 62: Xanthomonas cynarae , CFBP 4188), 63: Xanthomonas euvesicatoria , ICMP 109), 64: Xanthomonas fragariae ( CFBP 2157), 65: Xanthomonas fuscanns pv. Fucans ( Xanthomonas fuscans pv . Fuscans , ICMP 239), 66: Xanthomonas Fuscans subsp. Orantipoli ( Xanthomonas fuscans subsp . Aurantifolii , ICMP15806), 67: Xanthomonas gardnery ( Xanthomonas gardneri , NCPPB 881), 68: Xanthomonas Hortorum pv. Carotene ( Xanthomonas hortorum pv . carotae , CFBP 4997), 69: Xanthomonas Hortorum pv. Heiderae ( Xanthomonas hortorum pv . hederae , CFBP 4925), 70: xanthomonas hortorum pv. Pelargoni ( Xanthomonas hortorum pv . pelargonii , CFBP 2533), 71: xanthomonas hortorum pv. Fallen sashimi ( Xanthomonas hortorum pv . taraxaci , CFBP 410,), 72: xanthomonas hortorum pv. Vitians ( Xanthomonas hortorum pv . vitians LMG 938), 73: Xanthomonas hyacinthi ( LMG 739), 74: Xanthomonas Melonis melonis , CFBP 4644), 75: xanthomonas duck pv. Duck ( Xanthomonas oryzae pv . oryzae , ICMP 3125), 76: Xanthomonas duckwood pv. Original Cola ( Xanthomonas oryzae pv . oryzicola , LMG 797), 77: Xanthomonas perforans ( NCPPB 4321), 78: Xanthomonas pisi , ICMP 570, 79: Xanthomonas sacchari , CFBP 4641), 80: Xanthomonas theicola ( CFBP 4691), 81: Xanthomonas translucense pv. Arhenateri ( Xanthomonas translucens pv. arrhenatheri , CFBP 2056), 82: Xanthomonas translucens pv . cerealis , CFBP 2541), 83: Xanthomonas translucens pv. Graminis ( Xanthomonas translucens pv . graminis , CFBP 2053), 84: xanthomonas translucense pv. Graminis ( Xanthomonas translucens pv . phlei , CFBP 2062), 85: Xanthomonas translucense pv. Playpratensis ( Xanthomonas translucens pv . phleipratensis , ICMP 5744 ), 86: Xanthomonas translucens pv. Pore ( Xanthomonas translucens pv . poae , CFBP 2057), 87: xanthomonas translucense pv. Sepalis ( Xanthomonas translucens pv . secalis , CFBP 2539), 88: xanthomonas translucence pv. Translucent ( Xanthomonas translucens pv . translucens , CFBP 2054) 89: Xanthomonas translucens pv. Underrosa ( Xanthomonas translucens pv . undulosa , ICMP 5755), 90: Xanthomonas Bashicola pv. Whole Cola ( Xanthomonas) vasicola pv . holcicola , CFBP 2543), 91: Xanthomonas vesicatoria , CFBP 2537, 92: Erwinia amylovora , ATCC 15580 ) , 93: Erwinia psidii, ATCC 49406), 94: Earl Winiah rapon DC (Erwinia rhapondici, ATCC 29283), 95: Oh, I know the panto-Tees (Pantoea ananatis , ATCC 33244) , 96: Pectobacterium betavascolorum , ATCC 43762) , 97: Pectobacterium cypripedii ( LMG 1268) , 98: Pectobacterium chrysanthemi ( ATCC 11663) , 99: Brenneria nigrifluens nigrifluens , LMG 2696) , 100: Brenneria salissis salicis, LMG2698), 101: Enterobacter disol Bence (Enterobacter dissolvens, LMG 2683), 102: Enterobacter trad K pillars (Enterobacter tracheiphila , ATCC 33245) , 103: Pantoea Dispulsar dispersa , LMG 2603) , 104: Pantoea stewarti subsp. Stewarti ( Pantoea stewartii subsp. sewartii , ATCC 8199), 105: Pseudomonas syringae pv. syringae , ICMP 3319) , 106: Pseudomonas savastonnoy pv. Pseudomonas savastonoi pv. savastonoi , CFBP 1670) , 107: Pseudomonas tolaasii ( CFBP 2068), 108: Pseudomonas cichorii , LMG 2162), 109: Pseudomonas bilidiflava Pseudomonas viridiflava , CFBP 2107), 110: Pseudomonas corrugata ( CFBP 2431), 111: Dickeya dianthicola , CFBP 1200), 112: Acidovorax konjaci, LMG 5691), 113: Central Stony Solana Seah Ah Rum (Ralstonia solanasearum, LMG2297), 114 : Agrobacterium non-Tees (Agrobacterium vitis, BC3096), 115: ruby Agrobacterium (Agrobacterium rubi , BC3097), 116: Agrobacterium rhizogenes ( BC3098), 117: Agrobacterium tumerfaciens ( Agrobacterium tumefaciens , BC3099).

(i) separating the bacteria in a Trypticase Soy Agar (TSA), and then using a sterile water to make a suspension (OD ₆₀₀ = 0.1) or (ii) quinazinic acid in the isolated bacteria Use 20ng of nucleic acid to separate the nucleic acid with Qiagen Genomic DNA purification kit, or (i) crush the diseased area in 0.5 mL of sterile water at the suspected plant area, and then put the grinding solution into the eppendol tube and heat it. 2 μl was used.

In addition, to prepare a composition for the polymerase chain reaction of the bacteria. The composition was prepared in a total of 50 μl, including Tris HCl 50 mM, KCl 20 mM, Taq DNA polymerase ₂ units, dNTP 2.0 mM, MgCl ₂ 1.5 mM and 20 pmol each of the primers prepared in Example 1 above. The components and contents of the composition are shown in Table 2 below.

Components in the Composition density Tris HCl (pH 8.3) 10 mM KCl 50 mM dNTP 0.2 mM Taq DNA Polymerase 2 U MgCl ₂ 1.5 mM primer 20 pmol DNA 20 ng

< Example  3>

Performing Polymerase Chain Reaction

In order to confirm the specificity of the Xan23SF5 and Xan23SR6 primer pair of the present invention for the genus Xanthomonas, polymerase chain reaction (PCR) was carried out using the primer pair of Example 1 and the bacterial sample and composition of Example 2. Was performed.

PCR was performed by adding 20 ng of the nucleic acid of the bacterium of Example 1, (i) denaturation at 95 ° C. for 60 seconds, (ii) annealing primers and target nucleic acids at 58 ° C. for 60 seconds, and (iii). 1) 30 cycles of one cycle consisting of a process of elongation at 72 ° C. for 180 seconds, and (iii) further elongation at 72 ° C. for 10 minutes.

In addition, to confirm the PCR product, 5 μl of the reaction solution was loaded on a 1.5% NuSieve: GTG agarose gel after the completion of the PCR, followed by electrophoresis at 70V. The electrophoretic agarose gel was stained with EtBr, and then irradiated with a UV trans-illuminator to confirm the presence or absence of bands and sizes of PCR products.

As a result, as shown in Figure 4, the band appeared only for the microorganisms 1 to 91 belonging to the genus Xanthomonas, the band did not appear in the 92 to 117 microorganisms other than the remaining genus of xanthomonas. This result shows that the primer pair of the present invention specifically amplifies only 23S rRNA of the genus Xanthomonas, and does not bind to other genus microorganisms.

The present invention relates to a pair of PCR primers for detecting microorganisms belonging to the genus Xanthomonas, more specifically, PCR primer pairs represented by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and SEQ ID NO: 4, Xanthomonas comprising the primer pair The present invention relates to a composition for detecting genus microorganisms and a kit for detecting the genus microorganisms and a method for detecting genus microorganisms using xanthomonas. Since the primer pair of the present invention has a sequence specific to a gene specific to the genus Xanthomonas, it may be used for detecting the microorganisms included in the genus Xanthomonas.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Primer set of polymerase chain reaction for detecting the genus          Xanthomonas and method for detecting the genus Xanthomonas using          the same <130> NP10-0051 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 27 <212> DNA <213> Xanthomonas <400> 1 tggtcaagcc gcacggatca ttagtat 27 <210> 2 <211> 25 <212> DNA <213> Xanthomonas <400> 2 acgtggatag cctgcgaaaa gtgtc 25 <210> 3 <211> 27 <212> DNA <213> Xanthomonas <400> 3 accagttcgg cgtgcctagt aatcata 27 <210> 4 <211> 25 <212> DNA <213> Xanthomonas <400> 4 tgcacctatc ggacgctttt cacag 25

Claims (6)

PCR primer pair for detecting Xanthomonas genus microorganism represented by SEQ ID NO: 1 and SEQ ID NO: 2.
PCR primer pairs for detecting the genus Xanthomonas represented by SEQ ID NO: 3 and SEQ ID NO: 4.
The method according to claim 1 or 2, wherein the genus Xanthomonas microorganism is Xanthomonas albilineans , ICMP 196), Xanthomonas campestris pv. Inca ( Xanthomonas campestris pv . incanae , CFBP 2527), xanthomonas alfalfa subsp. Alfalfa ( Xanthomonas alfalfae subsp. alfalfae , ICMP 5718), Xanthomonas alfalfa subsp. Citromelonis ( Xanthomonas alfalfae subsp. citromelonis, ICMP15808), Xanthomonas arboricola pv. Selevensis ( Xanthomonas arboricola pv. celebennsis, CFBP 3523), Xanthomonas arboricola pv. Coralina ( Xanthomonas arboricola pv. corylina, CFBP 1159), Xanthomonas arboricola pv. Pragaria ( Xanthomonas arboricola pv. fragariae, CFBP 6771) , xanthomonas arboricola pv. Jugglandis ( Xanthomonas arboricola pv. juglandis , LMG 747), Xanthomonas arboricola pv. Shetty point cola (Xanthomonas arboricola pv. Poinsettiicola, ICMP 6274), Pseudomonas janto ahreubo Ricola pv. Four pyulri (Xanthomonas arboricola pv. Populi, CFBP 3123), Pseudomonas janto ahreubo Ricola pv. Pruny ( Xanyhomonas arboricola pv. pruni , ICMP 51), Xanthomonas axonopodis pv. Axonopodis ( Xanthomonas axonopodis pv. axonopodis , LMG 982), xanthomonas axonopodis pv. Bauhinia ( Xanthomonas axonopodis pv. bauhiniae ICMP 5720), Xanthomonas axonopodis pv. Begonia ( Xanthomonas axonopodis pv. begoniae CFBP 2524), Xanthomonas axonopodis pv. Kazani ( Xanthomonas axonopodis pv. cajani , LMG 558), Xanthomonas axonopodis pv. Casabae (X anthomonas axonopodis pv. cassavae , LMG 8049), Xanthomonas axonopodis pv. Cassia ( Xanthomonas axonopodis pv. cassiae , LMG 675), Xanthomonas axonopodis clitoris ( Xanthomonas axonopodis pv. clitoriae , LMG 9045 ), Xanthomonas axonopodis pv. Coracao ( Xanthomonas axonopodis pv. coracanae LMG 686), Xanthomonas axonopodis pv. Cyamopsidis , LMG 686), Xanthomonas axonopodis pv. Desmody ( Xanthomonas axonopodis pv. Desmodii , LMG 692), Xanthomonas axonopodis pv. Xanthomonas axonopodis pv. Desmodiigangetici , LMG 693), Xanthomonas axonopodis pv. Des mododilacciflori ( Xanthomonas axonopodis pv. desmodiilaxiflor , LMG 9046), Xanthomonas axonopodis pv. Desmododitondipoli ( Xanthomonas axonopodis pv. desmodiirotundifolii , LMG 694), Xanthomonas axonopodis pv. Diefenbachia ( Xanthomonas axonopodis pv. dieffenbachiae , LMG 695), Xanthomonas axonopodis pv. In the Eritry ( Xanthomonas axonopodis pv. erythrinae , LMG 698), Xanthomonas axonopodis pv. Glycines ( Xanthomonas axonopodis pv. glycines , ICMP 5732), xanthomonas axonopodis pv. Lespedes ( Xanthomonas axonopodis pv. lespedezae , LMG 757), Xanthomonas axonopodis pv. Manihortis ( Xanthomonas axonopodis pv. manihotis , ICMP 5741), Xanthomonas axonopodis pv. Nakata- Cocor ( Xanthomonas axonopodis pv. nakataecorchori , LMG 786), Xanthomonas axonopodis pv. Parteri ( Xanthomonas axonopodis pv. patelii , LMG 811), Xanthomonas axonopodis pv. Passery ( Xanthomonas axonopodis pv. phaseoli , ICMP 5834), Xanthomonas axonopodis pv. Pilanti ( Xanthomonas axonopodis pv. phyllanthi , LMG 844), Xanthomonas axonopodis pv. Fisalidicola ( Xanthomonas axonopodis pv. physalidicola , LMG 845), xanthomonas axonopodis pv. Poinseticola ( Xanthomonas axonopodis pv. poinsettiicola , LMG 849), Xanthomonas axonopodis pv. Lincosea ( Xanthomonas axonopodis pv. rhynchosiae , LMG 8021), Xanthomonas axonopodis pv. Richie ( Xanhomonas axonopodis pv. ricini, LMG 861), Pseudomonas janto liquid discharge port Sono process varnishes Ke (Xanthomonas axonopodis pv. sesbaniae, ICMP 367), Pseudomonas janto liquid discharge port Sono pv. Tamarindi ( Xanthomonas axonopodis pv. Tamarindi , LMG 955), Xanthomonas axonopodis pv. Bath particulate rarum (Xanthomonas axonopodis pv. Vascularum, CFBP 5823), Pseudomonas janto pv. Nonnadiadia ( Xanthomonas axonopodis pv.vignaeradiatae , LMG 936), xanthomonas axonopodis pv. Vinicola , ( Xanthomonas axonopodis pv. Vignicola , LMG 8752), xanthomonas axonopodis pv. Vitians ( Xanthomonas axonopodis pv . Vitians , CFBP 2538), Xanthomonas bromi X. bromi , LMG 947), Xanthomonas campestris pv. Everance ( Xanthomonas campestris pv . aberrans , CFBP 6865) , xanthomonas campestris pv. Amadorasiea ( X. campestris pv . armoracieae , CFBP 3838), Xanthomonas Campestris pv. Creeper ( Xanthomonas campestris pv . barbarae , CFBP 5825), Xanthomonas campestris pv. Campestris ( X. campestris pv . campestris , ICMP 13), Xanthomonas campestris pv. Fish ( X. campestris pv . Fici , LMG 701), Xanthomonas populii ( Xanthomonas populi , CFBP 1817), Xanthomonas Campestris pv. Mori ( Xanthomonas campestris pv . mori , ICMP 13199), Xanthomonas Campestris pv. Nigromaculans ( Xanthomonas campestris pv . nigromaculans , LMG 787), Xanthomonas campestris pv. Paulineia ( Xanthomonas campestris pv . paulliniae , CFBP 5862), Xanthomonas Campestris pv. Raphani ( Xanthomonas campestris pv . raphani , ICMP 1404), Xanthomonas Campestris pv. Sesame ( Xanthomonas campestris pv . sesami , ICMP 621), Xanthomonas Campestris pv. Viticola ( X. campestris pv . viticola , ICMP 3867), Xanthomonas cassavae , LMG 673), xanthomonas citri supsp. Citri ( Xanthomonas citri subsp . citri , ICMP15804) , xanthomonas citri subsp. Malvaserum ( Xanthomonas citri subsp . malvaceaerum , ICMP 217), Xanthomonas codiaei ( CFBP 4690), Xanthomonas cucurbita ( Xanthomonas cucurbitae , ICMP 2299), Xanthomonas sina ( Xanthomonas cynarae, CFBP 4188), Pseudomonas janto Juve Brassica thoria (Xanthomonas euvesicatoria, ICMP 109), Pseudomonas janto plastic cover Ke (Xanthomonas fragariae , CFBP 2157) , xanthomonas fuscans pv. Fuscans ( Xanthomonas fuscans pv . fuscans , ICMP 239), Xanthomonas fuskans subsp. Orantipoli ( Xanthomonas fuscans subsp . aurantifolii , ICMP15806), Xanthomonas gardneri ( NCPPB 881), xanthomonas hortorum pv. Carotene ( Xanthomonas hortorum pv . carotae, CFBP 4997) , xanthomonas hortorum pv. Heiderae ( Xanthomonas hortorum pv . hederae , CFBP 4925) , xanthomonas hortorum pv. Pelargoni ( Xanthomonas hortorum pv . pelargonii , CFBP 2533) , xanthomonas hortorum pv. Fallen sashimi ( Xanthomonas hortorum pv . taraxaci , CFBP 410,) , xanthomonas hortorum pv. Vitians ( Xanthomonas hortorum pv . vitians LMG 938), Xanthomonas hyacinthi ( LMG 739), Xanthomonas Melonis ( Xanthomonas melonis , CFBP 4644) , xanthomonas duckwood pv. Duck ( Xanthomonas oryzae pv . oryzae , ICMP 3125), Xanthomonas duckwood pv. Original Cola ( Xanthomonas oryzae pv . oryzicola , LMG 797), Xanthomonas perforans ( NCPPB 4321), Xanthomonas fish ( Xanthomonas pisi , ICMP 570), Xanthomonas sacchari ( CFBP 4641), Xanthomonas teicola ( Xanthomonas theicola , CFBP 4691) , xanthomonas translucence pv. Arhenateri ( Xanthomonas translucens pv . arrhenatheri , CFBP 2056), Xanthomonas translucense serialis ( Xanthomonas translucens pv . cerealis , CFBP 2541) , xanthomonas translucence pv. Graminis ( Xanthomonas translucens pv . graminis , CFBP 2053) , xanthomonas translucense pv. Frey ( Xanthomonas translucens pv . phlei , CFBP 2062) , xanthomonas translucens pv. Playpratensis ( Xanthomonas translucens pv . phleipratensis , ICMP 5744 ), xanthomonas translucens pv. Pore ( Xanthomonas translucens pv . poae , CFBP 2057) , xanthomonas translucens pv. Sepalis ( Xanthomonas translucens pv . secalis , CFBP 2539) , xanthomonas translucense pv. Translucent ( Xanthomonas translucens pv . translucens , CFBP 2054) xanthomonas translucens pv. Underrosa ( Xanthomonas translucens pv . undulosa , ICMP 5755), Xanthomonas Bashicola pv. Whole Cola ( Xanthomonas) vasicola pv . holcicola , CFBP 2543), Xanthomonas vesicatoria ( CFBP 2537), a pair of primers for detecting the genus of xanthomonas , characterized in that selected from the group consisting of.
A composition for detecting microorganisms of the genus Xanthomonas comprising the primer pair of claim 1 or claim 2.
Xanthomonas genus microorganism detection kit comprising a primer pair of claim 1 or 2.
(a) isolating DNA from the sample;
(b) amplifying the DNA extracted in step (a) by PCR using the primer pair of claim 1 or 2; And
(C) Xanthomonas genus detection method comprising the step of confirming the amplified PCR product of step (b) by electrophoresis.

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