KR20150074845A - Method for making prove or primer for detecting Xanthomonas campestris pv. lespedezae - Google Patents
Method for making prove or primer for detecting Xanthomonas campestris pv. lespedezae Download PDFInfo
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Abstract
Description
본 발명은 식물 병원세균인 Xanthomonas campestris pv. lespedezae를 용이하게 검출할 수 있는 프로브 또는 프라이머의 제조 방법에 관한 것이다. The present invention relates to a plant pathogenic bacterium, Xanthomonas campestris pv. to a method for producing a probe or a primer capable of easily detecting lespedezae .
식물 병원세균의 유전자 진단방법으로는 Dot-blot, 중합효소 연쇄반응(PCR) 등의 방법이 이용된다. 특히 PCR 은 Dot-blot 방법보다 시간, 노력, 인력 등이 경제적이기 때문에 최근 널리 사용되고 있는 방법이다. PCR(중합효소 연쇄반응, polymerase chain reaction) 진단 방법은 10~20bp로 구성된 특정 DNA 단편(프라이머)을 세균의 게놈유전자(genomic DNA)에 접촉(annealing)한 후 내열성 DNA 중합효소(Taq polymerase)를 첨가하고 합성반응을 반복적으로 행하게 되면, 프라이머가 결합된 영역이 급속히 증폭하게 되며, 그 PCR 증폭산물을 아가로스 젤(agarose gel) 또는 아크릴아마이드 젤(acrylamide gel)에 전기영동하고 에티디움 브로마이드(ethidium bromide) 및 은 염색(silver stain)으로 DNA를 염색한 후 나타나는 DNA 밴드의 유무 혹은 형태의 차이와 같은 세균종의 DNA 다형성을 검출 진단하는 방법이다. 현재, 이러한 PCR에 의한 세균의 진단법은 인체나 동물의 세균성 질병을 진단에 주로 이용되나, 최근에는 식물 병원균을 진단할 수 있도록 PCR 진단법이 개발되어 병의 진단과 함께 수출입 농산물의 검역에 이용되고 있다. 또한, PCR 진단 방법은 다른 방법에 비해 소요되는 시간이 짧고, 진단에 소요되는 비용이 저렴하며 또한, 많은 샘플을 동시에 검정할 수 있어 경제적이다. 이에 최근에는 잔토모나스(Xanthomonas) 검출방법 중에서 유전물질인 핵산을 증폭하는 PCR 진단 방법이 가장 선호되고 있다.Dot-blot and polymerase chain reaction (PCR) are used for the genetic diagnosis of plant pathogenic bacteria. In particular, PCR is a widely used method because it is economical in terms of time, effort, and manpower than the dot-blot method. PCR (polymerase chain reaction) is a diagnostic method in which a specific DNA fragment (primer) composed of 10-20 bp is annealed to a genomic DNA of a bacterium and then a heat-resistant DNA polymerase The PCR amplification product is electrophoresed on an agarose gel or an acrylamide gel and ethidium bromide is added to the reaction mixture, The DNA polymorphism of bacterium species such as the presence or absence of DNA bands after DNA staining with bromide and silver stain is detected and diagnosed. Currently, the PCR method is mainly used to diagnose bacterial diseases in humans and animals. Recently, PCR diagnosis method has been developed for diagnosis of plant pathogens, and is used for quarantine of agricultural products in addition to diagnosis of diseases . In addition, the PCR diagnosis method is shorter in time than other methods, is low in cost for diagnosis, and is also economical because many samples can be assayed at the same time. Recently, a PCR method for amplifying nucleic acid as a genetic material among Xanthomonas detection methods is most preferred.
잔토모나스(Xanthomonas) 속 미생물은 그람 양성의 간상 세균으로서 토양질소의 탈질에 관여하며, 복숭아나무 세균성구멍병, 무 검은빛썩음병, 감귤 궤양병, 벼흰빛잎마름병, 강남콩 불마름병 등 다양항 식물병을 유발한다.Microorganisms in Xanthomonas are gram-positive pathogenic microorganisms and are involved in the denitrification of soil nitrogen and cause various plant diseases such as peach tree bacterial pericarditis, non-black rot, citrus ulcer disease, do.
잔토모나스 속 미생물에 의해 감염된 식물의 경우, 병든 식물에서 세균을 진단 및 동정을 위하여 병든 부분 표면살균, 고체배양기 위에서 배양을 위하여 2일 이상이 소요되며, 배양된 병원세균을 순수 분리를 위하여 3회의 순수 분리과정에 3일 이상이 소요되며, 순수 분리된 미생물을 상업화된 프로그램인 Biolog에 의해 분석하거나 혹은 지방산 분석방법으로 분석하는데 2일 이상이 소요된다. In the case of plants infected by microorganisms of the genus Zanthomas, it takes more than 2 days to cultivate on diseased part surface sterilization and solid culture for the diagnosis and identification of bacteria in diseased plants, It takes more than three days for the pure separation process and it takes more than two days to analyze purely isolated microorganisms by the commercial program Biolog or by fatty acid analysis method.
기존의 잔토모나스 속 미생물 중, 벼흰잎마름병원균(Xanthomonas oryzae pv. oryzae), 세균성점무늬병원균(Xanthomonas campestris pv. vesicatoria), 십자화과 흑부병원균(Xanthomonas campestris pv. campestris), 감귤류 궤양병 병원균(Xanthomonas axonopodis pv. citri) 및 콩불마름병원균(Xanthomonas axonopodis pv. glycines) 등의 검출에 사용되는 개별적인 PCR 프라이머에 대한 연구는 이루어졌으나, 잔토모나스 속 가운데 Xanthomonas campestris pv. lespedezae에 특이적 프라이머에 대하여는 아직까지 보고된 바 없다.Among the existing microorganisms of the genus Zanthomas, the pathogenic bacteria such as Xanthomonas oryzae pv. oryzae ), bacterial spotty pathogen ( Xanthomonas campestris pv. vesicatoria ), cruciferous black pathogens ( Xanthomonas campestris pv. campestris ), citrus ulcerative pathogen ( Xanthomonas axonopodis pv. citri and Xanthomonas axonopodis pv. glycines , etc., have been studied. However, in the case of Xanthomonas campestris pv. No specific primers for lespedezae have been reported yet.
그러므로 Xanthomonas campestris pv. lespedezae를 쉽고 빠르게 진단할 수 있도록, PCR 진단이나 DNA 칩에 이용할 수 있는 Xanthomonas campestris pv. lespedezae에 특이적인 프라이머 또는 프로브의 개발이 요구되는 실정이다.Therefore, Xanthomonas campestris pv. In order to diagnose lespedezae easily and quickly, Xanthomonas which can be used for PCR diagnosis and DNA chip campestris pv. development of primers or probes specific for lespedezae is required.
이에 본 발명이 해결하고자 하는 과제는, Xanthomonas 속 식물 병원세균 가운데 Xanthomonas campestris pv. lespedezae를 용이하게 검출할 수 있는 프라이머 또는 프로브를 제공하는 것이다.Accordingly, a problem to be solved by the present invention is to prevent Xanthomonas campestris pv. the present invention provides a primer or a probe capable of easily detecting a lespedezae .
상기 기술적 과제를 달성하기 위하여, 본 발명에서는 서열번호 1로 표시되는 Xanthomonas campestris pv. lespedezae 23S rRNA의 염기서열을 이용하는 것을 특징으로 하는 Xanthomonas campestris pv. lespedezae 검출용 프라이머 제조 방법을 제공한다.According to an aspect of the present invention, Xanthomonas campestris pv. < RTI ID = 0.0 > Xanthomonas < / RTI > campestris pv. A method for producing a primer for detecting lespedezae is provided.
또한, 본 발명은 서열번호 1로 표시되는 Xanthomonas campestris pv. lespedezae 23S rRNA의 염기서열과 동일하거나 그에 상보적인 염기서열을 포함하는 Xanthomonas campestris pv. lespedezae 검출용 프로브 제조 방법을 제공한다.In addition, the present invention relates toXanthomonas campestris pv.lespedezae 23S < / RTI > rRNA, which comprises a nucleotide sequence identical or complementary to the nucleotide sequence of < RTI ID =Xanthomonas campestris pv.lespedezae A method for producing a detection probe is provided.
또한, 본 발명은 (a) 시료로부터 DNA를 추출하는 단계; (b) 상기 (a)단계에서 추출된 DNA를 서열번호 1로 표시되는 Xanthomonas campestris pv. lespedezae 23S rRNA의 염기서열을 이용하는 Xanthomonas campestris pv. lespedezae 검출용 프라이머 제조 방법으로 제조된 프라이머를 이용하여 PCR로 증폭하는 단계; 및 (c) 상기 (b)단계의 증폭된 PCR 산물을 전기영동으로 확인하는 단계;로 이루어진 Xanthomonas campestris pv. lespedezae 검출 방법을 제공한다.(A) extracting DNA from a sample; (b) contacting the DNA extracted in the step (a) with Xanthomonas campestris pv. Xanthomonas using the base sequence of lespedezae 23S rRNA campestris pv. a step of PCR amplification using a primer prepared by a method for producing a primer for detection of lespedezae ; And (c) confirming the amplified PCR product of step (b) by electrophoresis. The Xanthomonas campestris pv. lespedezae detection method.
또한, 본 발명은 (a) 시료로부터 DNA를 추출하여 중합효소연쇄반응(PCR)을 수행하여 PCR 산물을 얻는 단계; (b) 상기 PCR 산물을 서열번호 1로 표시되는 Xanthomonas campestris pv. lespedezae 23S rRNA의 염기서열과 동일하거나 그에 상보적인 염기서열을 포함하는 Xanthomonas campestris pv. lespedezae 검출용 프로브에 결합시키는 단계; 및 (c) 상기 결합 여부에 따라 상기 시료의 Xanthomonas campestris pv. lespedezae를 판별하는 단계;를 포함하는 Xanthomonas campestris pv. lespedezae 검출 방법을 제공한다.(A) extracting DNA from a sample and performing a polymerase chain reaction (PCR) to obtain a PCR product; (b) The PCR product was digested with Xanthomonas campestris pv. Xanthomonas < / RTI > containing the nucleotide sequence identical or complementary to the base sequence of the lespedezae 23S rRNA campestris pv. binding to a probe for detection of lespedezae ; And (c) determining whether or not the Xanthomonas campestris pv. determining the lespedezae , including Xanthomonas campestris pv. lespedezae detection method.
본 발명의 Xanthomonas campestris pv. lespedezae 23S rRNA의 특이적 염기 및 염기조합을 이용해 Xanthomonas campestris pv. lespedezae 및 이의 병원형을 용이하게 검출할 수 있는 프로브 또는 프라이머를 제조할 수 있다.The Xanthomonas campestris pv. Using specific base and base combinations of lespedezae 23S rRNA, Xanthomonas campestris pv. a probe or a primer capable of easily detecting lespedezae and its pathotype can be produced.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.
Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.
XanthomonasXanthomonas campestriscampestris pvpv . . lespedezaelespedezae 식물 병원세균의 23S 23S of botanical hospital bacteria rRNArRNA 유전자 분석 Gene analysis
하기 실험 방법을 통하여, 서열번호 1로 표시되는 Xanthomonas campestris pv. lespedezae 23S rRNA의 염기서열을 Xanthomonas campestris pv. campestris 23S rRNA의 염기서열과 비교하였다.
Through the following experimental procedure, Xanthomonas < RTI ID = 0.0 > campestris pv. The base sequence of lespedezae 23S rRNA was identified as Xanthomonas campestris pv. were compared with the nucleotide sequences of campestris 23S rRNA.
RTRT -- PCRPCR 을 통한 through cDNAcDNA 증폭 Amplification
서열번호 1로 표시되는 Xanthomonas campestris pv. lespedezae 23S rRNA를 cDNA로 만들어 증폭하기 위해 한국 공개특허공보 10-2012-0053792에 공개된 Xan23SF5(5-TGGTCAAGCCGCACGGATCATTAGTAT-3) 및 Xan23SR6(5-ACGTGGATAGCCTGCGAAAAGTGTC-3)를 프라이머로 이용하였다. Xanthomonas < RTI ID = 0.0 > campestris pv. Xan23SF5 (5-TGGTCAAGCCGCACGGATCATTAGTAT-3) and Xan23SR6 (5-ACGTGGATAGCCTGCGAAAAGTGTC-3) disclosed in Korean Patent Publication No. 10-2012-0053792 were used as primers to amplify and amplify lespedezae 23S rRNA into cDNA.
시료로부터 RNA를 분리하여 RT-PCR을 수행하였다. 단일 cDNA 사슬을 주형으로 상기 프라이머와 Taq 폴리머라제(Pyrobest, Takara)를 사용하여 RT-PCR 반응을 수행하였다. 증폭된 cDNA 절편을 클로닝하고 전체 염기서열을 결정하였다.
RNA was isolated from the samples and subjected to RT-PCR. The RT-PCR reaction was performed using the above primers and Taq polymerase (Pyrobest, Takara) with a single cDNA chain as a template. The amplified cDNA fragment was cloned and the entire nucleotide sequence was determined.
염기서열의 정렬(Alignment of the base sequence ( alignmentalignment ))
서열번호 1로 표시되는 Xanthomonas campestris pv. lespedezae 23S rRNA를 GenBank에 보고된 X. campestris pv. campestris의 23S rRNA (NC010688.1)와 비교하였다. 비교한 결과, 78, 86, 698, 740, 1177, 1370, 1725~1726, 1735, 1966~1967, 1969, 1973, 1977~1979, 2048~2050, 2053, 2056, 2058, 2060~2061, 2626, 2649, 2715, 2737의 염기 G, C, C, A, C, A, (CG)결손, T, AT, C, C, CTC, GAG, G, A, A, AT, C, T, C, G 유전자 염기 및 유전자 염기 조합과 이의 상보적 염기 및 염기조합으로 X. campestris pv. lespedezae를 구별할 수 있다.1 < / RTI > Xanthomonas campestris pv. lespedezae 23S rRNA in X. campestris pv. Compared with 23S rRNA of campestris (NC010688.1). As a result of comparison, it was found that the values of 78, 86, 698, 740, 1177, 1370, 1725 to 1726, 1735, 1966 to 1967, 1969, 1973, 1977 to 1979, 2048 to 2050, 2053, 2056, 2058, 2060 to 2061, 2626, G, C, C, A, C, A, (CG) deletion, T, AT, C, C, CTC, GAG, G, A, A, AT, C, T, C, G gene base and gene base combination and its complementary base and base combination, X. campestris pv. lespedezae .
<110> Rural development administration <120> Method for making prove or primer for detecting Xanthomonas campestris pv. lespedezae <130> 2013-0281-10-A (P13-277) <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 2714 <212> RNA <213> Xanthomonas campestris pv. lespedezae 23S rRNA <400> 1 acacacctga cctatcaacc acgtagtcta catggttcct ttagggggct tgtgccccgg 60 gaagtctcat cttgaggcgc gcttcccgct tagatgcttt cagcggttat cgcttccgaa 120 catagctacc cggcaatgcc actggcgtga caaccggaac accagaggtt cgtccactcc 180 ggtcctctcg tactaggagc agcccctctc aaacttccaa cgcccatggc agatagggac 240 cgaactgtct cacgacgttc tgaacccagc tcgcgtacca ctttaaatgg cgaacagcca 300 tacccttggg accgactaca gccccaggat gtgatgagcc gacatcgagg tgccaaacac 360 cgccgtcgat atgaactctt gggcggtatc agcctgttat ccccggagta ccttttatcc 420 gttgagcgat ggcccttcca tacagaacca ccggatcact aagacctact ttcgtacctg 480 cttgatccgt cgatcttgca gtcaagcacg cttatgcctt tgcacacagt gcgcgatgtc 540 cgaccgcgct gagcgtacct tcgtgctcct ccgttactct ttaggaggag accgccccag 600 tcaaactacc caccatacac ggtccctgat ccggataacg gacctaggtt agaacgtcaa 660 gcacgacagg gtggtatttc aaggatggct ccactgcagc tagcgccaca gtttcatagc 720 ctcccaccta tcctacacag acgaactcaa cgttcagtgt aaagctatag taaaggttca 780 cggggtcttt ccgtcttgcc acgggaacgc tgcatcttca cagcgatttc aatttcactg 840 agtctcgggt ggagacagcg ccgctgtcgt tacgccattc gtgcaggtcg gaacttaccc 900 gacaaggaat ttcgctacct taggaccgtt atagttacgg ccgccgttta ctggggcttc 960 gatcaagagc ttcgccttgc ggctgacccc atcaattaac cttccagcac cgggcaggcg 1020 tcacacccta tacgtccact ttcgtgtttg cagagtgctg tgtttttgat aaacagtcgc 1080 agcggcctgg tttctgcgac cctcttcagc tatagctcgc acgagccacc aaaaagggtg 1140 caccttctcc cgaagttacg gtgccatgtt gcctagttcc ttcacccgag ttctctcaag 1200 cgcctgagaa ttctcatcct acccacctgt gtcggtttac ggtacggtct tcgtgagctg 1260 aagcttagga gcttttcctg gaagcgtggt atcagtgact tcgccataaa ggctagtctc 1320 ggtgctcggt cttaaaggat cccggatttg ccaaagatcc aaacctaccg cctttccccg 1380 ggacaaccaa cgcccggtac acctaacctt ctccgtccct ccatcgcact cacgcgaggt 1440 gcaggaatat taacctgctt cccatcgact acggctttcg ccctcgcctt agggaccgac 1500 taaccctgcg tcgattaacg ttgcgcaagg aaaccttggg ctttcggcgt gcgggctttt 1560 cacccgcatt atcgttactc atgtcagcat tcgcacttcc gatacctcca gcagacttct 1620 caatccacct tcgcaggctt acggaacgct cctctaccgc gcataaaaca agttttatgc 1680 accccaagct tcggttcact gcttagcccc gttaaatctt ccgcgcagac cgactcgacc 1740 agtgagctat tacgctttct ttaaagggtg gctgcttcta agccaacctc ctggctgtct 1800 atgcctttcc acatcgtttt ccacttagca gtgaatttgg gaccttagct gtgggtctgg 1860 gttgtttccc ttttcacgac ggacgttagc acccgccgtg tgtctcccat acagtccgtc 1920 tcggtattcg gagtttgcaa tggtttggta agtcgcgatg accccctagc cataacagtg 1980 ctctaccccc gagaggatac atatgaggcg ctacctaaat agctttcgag gagaaccagc 2040 tatctccggg ttcgattagc ttttcactcc taatcacagc tcatccccgt cttttgcaac 2100 agacgtgggt tcgggcctcc agtacctgtt acggcacctt caccctggcc atgactagat 2160 cacccggttt cgggtctact gcccgcgact atgcgccctt atcagactcg gtttcccttc 2220 gcctccccta tacggttaag cttgccacga acagtaagtc gctgacccat tatacaaaag 2280 gtacgcagtc actcttgcga gctcctactg cttgtacgca cacggtttca ggatctattt 2340 cactcccctc tccggggttc ttttcgcctt tccctcacgg tactggttca ctatcggtcg 2400 gtcaggagta tttagccttg gaggatggtc cccccatatt cagacagggt ttcacgtgcc 2460 ccgccctact cgtcttcact ggagtggccc ttttaaatac agggctatca ccttctatgg 2520 ccaatctttc cagattgttt ttctaaagcc attccagctt aagggctgct ccccgttcgc 2580 tcgtcactac ttagggaatc tcggttgatt tcttttcctc cggttactta gatatttcag 2640 ttcaccgggt tcgcttccag cagctatgaa ttcactgcag gatactgccg aagcagtggg 2700 tttccccatt cgga 2714 <110> Rural development administration <120> Method for making prove or primer for detecting Xanthomonas campestris pv. lespedezae <130> 2013-0281-10-A (P13-277) <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 2714 <212> RNA <213> Xanthomonas campestris pv. lespedezae 23S rRNA <400> 1 acacacctga cctatcaacc acgtagtcta catggttcct ttagggggct tgtgccccgg 60 gaagtctcat cttgaggcgc gcttcccgct tagatgcttt cagcggttat cgcttccgaa 120 catagctacc cggcaatgcc actggcgtga caaccggaac accagaggtt cgtccactcc 180 ggtcctctcg tactaggagc agcccctctc aaacttccaa cgcccatggc agatagggac 240 cgaactgtct cacgacgttc tgaacccagc tcgcgtacca ctttaaatgg cgaacagcca 300 tacccttggg accgactaca gccccaggat gtgatgagcc gacatcgagg tgccaaacac 360 cgccgtcgat atgaactctt gggcggtatc agcctgttat ccccggagta ccttttatcc 420 gttgagcgat ggcccttcca tacagaacca ccggatcact aagacctact ttcgtacctg 480 cttgatccgt cgatcttgca gtcaagcacg cttatgcctt tgcacacagt gcgcgatgtc 540 cgaccgcgct gagcgtacct tcgtgctcct ccgttactct ttaggaggag accgccccag 600 tcaaactacc caccatacac ggtccctgat ccggataacg gacctaggtt agaacgtcaa 660 gcacgacagg gtggtatttc aaggatggct ccactgcagc tagcgccaca gtttcatagc 720 ctcccaccta tcctacacag acgaactcaa cgttcagtgt aaagctatag taaaggttca 780 cggggtcttt ccgtcttgcc acgggaacgc tgcatcttca cagcgatttc aatttcactg 840 agtctcgggt ggagacagcg ccgctgtcgt tacgccattc gtgcaggtcg gaacttaccc 900 gacaaggaat ttcgctacct taggaccgtt atagttacgg ccgccgttta ctggggcttc 960 gatcaagagc ttcgccttgc ggctgacccc atcaattaac cttccagcac cgggcaggcg 1020 tcacacccta tacgtccact ttcgtgtttg cagagtgctg tgtttttgat aaacagtcgc 1080 agcggcctgg tttctgcgac cctcttcagc tatagctcgc acgagccacc aaaaagggtg 1140 caccttctcc cgaagttacg gtgccatgtt gcctagttcc ttcacccgag ttctctcaag 1200 cgcctgagaa ttctcatcct acccacctgt gtcggtttac ggtacggtct tcgtgagctg 1260 aagcttagga gcttttcctg gaagcgtggt atcagtgact tcgccataaa ggctagtctc 1320 ggtgctcggt cttaaaggat cccggatttg ccaaagatcc aaacctaccg cctttccccg 1380 ggacaaccaa cgcccggtac acctaacctt ctccgtccct ccatcgcact cacgcgaggt 1440 gcaggaatat taacctgctt cccatcgact acggctttcg ccctcgcctt agggaccgac 1500 taaccctgcg tcgattaacg ttgcgcaagg aaaccttggg ctttcggcgt gcgggctttt 1560 cacccgcatt atcgttactc atgtcagcat tcgcacttcc gatacctcca gcagacttct 1620 caatccacct tcgcaggctt acggaacgct cctctaccgc gcataaaaca agttttatgc 1680 accccaagct tcggttcact gcttagcccc gttaaatctt ccgcgcagac cgactcgacc 1740 agtgagctat tacgctttct ttaaagggtg gctgcttcta agccaacctc ctggctgtct 1800 atgcctttcc acatcgtttt ccacttagca gtgaatttgg gaccttagct gtgggtctgg 1860 gttgtttccc ttttcacgac ggacgttagc acccgccgtg tgtctcccat acagtccgtc 1920 tcggtattcg gagtttgcaa tggtttggta agtcgcgatg accccctagc cataacagtg 1980 ctctaccccc gagaggatac atatgaggcg ctacctaaat agctttcgag gagaaccagc 2040 tatctccggg ttcgattagc ttttcactcc taatcacagc tcatccccgt cttttgcaac 2100 agacgtgggt tcgggcctcc agtacctgtt acggcacctt caccctggcc atgactagat 2160 cacccggttt cgggtctact gcccgcgact atgcgccctt atcagactcg gtttcccttc 2220 gcctccccta tacggttaag cttgccacga acagtaagtc gctgacccat tatacaaaag 2280 gtacgcagtc actcttgcga gctcctactg cttgtacgca cacggtttca ggatctattt 2340 cactcccctc tccggggttc ttttcgcctt tccctcacgg tactggttca ctatcggtcg 2400 gtcaggagta tttagccttg gaggatggtc cccccatatt cagacagggt ttcacgtgcc 2460 ccgccctact cgtcttcact ggagtggccc ttttaaatac agggctatca ccttctatgg 2520 ccaatctttc cagattgttt ttctaaagcc attccagctt aagggctgct ccccgttcgc 2580 tcgtcactac ttagggaatc tcggttgatt tcttttcctc cggttactta gatatttcag 2640 ttcaccgggt tcgcttccag cagctatgaa ttcactgcag gatactgccg aagcagtggg 2700 tttccccatt cgga 2714
Claims (4)
(b) 상기 (a)단계에서 추출된 DNA를 서열번호 1로 표시되는 Xanthomonas campestris pv. lespedezae 23S rRNA의 염기서열을 이용하는 Xanthomonas campestris pv. lespedezae 검출용 프라이머 제조 방법으로 제조된 프라이머를 이용하여 PCR로 증폭하는 단계; 및
(c) 상기 (b)단계의 증폭된 PCR 산물을 전기영동으로 확인하는 단계;로 이루어진 Xanthomonas campestris pv. lespedezae 검출 방법.(a) extracting DNA from a sample;
(b) contacting the DNA extracted in step (a) with Xanthomonas campestris pv. Xanthomonas campestris pv. using the base sequence of lespedezae 23S rRNA. a step of PCR amplification using a primer prepared by a method for producing a primer for detection of lespedezae ; And
Xanthomonas consisting of; (c) identifying the amplified PCR product of the step (b) by electrophoresis campestris pv. lespedezae detection method.
(b) 상기 PCR 산물을 서열번호 1로 표시되는 Xanthomonas campestris pv. lespedezae 23S rRNA의 염기서열과 동일하거나 그에 상보적인 염기서열을 포함하는 Xanthomonas campestris pv. lespedezae 검출용 프로브에 결합시키는 단계; 및,
(c) 상기 결합 여부에 따라 상기 시료의 Xanthomonas campestris pv. lespedezae를 판별하는 단계;를 포함하는 Xanthomonas campestris pv. lespedezae 검출 방법.(a) extracting DNA from a sample and performing PCR to obtain a PCR product;
(b) The PCR product was digested with Xanthomonas campestris pv. Xanthomonas campestris pv. < / RTI > containing the nucleotide sequence identical or complementary to the base sequence of the lespedezae 23S rRNA. binding to a probe for detection of lespedezae ; And
(c) determining whether or not the Xanthomonas campestris pv. determining the lespedezae , including Xanthomonas campestris pv. lespedezae Detection method.
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| CN111057777B (en) * | 2019-12-30 | 2022-07-12 | 北京市农林科学院 | Molecular marker and primer pair for identifying xanthomonas campestris 9 physiological race and application thereof |
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