种传番茄溃疡病菌直接PCR和免疫捕捉PCR检测方法之比较.pdfVIP

2010年第4期 植物检疫PLANTQUARANTINE Vo1．24No．4 种传番茄溃疡病菌直接 PCR和免疫 捕捉 PCR检测方法之 比较 闵现华 ，韩跃武 ，刘 箐 ，王军平 ，刘雅莉 ，文朝慧 ，刘姝彤 ，宋 蕤 ，施颖波 (1．兰州大学基础医学院医学生物化学与分子生物学研究所 甘肃兰州 730000；2．甘肃出入境检验检疫局； 3．兰州大学生命科学院细胞生物学研究所；4．甘肃农业大学食品科学与工程学院) Evaluation ofdirect——PCR and immunocapture——PCR fordetection seed——borneinfection bytomatoes bacterialcankerpathogens，Clavibactermichiganensissubsp．M ichiganensis．MinXianhua ，HanYuewu ， LiuQing ，WangJunping，LiuYali，WenZhaohui，LiuShutong。，SongRui。，ShiYingbo(1．Instituteof MedicalBiochemistry and MolecularBiology，SchoolofBasic MedicalSciences，Lanzhou University，Lanzhou 730000，China；2．GansuEntry—exitInspectionandQuarantineBureau；3．InstituteofCellBiology，Collegeof LifeScience，LanzhouUniversity；4．SchoolofFoodScienceandEngineering，GansuAgriculturalUniversity) Abstract Clavibactermichiganensissubsp．michiganensisculturewere10一folddilutedinPBS，thendraw some dilutionastemplatefordirect～PCR andimmunocapture—PCR．Seedextractsampleswerepreparedusingprevi— ouslytested noninfected commercialtomato seeds；uncontaminated seed extractswere spiked with Clavibacter michiganensissubsp．michiganensisasdescribedabove．Bacterialsuspensions，tomato seed extractsspikedwith Clavibactermichiganensissubsp．michiganensisweredetectedbyimmunocapture—PCR．Theseresuhswerecom— paredwithdirect—PCR tofindahighspecificity，highsensitivity，highsensitivity，simpleandfastmethodforde- tection ofClavibactermichiganensissubsp．michiganensisintomatoseeds．Thedirect—PCR providedpositivere- suhsofr10 cfu／mLinbacterialsuspensionsand10 cfu／mLinseedextractsrespectively．Asanew method．Immu． nocaptre—PCR providedpositiveresultsforl0efu／mLinbacterialsuspensionsand10 cfu／mLinseedextractre— spectively，Thesensitivity is100一foldthandirect—PCR．Immunocapture—PCR provided thebetterresultsre- gardingsensitivityand specificityofrClavibactermichiganensissubsp．michiganensisdetection，thesens