Nested-PCR检测种传番茄细菌性溃疡病菌  

Nested-PCR for Detection of Seed-borne Pathogen of Tomato Bacterial Canker,Clavibacter michiganensis subsp.michiganensis

作　　者：闵现华[1,2] 刘箐[2] 韩跃武[1] 文朝慧[2] 王军平[2] 刘姝彤[2] 刘雅莉[2] 宋蕤[2] 施颖波[2] 

机构地区：[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,兰州730000 [2]甘肃出入境检验检疫局,国家质检总局外繁种子检疫重点实验室,兰州730020

出　　处：《西北农业学报》2011年第7期28-31,共4页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：甘肃省科技厅中小企业创新基金计划(0802NCCA028);国家质检总局项目(2007IK259,2009IK276)

摘　　要：利用番茄溃疡病菌特异性引物对纯菌液、模拟带菌种子及自然感染种子提取液分别进行Direct-PCR和Nested-PCR检测。结果在纯菌液中,Direct-PCR的检测灵敏度为105cfu/mL,Nested-PCR为102cfu/mL;在模拟带菌种子提取液中,Direct-PCR的检测灵敏度为107cfu/mL,Nested-PCR为104cfu/mL;在自然感染种子提取液中,稀释104倍后Nested-PCR仍然可以检出。建立的番茄种子Nested-PCR检测技术,无需经过核酸提取步骤,可以在8 h内完成整个检测过程,方便快捷,成本低廉,可作为番茄种子带菌的常规检测方法。Tomato bacterial canker is the most important bacterial diseases which caused substantial economic losses in the worldwide.Seeds were the main pathway of long-distance spread and seed-borne Clavibacter michiganensis subsp.michiganensis（Cmm） were difficult to detect by current methods because of they were low rate of infected.Direct-PCR and Nested-PCR were performed for the detection of Cmm in bacterial suspensions,artificial contaminated seed extracts and naturally infected seed extracts,respectively.The detection sensitivity of Direct-PCR is 105 cfu/mL and 107 cfu/mL in bacterial suspensions and artificial contaminated seed extracts,respectively.But the sensitivity of Nested-PCR was 102 cfu/mL and 104 cfu/mL respectively.And Nested-PCR could provide positive result in 104 fold dilution in naturally infected seed extracts.The Nested-PCR developed in our study was convenient and low-cost,which could be used a routine method for the detection of tomato bacterial canker.

关 键 词：番茄溃疡病 番茄种子 Direct-PCR NESTED-PCR 

分 类 号：S436.412[农业科学—农业昆虫与害虫防治]