种传番茄溃疡病菌直接PCR和免疫捕捉PCR检测方法之比较  

Evaluation of direct-PCR and immunocapture-PCR for detection seed-borne infection by tomatoes bacterial canker pathogens,Clavibacter michiganensis subsp. Michiganensis

作　　者：闵现华[1,2] 韩跃武[1] 刘箐[2,3] 王军平 刘雅莉[4] 文朝慧 刘姝彤 宋蕤 施颖波 

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,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

出　　处：《植物检疫》2010年第4期12-16,共5页Plant Quarantine

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

摘　　要：用PBS和种子研磨后的普通病原提取液稀释番茄溃疡病菌纯菌液,并以梯度纯菌液和带菌种子提取液作为模板进行直接PCR和免疫捕捉PCR,比较2种方法在纯菌液和带菌种子提取液中的检测灵敏度,找到一种高特异性、高灵敏度、简便快捷的方法用于检测番茄种子携带的番茄溃疡病菌。结果显示在纯菌液中直接PCR灵敏度为104cfu/mL,免疫捕捉PCR为102cfu/mL;在带菌种子提取液中直接PCR灵敏度为106cfu/mL,免疫捕捉PCR为104cfu/mL;免疫捕捉PCR比直接PCR灵敏度高100倍,尤其在实际种子检测中优势更明显,而且检出时间短,重复性好。Clavibacter michiganensis subsp.michiganensis culture were 10-fold diluted in PBS,then draw some dilution as template for direct-PCR and immunocapture-PCR.Seed extract samples were prepared using previously tested noninfected commercial tomato seeds;uncontaminated seed extracts were spiked with Clavibacter michiganensis subsp.michiganensis as described above.Bacterial suspensions,tomato seed extracts spiked with Clavibacter michiganensis subsp.michiganensis were detected by immunocapture-PCR.These results were compared with direct-PCR to find a high specificity,high sensitivity,high sensitivity,simple and fast method for detection of Clavibacter michiganensis subsp.michiganensis in tomato seeds.The direct-PCR provided positive results for 10^4cfu/mL in bacterial suspensions and 10^6cfu/mL in seed extracts respectively.As a new method,Immunocaptre-PCR provided positive results for10^2cfu/mL in bacterial suspensions and 10^4cfu/mL in seed extract respectively,The sensitivity is 100-fold than direct-PCR.Immunocapture-PCR provided the better results regarding sensitivity and specificity for Clavibacter michiganensis subsp.michiganensis detection,the sensitivity and specificity indicated that the immunocapture-PCR developed in our study could be used for routine seed born diseases detection.

关 键 词：番茄溃疡病 番茄种子 直接PCR 免疫捕捉PCR 

分 类 号：S436.412[农业科学—农业昆虫与害虫防治]