木薯细菌性萎蔫病菌的LAMP快速检测方法  

Loop-mediated isothermal amplification(LAMP)for rapid detection of Xanthomonas axonopodis pv.manihotis

作　　者：封立平[1] 李洪林 倪新 王简[1] 纪瑛[1] 甘琴华[1] 

机构地区：[1]山东出入境检验检疫局,青岛266002 [2]滨州出入境检验检疫局,滨州256603 [3]临沂出入境检验检疫局综合技术服务中心,临沂276034

出　　处：《植物保护》2015年第3期93-99,共7页Plant Protection

基　　金：国家质检总局科技计划项目(2009IK254;2011IK286);"十二五"国家科技支撑计划(2012BAK11B02);山东出入境检验检疫局科研项目(2005SK09)

摘　　要：建立了一种木薯细菌性萎蔫病菌的环介导恒温扩增快速检测方法,为木薯细菌性萎蔫病的快速检测提供有力的技术支持。针对木薯细菌性萎蔫病菌TAL效应器蛋白质(pthBXam)靶序列的6个位点设计4条特异性引物,并对反应温度和内引物浓度等参数进行了优化,设计的引物与试验中提供的其他黄单胞近缘种都没有扩增反应,表现了较好的特异性。LAMP方法对木薯细菌性萎蔫病菌菌株DNA的检测下限为1pg/μL,比常规PCR灵敏度高100倍。该方法采用SYBR Green I染料法对扩增产物闭管检测,裸眼观察颜色变化判断反应结果,能快速、准确地对田间样品进行检测,没有出现假阳性和假阴性。与其他检测方法相比,LAMP方法检测时间短,效率高,降低了设备投入,易于操作,适合木薯细菌性萎蔫病菌的现场检疫和大规模监测。A rapid loop-mediated isothermal amplification （LAMP） method was established for quick detection of Xanthornonas axonopodis pv. manihotis. The target sequence pthBXam was amplified by a set of four specific primers that recognize six distinct target sequences. The parameters, including the reaction temperature and the inner primer concentrations, were optimized. The LAMP primers did not react with other closely related Xan- thomonas in this test. The lower limit of detection of this LAMP assay was about 1 pg/t^L DNA, which was 100 times more sensitive than PCR method. The amplification products could be checked by naked-eye visualization by a closed-tube detection technique with SYBR Green I staining. The LAMP method is simple and rapid method for detection of X. axonopodis pv. rnanihotis from the infected cassava sampled in the field. The LAMP assay has ad- vantage of short detection time, high efficiency and low cost, which provides a new tool for on-site quarantining and large-scale monitoring of this pathogen.

关 键 词：木薯细菌性萎蔫病菌 环介导等温扩增 检测 

分 类 号：S432.41[农业科学—植物病理学] S436.43[农业科学—农业昆虫与害虫防治]