马铃薯S病毒PVS^O株系RT-LAMP检测方法的建立及应用  

Establishment of Loop-mediated Isothermal Amplification Assay for Detection of Potato virus S Ordinary Strain

作　　者：李华伟[1] 许泳清[1] 罗文彬[1] 纪荣昌[1] 刘中华[1] 许国春 张鸿[1] 李国良[1] 林赵淼[1] 邱永祥[1] 邱思鑫[1] 汤浩[1] LI Huawei;XU Yongqing;LUO Wenbin;JI Rongchang;LIU Zhonghua;XU Guochun;ZHANG Hong;LI Guoliang;LIN Zhaomiao;QIU Yongxiang;QIU Sixin;and TANG Hao＊(Crop Research Institute,Fujian Academy of Agricultural Sciences,Scientific Observing and Experimental Station of Tuber and Root Crops in South China,Ministry of Agricultrue,P R.China,Fuzhou 350013,China)

机构地区：[1]福建省农业科学院作物研究所农业部南方薯类科学观测实验站,福州350013

出　　处：《园艺学报》2018年第8期1613-1620,共8页Acta Horticulturae Sinica

基　　金：福建省公益类科研院所基本科研专项(2016R1025-8);国家现代农业产业技术体系建设专项资金项目(CARS-10-ES09);国家重点研发计划课题(2018YFD0200809);福建省农业科学院创新团队项目(STIT2017-2-3)

摘　　要：为建立一种马铃薯S病毒普通株系PVS^O快速、灵敏的RT-LAMP可视化检测方法。根据马铃薯S病毒普通株系的CP核苷酸序列设计4条引物,以感染PVS的马铃薯叶片总RNA为模板,采用一步法RT-LAMP,在62℃下反应60 min,扩增产物利用琼脂糖电泳分析和钙黄绿素染料显色判断结果,同时对其特异性和灵敏性进行测定。结果表明,建立的RT-LAMP方法可特异地对PVS^O进行检测,检测灵敏度为RT-PCR的100倍。对33份马铃薯样品检测表明,RT-LAMP与RT-PCR方法检测结果基本一致。因此建立的PVS^O的RT-LAMP可视化检测方法简便,特异性强,灵敏度高,适合PVS^O的快速检测。The object of this study is to develop a One-step reverse transcription loop-mediated isothermal amplification（RT-LAMP）assay for rapid and sensitive detection of Potato virus S ordinary strain（PVS^O）. Four RT-LAMP primers were designed on the basis of the coat protein（CP）gene sequences of PVS^O,and template RNA from infected leaves were used for One-step RT-LAMP which were carried out under isothermal conditions at 62 ℃ for 60 minutes. RT-LAMP products were analyzed by electrophoresis in agarose gels followed by staining with Calcein. Then the specificity and sensitivity of RT-LAMP were testified. A rapid and specific RT-LAMP method for detection of PVS^O was established. Sensitivity of the RT-LAMP assay was 100-fold higher than ordinary RT-PCR method,and the testing results of 33 potato samples were basically consistent with RT-PCR. The RT-LAMP described in this study represents a sensitive,specific and rapid assay for the detection of PVS^O.

关 键 词：马铃薯 马铃薯S病毒 环介导等温扩增 钙黄绿素 

分 类 号：S641[农业科学—蔬菜学]