逆转录交叉引物等温扩增检测南方菜豆花叶病毒方法的建立  

Development of a method of reverse transcription-cross-priming amplification for the detection of southern bean mosaic virus

作　　者：王道 张蓬军 胡学难 魏霜 孙凯 杨倩倩 俞晓平 Wang Dao;Zhang Pengjun;Hu Xuenan;Wei Shuang;Sun Kai;Yang Qian-qian;Yu Xiaoping(Zhejiang Provincial Key Laboratory of Biometrology and Inspection&Quarantine,College of Life Sciences,China Jiliang University,Hangzhou 310018,China;Guangzhou Customs Technology Center)

机构地区：[1]计量大学生命科学学院浙江省生物计量及检验检疫技术重点实验室,浙江杭州310018 [2]广州海关技术中心

出　　处：《植物检疫》2022年第1期39-44,共6页Plant Quarantine

基　　金：国家重点研发计划项目(2018YFC0809200,2017YFF0210200)。

摘　　要：南方菜豆花叶病毒(southern bean mosaic virus,SBMV)是我国进境植物检疫性病毒,可随豆类种子远距离传播。本研究针对SBMV的外壳蛋白(coat protein,CP)基因序列设计特异引物组,建立了一种反转录(reverse transcription,RT)交叉引物扩增(cross-priming amplification,CPA)快速检测方法,并分别结合实时荧光和侧向层析试纸条(lateral flow dipstick,LFD)检测扩增产物构建了实时荧光RT-CPA和RT-CPA-LFD体系。结果显示,所建立的针对SBMV的RT-CPA检测方法特异性强,以其他4种可随豆类种传的植物检疫性病毒为对照,均无交叉反应。其中,实时荧光RT-CPA和RT-CPA-LFD检测SBMV灵敏度分别达5×10^(-4)ng/μL和5 ng/μL。所建立的RT-CPA检测SBMV方法具有快速、准确、可视化等优点,在口岸检测中具有良好的应用前景。Southern bean mosaic virus(SBMV)is listed as a quarantine virus by China,which can be spread over long distance by infected bean seeds.In this study,we designed a group of specific primers targeting on the coat protein gene of SBMV,and developed a fast diagnostic method of reverse transcription-cross-priming amplification coupled with the real-time fluorescent and lateral flow dipstick for SBMV detection.Specificity and sensitivity of the RT-CPA assays were further tested.The results showed that the RT-CPA assays for detecting SBMV were of high specificity.There was no cross reaction when applying the assay on other four species of plant quarantine virus that can be transported by bean seeds.The sensitivity of the real-time fluorescent RT-CPA and RT-CPA-LFD assays for detecting SBMV were5×10^(-4)ng/μL and 5 ng/μL,respectively.The method developed in this study demonstrated advantages in fast,accuracy and visualization,showing a good application prospect at ports.

关 键 词：南方菜豆花叶病毒 等温扩增技术 交叉引物扩增 检测 

分 类 号：S41[农业科学—植物保护]