微滴数字PCR(ddPCR)检测马铃薯S病毒(PVS)  

Detection of Potato virus S(PVS) by Droplet Digital PCR(ddPCR)

作　　者：张永江[1] 黄洁芳 王溪桥 周慧[4] 李晓宇 付伟[1] 朱鹏宇[1] 王晨光[1] 

机构地区：[1]中国检验检疫科学研究院,北京100176 [2]吴江出入境检验检疫局,吴江215200 [3]甘肃出入境检验检疫局综合技术中心,兰州730010 [4]海南出入境检验检疫局热带植物隔离中心,海口570311

出　　处：《农业生物技术学报》2017年第10期1721-1728,共8页Journal of Agricultural Biotechnology

基　　金：“十三五”国家重点研发计划项目(No.2016YFF0203203)

摘　　要：马铃薯(Solanum tuberosum)为世界重要农作物之一,受马铃薯S病毒(Potato virus S,PVS)侵染会导致其品质下降,产量降低;灵敏准确的检测技术将有助于提高PVS的检出率,进而防止其传播扩散危害马铃薯生产,保护马铃薯产业发展。为了建立PVS灵敏准确的检测技术,本研究根据其外壳蛋白(coat protein,CP)基因保守序列设计了微滴数字PCR(droplet digital PCR,ddPCR)的引物探针,并进行了特异性、灵敏度及重复性验证。结果表明,所建立的方法对PVS具有良好的特异性,能够有效区分对照病毒马铃薯X病毒(Potato virus X,PVX)、马铃薯Y病毒(Potato virus Y,PVY)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和烟草花叶病毒(Tobacco mosaic virus,TMV),与传统qRT-PCR结果一致;检测灵敏度可达20拷贝/μL的RNA,比qRT-PCR灵敏度高10倍;3次重复实验微滴数的标准误差小于总微滴数的1.5%,表明所建立的ddPCR方法具有良好的重复性。本研究建立的ddPCR方法可用于PVS的准确检测,也为其他植物病毒的准确检测提供了借鉴。Potato （Solanum tuberosum） is one of the most important crops in the world and it can be infected by Potato virus S （PVS）, which lead to reduction of quality and yield. The high detection rate of PVS, which can be improved by the sensitive and accurate detection technology, will control the PVS threaten for potato production because its introduction and spread is prevented, and the potato industry can be protected. For developing a sensitive and accurate technique, the droplet digital PCR （ddPCR） primers and probe were designed according to the conserved sequence of PVS coat protein （CP） gene and tested through specificity, sensitivity and reproducibility experiment. The results showed that the established method was specific to PVS, and it could effectively distinguish PVS from the control viruses Potato virus X （PVX）, Potato virus Y （PVY）, Cucumber mosaic virus （CMV） and Tobacco mosaic virus （TMV）, which was same with the result of qRT-PCR. The detection sensitivity of ddPCR was up to 20 copies/μL of RNA which was 10-fold higher than that of qRT-PCR. The standard error of three repeats droplets was less than 1.5% of the total number of droplets, indicating the established ddPCR method had good reproducibility. The ddPCR method established in this study can be used for the accurate detection of PVS and also provides a reference for the accurate detection of other plant viruses.

关 键 词：马铃薯S病毒(PVS) 微滴数字PCR(ddPCR) 检测鉴定 特异性 灵敏度 

分 类 号：S41-30[农业科学—植物保护]