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当前位置: 首页 > 文章 > 马铃薯M病毒微滴数字PCR检测方法的建立 河南农业科学 2018 (5) 69-74
Position: Home > Articles > Establishment of Droplet Digital PCR(ddPCR) for Detection of Potato Virus M Journal of Henan Agricultural Sciences 2018 (5) 69-74

马铃薯M病毒微滴数字PCR检测方法的建立

作  者：
刘晗;黄洁芳;向均;燕照玲;张永江
关键词：
马铃薯;马铃薯M病毒;微滴数字PCR;实时荧光RT-PCR;检测
摘  要：
为了建立马铃薯M病毒(Potato virus M,PVM)的精准检测技术,根据PVM外壳蛋白基因核苷酸保守序列设计微滴数字PCR(dd PCR)的引物、探针,建立PVM的dd PCR检测技术。以田间采集并经RT-PCR验证的PVM及其他4种同样可侵染马铃薯的病毒(PVX、PVY、CMV、TMV)总RNA为模板,进行dd PCR特异性分析;对PVM的总RNA进行10倍梯度稀释并用作模板,进行dd PCR灵敏度测定。结果表明,建立的dd PCR方法可以特异性地检测马铃薯上的PVM,与实时荧光RT-PCR结果一致;最低可检测5.7 fg/μL的总RNA,比实时荧光RT-PCR灵敏度高100倍。说明建立的dd PCR检测方法可用于PVM的准确鉴定。
译  名：
Establishment of Droplet Digital PCR(ddPCR) for Detection of Potato Virus M
作  者：
LIU Han;HUANG Jiefang;XIANG Jun;YAN Zhaoling;ZHANG Yongjiang;Chinese Academy of Inspection and Quarantine;Wujiang Entry-Exit Inspection and Quarantine Bureau;Institute of Agricultural Economics and Information,Henan Academy of Agricultural Sciences;
单  位：
LIU Han%HUANG Jiefang%XIANG Jun%YAN Zhaoling%ZHANG Yongjiang%Chinese Academy of Inspection and Quarantine%Wujiang Entry-Exit Inspection and Quarantine Bureau%Institute of Agricultural Economics and Information,Henan Academy of Agricultural Sciences
关键词：
Potato;;Potato virus M(PVM);;Droplet digital PCR;;Real-time fluorescence RT-PCR;;Detection
摘  要：
For establishing a sensitive and accurate detection technique of potato virus M(PVM),the droplet digital PCR(ddPCR) primers and probe were designed according to the conserved nucleotide sequence of PVM coat protein gene.The ddPCR method was established through a specificity experiment using total RNAs of PVM,which was isolated from field potato and identified by RT-PCR,and other four potato-infecting viruses as templates and a sensitivity experiment using ten dilutions of PVM total RNA as templates.The results showed that the established method was specific for PVM,and it could effectively distinguish PVM from the control viruses,potato virus X(PVX),potato virus Y(PVY),cucumber mosaic virus(CMV) and tobacco mosaic virus(TMV),which was the same with the result of real-time fluorescence RT-PCR.The detection sensitivity of ddPCR was up to 5.7 fg/μL of total RNA,which was 100-fold higher than that of real-time fluorescence RT-PCR.The ddPCR method established in this study can be used for the accurate detection of PVM.

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