甜瓜内源病毒CmEV的TaqMan RT-qPCR检测方法研究

Studies on Detection of Cucumis Melo Endornavirus Using TaqMan RT-qPCR Method

甜瓜内源病毒(cucumis melo endornavirus,Cm EV)是近年来发现的寄主广泛、发病率高、种传率高的重要病毒。利用Cm EV基因组的序列信息设计特异性引物和探针,建立基于探针法的实时荧光定量PCR(Real-Time Fluorescent Quantitative PCR with Taq Man probes,Taq Man RT-q PCR)的检测方法。该方法可以检测出1×10^(-2)ng·μL^(-1)的RNA和1.8×10^(3)copies·μL^(-1)的质粒,灵敏度是普通PCR的100倍。利用该方法检测了在北京、山东、辽宁、海南瓜类种苗主产区的甜瓜、西瓜、黄瓜与南瓜砧木11份种苗带毒情况,发现山东和海南各有1份甜瓜种苗携带Cm EV。本研究建立的CmEV TaqMan RT-q PCR方法特异性好、灵敏度高,可为开展病毒精准鉴定、科学防控以及从源头遏制病毒传播提供技术支撑。

Cucumis melo endornavirus is an important plant virus that infects a wide range of hosts,occurs popularly and transmits highly in seeds.One set of specific primers and probes were designed according to the conserved sequence of Cm EV genome,and a detecting method of Cm EV was established using the Real-Time Fluorescent Quantitative PCR with TaqMan probes(TaqMan RT-qPCR).This detection method could detect the virus in 1×10^(-2)ng·μL^(-1)RNA samples and 1.8×10^(3)copies·μL^(-1)plasmids,the sensitivity of which was about one hundred times higher than traditional RT-PCR.We used the Cm EV TaqMan RT-qPCR technique to detect 11 seedling lots of Cucumis melo,Citrullus lanatus,Cucumis sativus,Cucurbita moschata stock that randomly sampled from the main cucurbit industries of Beijing,Shandong,Liaoning and Hainan provinces.It was found that Cucumis melo seedling from Shandong and Hainan industries carried Cm EV.The novel TaqMan RT-qPCR method was proved to be efficient in testing Cm EV,which could be a technical support for precisely detecting and scientific controlling of this virus,and could help preventing the virus spread from the source.