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

作　　者：赵立群[1] 张晓飞 邱艳红 刘慧 张建 杨静静 张海军 徐秀兰 温常龙[2,3] ZHAO Liqun;ZHANG Xiaofei;QIU Yanhong;LIU Hui;ZHANG Jian;YANG Jingjing;ZHANG Haijun;XU Xiulan;WEN Changlong(Beijing Agricultural Extension Station,Beijing 100029,China;Key Laboratory of Biology and Genetics Improvement of Horticultural Crops(North China),Institute of Vegetable Science,Beijing Academy of Agricultural and Forestry Sciences,Beijing 100097,China;Supervision,Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs,Beijing 100097,China)

机构地区：[1]北京市农业技术推广站,北京100029 [2]农业农村部华北地区园艺作物生物学与种质创制重点实验室,北京市农林科学院蔬菜研究所,北京100097 [3]农业农村部蔬菜种子质量监督检验测试中心,北京100097

出　　处：《园艺学报》2022年第11期2471-2478,共8页Acta Horticulturae Sinica

基　　金：国家重点研发计划项目(2017YFD0102004)。

摘　　要：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.

关 键 词：甜瓜内源病毒 TaqMan探针法荧光实时定量PCR 葫芦科作物 种苗带毒 

分 类 号：S652[农业科学—果树学]