黄瓜绿斑驳花叶病毒SYBR GreenⅠ实时荧光RT-PCR检测方法的建立  

Development of SYBR Green Ⅰ Real-time RT-PCR for the Detection of Cucumber green mottle mosaic virus

作　　者：田沂民 吴建祥[2] 陈磊 罗金燕 滕凯 叶军 杜伟 于翠 TIAN Yi-Min;WU Jian-Xiang;CHEN Lei;LUO Jin-Yan;TENG Kai;YE Jun;DUWei;YU Cui(Technical Center for Animal,Plant and Food Inspection and Quarantine of Shanghai Customs District,Shanghai 200135,China;College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China;Shanghai Agricultural Technology Extension Center,Shanghai 201103,China;Division of Animal and Plant Quarantine and Inspection of Shanghai Customs District,Shanghai 200082,China;Agricultural Technology Extension Station of Ningxia,Yinchuan 750001,China)

机构地区：[1]上海海关动植物与食品检验检疫技术中心,上海200135 [2]浙江大学农业与生物技术学院,杭州310058 [3]上海农业技术推广服务中心,上海201103 [4]上海海关动植物检疫监管处,上海200082 [5]宁夏回族自治区农业技术推广总站,银川750001

出　　处：《农业生物技术学报》2022年第8期1640-1648,共9页Journal of Agricultural Biotechnology

基　　金：上海市科技兴农项目(2019-02-08-00-08-F01150);宁夏回族自治区重点研发计划(2020BBF03004)。

摘　　要：黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)是我国重要的检疫性植物种传病毒,在世界各地均有发生,造成西瓜(Citrullus lanatus)、黄瓜(Cucumis sativus)、甜瓜(Cucumis melo)等作物的严重经济损失。CGMMV可由种子直接传播至下一代秧苗,为保障种子贸易顺利进行,有必要建立快速而准确的病毒检测方法。本研究以截获的带毒种子为材料,针对CGMMV建立了SYBR GreenⅠ实时荧光RT-PCR检测方法,其中设计筛选特异、灵敏的引物是关键。利用多次筛选得到的引物对建立了SYBR GreenⅠ实时荧光RT-PCR检测方法,实验结果表明该方法能够特异地检测CGMMV,不与其他侵染葫芦科植物的病毒发生交叉反应;灵敏度是RT-PCR方法的1000倍,是Taq Man探针实时荧光RT-PCR方法的100倍;且整个检测过程可在2.5 h内完成,是一种快速、灵敏、可靠的检测方法;应用该方法及Taq Man实时荧光RT-PCR方法同时对国内外198份种苗样品进行检测,结果显示kappa值大于0.82,说明两种方法具有高度一致性,本研究建立的SYBR GreenⅠ实时荧光RT-PCR方法有效、可靠,为种子快速通关提供新的技术手段。Cucumber green mottle mosaic virus(CGMMV) is an important quarantine plant virus and is also transmitted by seeds which often causes seriously damages to productions of watermelon(Citrullus lanatus),melon(Cucumis melo), cucumber(Cucumis sativus), and other cucurbits. To guarantee the smooth proceeding of seed trade, it is necessary to establish more rapidly and precisely detection methods. This study developed a SYBR Green Ⅰ real-time RT-PCR technique for detection of CGMMV in seeds. It is crucial to design and screen a pair of specific primers based on the conserved CGMMV gene sequences. The result showed that the development of SYBR GreenⅠreal time RT-PCR method in this study could detect CGMMV specially, and there was no cross-reactivity with other common cucurbits viruses. Sensitivity assay showed that the sensitivity of this method was 1 000 times that of RT-PCR and 100 times that of TaqMan RT-PCR. And the whole detection process could be completed within 2.5 h, indicating that the SYBR GreenⅠreal-time RT-PCR was a rapid, sensitive and simple detection method. Cucurbitaceae seeds, seedlings, and fruits imported and purchased from domestic market were employed to be detected by SYBR Green Ⅰ real-time RT-PCR and Taq Man real-time RT-PCR simultaneously. The result showed that the value of kappa of the two method was above 0.82, indicating that the novel SYBR GreenⅠreal-time RT-PCR method could be effective and reliable.The present study provides new technical means for rapid clearance of seeds.

关 键 词：黄瓜绿斑驳花叶病毒(CGMMV) SYBR GreenⅠ实时荧光RT-PCR 种子 灵敏度 特异性 

分 类 号：S41-30[农业科学—植物保护]