TaqMan探针法实时荧光定量RT-PCR检测番茄斑驳花叶病毒  

Detection of tomato mottle mosaic virus using TaqMan RT-qPCR method.

作　　者：黄迎波 王哲 莫瑾 张松柏 彭梓 周慧平 Huang Yingbo;Wang Zhe;Mo Jin;Zhang Songbai;Peng Zi;Zhou Huiping(Changsha Customs Technical Center.Changsha 410004,China;Plant Protection Institute of Hunan)

机构地区：[1]长沙海关技术中心,湖南长沙410004 [2]湖南省植物保护研究所

出　　处：《植物检疫》2023年第4期28-32,共5页Plant Quarantine

基　　金：长沙海关微创新项目(2022)。

摘　　要：番茄斑驳花叶病毒(tomato mottle mosaic virus,ToMMV)为近年来发现的一种烟草花叶病毒属新成员,是茄科作物上危害严重的主要病毒之一,近几年EPPO全球数据库报道多个国家从进境辣椒和番茄种子中截获该病毒。为防止番茄斑驳花叶病毒通过种子种苗进行传播扩散,本文根据该病毒GenBank中不同分离株外壳蛋白(coat protein,CP)基因保守序列设计特异性引物和探针,建立了TaqMan探针法实时荧光定量RT-PCR检测ToMMV的方法。结果表明,本检测方法特异性强,对烟草花叶病毒(tobacco mosaic virus,TMV)、番茄花叶病毒(tomato mosaic virus,ToMV)、番茄褐色皱果病毒(tomato brown rugose fruit virus,ToBRFV)、黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)、辣椒轻斑驳病毒(pepper mild mottle virus,PMMoV)、番茄环斑病毒(tomato ringspot virus,TomRSV)6种病毒均无交叉反应,且检测方法灵敏度高,对RNA模板最低检测限达到了0.08 pg/μL,与普通RT-PCR方法相比灵敏度提高了100倍。研究证明本文建立的TaqMan探针法实时荧光定量RT-PCR检测ToMMV技术特异性强、灵敏度高,适用于口岸和实验室开展ToMMV快速检测和精准鉴定。Tomato mottle mosaic virus(ToMMV)is a new member of the Tobamovirus genus in recent years.It is one of the most important viruses of Solanaceae crops.The EPPO Global Database has reported that several countries have intercepted the virus from imported seeds of pepper and tomato.To prevent the spread of ToMMV through seeds and seedlings,in this study,specific primers and probes were designed according to the conserved sequences of coat protein(CP)genes of different virus isolates in GenBank,and a real-time fluorescence quantitative RT-PCR method with TaqMan probe was developed for the detection of ToMMV.The results showed that the detection method had strong specificity,and there was no cross-amplification to tobacco mosaic virus(TMV),tomato mosaic virus(ToMV),tomato brown rugose fruit virus(ToBRFV),cucumber green mottle mosaic virus(CGMMV),pepper mild mottle virus(PMMoV)and tomato ringspot virus(TomRSV),the detection limit of RNA template was 0.08 pg/μL,which was 100 times higher than that of conventional RT-PCR.The research showed that the real-time fluorescence quantitative RT-PCR assay with TaqMan probe had high specificity and sensitivity,and it was suitable for rapid detection and accurate identification of ToMMV at ports and laboratories.

关 键 词：TAQMAN探针 荧光定量RT-PCR 番茄斑驳花叶病毒 

分 类 号：S41[农业科学—植物保护]