西番莲病毒可视化多重RT-LAMP检测技术的建立  

Establishment of Visual Multiplex RT-LAMP Assay for Simultaneous Detection of Passion Fruit Viruses

作　　者：谢丽雪[1] 陈细红 张小艳[1] 陈江姗 张立杰[1] 高芳銮[3] 沈建国 李韬[1] XIE Lixue;CHEN Xihong;ZHANG Xiaoyan;CHEN Jiangshan;ZHANG Lijie;GAO Fangluan;SHEN Jianguo;LI Tao(Fruit Research Institute,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China;Fujian Key Laboratory for Technology Research of Inspection and Quarantine,Technology Center of Fuzhou Customs District,Fuzhou 350001,China;Institute of Plant Virology,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建省农业科学院果树研究所,福州350013 [2]福州海关技术中心/福建省检验检疫技术研究重点实验室,福州350001 [3]福建农林大学植物病毒研究所,福州350002

出　　处：《病毒学报》2023年第6期1682-1692,共11页Chinese Journal of Virology

基　　金：国家重点研发计划项目(项目号:2016YFF020320),题目:高频跨境细菌和病毒高精准检测技术研究;福建省公益类科研院所专项(项目号:2020R1028005),题目:福建省西番莲重要病毒RT-LAMP分子检测技术研究;福建省农业科学院引导性科技创新项目(项目号:YDXM202202),题目:福建省西番莲主要病毒的多重快速检测技术研究;福州海关科研项目(项目号:FK2020-10),题目:西番莲重要病毒分子快速检测技术的研究。

摘　　要：东亚西番莲病毒(East Asian Passiflora virus,EAPV)、夜来香花叶病毒(Telosma mosaic virus,TeMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)是当前危害我国西番莲的主要病毒种类。为建立同时快速检测西番莲上EAPV、TeMV和CMV的可视化多重逆转录环介导等温核酸扩增(Multiplex reverse transcription loop-mediated isothermal amplification,multiplex RT-LAMP)技术,本研究根据GenBank已报道的EAPV、TeMV和CMV外壳蛋白(Coat protein,CP)基因序列保守区域分别设计、筛选特异性引物,并通过反应体系和反应条件的优化,建立了可同时快速检测EAPV、TeMV和CMV的多重RT-LAMP技术,并进行了特异性、灵敏度测定及西番莲果园样品的检测。结果表明,建立的多重RT-LAMP方法特异性良好,可对EAPV、TeMV和CMV同时进行检测,而与西番莲上其他病毒及阴性对照无交叉反应;多重RT-LAMP的灵敏度最低可检测稀释至145×10-3 ng/μL总RNA,与一步法RT-PCR方法的灵敏度相当;西番莲果园样品3种病毒的多重RT-LAMP检测结果与一步法RT-PCR检测结果一致。本研究建立的多重RT-LAMP方法操作简便、特异性强、灵敏度高,适用于基层实验室和现场对西番莲上EAPV、TeMV和CMV的快速初筛。East Asian Passiflora virus(EAPV),telosma mosaic virus(TeMV) and cucumber mosaic virus(cucumber mosaic virus,CMV) are the major viruses that endanger passion fruit in China currently.To establish a visual multiplex reverse transcription loop-mediated isothermal amplification(multiplex RT-LAMP) assay for simultaneous and rapid detection of East Asian Passiflora virus(EAPV),telosma mosaic virus(TeMV) and cucumber mosaic virus(CMV),specific primers were designed and screened according to the conserved region of coat protein(CP) gene sequences of EAPV,TeMV and CMV reported in GenBank.After the optimization of reaction system and reaction conditions,the multiplex RT-LAMP assay was established for rapid detection of EAPV,TeMV and CMV simultaneously.Specificity and sensitivity of the multiplex RT-LAMP were determined and the method was then applied to detect samples collected from passion fruit orchards.The results showed that the multiplex RT-LAMP assay had high specificity for the simultaneous detection of EAPV,TeMV and CMV,while no cross-reactivity was observed with other viruses or the negative control.The detection limit of total RNA detected by the multiplex RT-LAMP assay was diluted up to 145×10-3 ng/μL,which was equivalent to that of the one-step RT-PCR.The detection results of orchard samples showed that the multiplex RT-LAMP was consistent with that of the one-step RT-PCR,with 100%coincidence rate.The established multiplex RT-LAMP assay has the advantages of simple operation,strong specificity and high sensitivity,making it especially suitable for rapid screening of EAPV,TeMV and CMV in both basic laboratory settings and on-site applications.

关 键 词：西番莲 东亚西番莲病毒 夜来香花叶病毒 黄瓜花叶病毒 多重逆转录环介导等温核酸扩增 病毒检测 

分 类 号：S41-30[农业科学—植物保护]