马铃薯卷叶病毒RT-LAMP检测方法的建立  

Establishment of the Detection Method for Potato Leafroll Virus by RT-LAMP

作　　者：高彦萍[1,3] 吕和平 张武[1,3] 王国祥 吴雁斌[1,3] 梁宏杰 GAO Yanping;LYU Heping;ZHANG Wu;WANG Guoxiang;WU Yanbin;LIANG Hongjie(Potato Institute,Gansu Academy of Agricultural Sciences,Lanzhou,Gansu 730070;Institute of Chinese Herbal Medicines,Gansu Academy of Agricultural Sciences,Lanzhou,Gansu 730070;Gansu Potato Seed(seedling)Virus Detection and Evaluation of Engineering Technology Research Center,Lanzhou,Gansu 730070)

机构地区：[1]甘肃省农业科学院马铃薯研究所,甘肃兰州730070 [2]甘肃省农业科学院中药材研究所,甘肃兰州730070 [3]甘肃省马铃薯脱毒种薯(种苗)病毒检测及安全评价工程技术研究中心,甘肃兰州730070

出　　处：《核农学报》2020年第9期1943-1950,共8页Journal of Nuclear Agricultural Sciences

基　　金：甘肃省农业生物技术研究与应用开发项目(GNSW-2016-15);国家重点研发计划项目(2017YFD0201602-4);甘肃省农业科学院院列项目(2019GAAS04、2017GAAS29)。

摘　　要：为建立一种快速准确检测马铃薯卷叶病毒(potato leafroll virus,PLRV)的方法,本研究以PLRV CP(coat protein,CP)基因ORF3保守序列为靶标,建立了PLRV反转录环介导等温扩增(RT-LAMP)检测体系,并对建立的RT-LAMP检测体系的特异性、灵敏度、实际应用效果进行评估。结果表明,建立的PLRV RT-LAMP检测方法,在62℃恒温下扩增50 min,扩增产物中加入双链嵌合荧光染色(SYBR GreenⅠ),阳性样品颜色变为绿色,阴性样品颜色仍为褐色,可直接目测判断待检测样品是否感染PLRV。该方法只特异性检测PLRV,不与马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯S病毒(PVS)、马铃薯A病毒(PVA)和马铃薯M病毒(PVM)等其他5种马铃薯主要病毒发生交叉反应。此外,建立的PLRV RT-LAMP检测方法的灵敏度较反转录PCR(RT-PCR)方法高100倍。田间样品检测验证,该方法与标准RT-PCR方法符合率达100%。本研究建立的以SYBR GreenΙ为颜色指示的PLRV可视化检测RT-LAMP方法,特异、灵敏、便捷,成本低,可满足科研、基层单位对该病毒快速诊断和检测的需要。The objective of this study is to establish a quick and accurate detection technique for Potato leafroll virus(PLRV)by reverse transcription loop-mediated isothermal amplification(RT-LAMP).The RT-LAMP detection system of PLRV that based on the conserved region ORF3 of PLRV CP gene was developed,and the specificity,sensitivity and practical detection were evaluated.The results showed that with the established RT-LAMP method,after amplification at 62℃for 50 min,the products was detected according to the color by adding SYBR Green I to the reaction,the positive sample showed green and the negative sample appeared brown.This visualized method suggested that it can be used to detect PLRV directly.The developed RT-LAMP showed highly specific for PLRV by using different RNA templates from PLRV,and had no cross reaction with Potato virus X(PVX),Potato virus Y(PVY),Potato virus S(PVS),Potato virus A(PVA),Potato virus M(PVM)and the healthy control.Furthermore,the sensitivity of RT-LAMP was 100-fold higher than that of conventional RT-PCR.When detecting 90 field samples,the result obtained by RT-LAMP was totally coincident with that by RT-PCR.The visualized RT-LAMP detection for PLRV using SYBR Green as color indicator is specific,sensitive,simple and low-cost,and can meet the needs of scientific research and grass-roots units for the rapid diagnosis and detection of PLRV.

关 键 词：马铃薯卷叶病毒 反转录环介导等温扩增 反转录聚合酶链式扩增 检测 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]