葡萄卷叶伴随病毒-3 RT-LAMP检测体系的建立与应用  

Development and Application of a RT-LAMP Approach for Detection of Grapevine Leafroll-Associated Virus-3

作　　者：张强强 王雯雯 闫思远 李玲[1] 王若彤 顾沛雯[1,2] ZHANG Qiangqiang;WANG Wenwen;YAN Siyuan;LI Ling;WANG Ruotong;GU Peiwen(School of Forestry and protoculture,Ningxia University,Yinchuan,Ningxia 750021;School of Agricultural,Ningxia University,Yinchuan,Ningxia 750021)

机构地区：[1]宁夏大学林业与草业学院,宁夏银川750021 [2]宁夏大学农学院,宁夏银川750021

出　　处：《核农学报》2023年第11期2166-2174,共9页Journal of Nuclear Agricultural Sciences

基　　金：国家重点研发专项子课题(2019YFD1002502);宁夏回族自治区科技重大专项(2019BBF02013)。

摘　　要：为实现葡萄生产中对葡萄卷叶伴随病毒-3(GLRaV-3)的快速检测。本研究以GLRaV-3的CP基因序列(GenBank登录号:KC477128.1)为靶序列设计并合成了3对逆转录环介导恒温扩增技术(RTLAMP)引物,建立并优化GLRaV-3的RT-LAMP检测方法。结果表明,该方法能够实现在恒温64℃下反应1 h完成对GLRaV-3的检测,引物GLRaV3-LAMP-Ⅰ特异性高,不与GLRaV-1~2、葡萄扇叶病毒、沙地葡萄茎痘相关病毒、葡萄病毒A、灰皮诺葡萄病毒和葡萄浆果内坏死病毒等7种葡萄常见病毒发生交叉反应;RNA最低检测限为10 pg·μL^(-1),灵敏度是逆转录PCR(RT-PCR)的10倍。田间样品检测验证结果表明,该方法与常规RT-PCR法检测一致率为100%。综上,本研究建立的GLRaV-3 RT-LAMP检测体系具有高特异性、高灵敏度和实用性强等特点,可应用于田间葡萄卷叶病样的检测。In order to achieve the rapid detection of grapevine leafroll-associated virus-3(GLRaV-3)in grape production.In this study,three pairs of Reverse transcription loop-mediated isothermal amplification(RTLAMP)primers were designed and synthesized according to the CP gene sequence(GenBank login id:KC477128.1)of GLRaV-3,and the RT-LAMP detection method for GLRaV-3 was established and optimized.The results showed that with this method,GLRaV-3 could be detected at a constant temperature of 64℃for 1 h.The primer GLRaV3-LAMP-Ⅰshowed high specificity,which avoided cross-reacting with seven common grape viruses including GLRaV-1~2,grapevine fan leaf virus,grapevine virus A,grapevine rupestris stem pitting-associated virus,grapevine pinot gris virus and grapevine berry inner necrosis virus.The minimum detection limit of RNA was 10 pg·μL^(-1),which was 10 times more sensitive than reverse transcription-polymerase chain reaction(RT-PCR).The validation results with the field sample demonstrated that the method created in this paper was in 100%agreement with the conventional RT-PCR method.In summary,the GLRaV-3 RT-LAMP detection system established in this study has high specificity,sensitivity and practicality for the detection of grapevine leafroll samples in the field.

关 键 词：葡萄卷叶伴随病毒3 RT-LAMP 引物筛选 反应条件优化 田间检测 

分 类 号：S436.631[农业科学—农业昆虫与害虫防治]