猕猴桃褪绿环斑相关病毒RT-LAMP-LFD可视化检测方法的建立  

Establishment of reverse transcription loop-mediated isothermal amplification combined with lateral flow dipstick visual detection method for Actinidia chlorotic ringspot-associated virus

作　　者：邹佳伶 彭期定 王韵茹 席德慧[1] ZOU Jia-Ling;PENG Qi-Ding;WANG Yun-Ru;XI De-Hui(Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,College of Life Sciences,Sichuan University,Chengdu 610065,China)

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610065

出　　处：《四川大学学报（自然科学版）》2022年第1期189-196,共8页Journal of Sichuan University(Natural Science Edition)

基　　金：国家自然科学基金(32070167,31772131);四川省应用基础项目(2019YJ0135);成都市科技局国际合作项目(2020-GH02-00026-HZ)。

摘　　要：猕猴桃褪绿环斑相关病毒(Actinidia chlorotic ringspot-associated virus,AcCRaV)是侵染猕猴桃的主要病毒之一.为实现对猕猴桃中AcCRaV的快速检测和监控,本研究建立了一种逆转录环介导等温扩增(RT-LAMP)结合横向流动试纸条(LFD)的检测方法,可实现快速、可视化检测猕猴桃中AcCRaV.本研究基于AcCRaV外壳蛋白基因保守区域,设计了一套LAMP特异性引物,优化后的反应体系在62℃恒温条件下,反应60 min,再进行5 min试纸条显色反应,即可完成样品的检测.检测灵敏性实验结果表明,建立的RT-LAMP-LFD方法具有较高的灵敏性,其检测灵敏度是常规RT-PCR的100倍.二者检测结果一致,LFD试纸条实现了检测结果的可视化.总之,该检测方法可为检测猕猴桃中AcCRaV感染状况提供了一种省时、高效的诊断方法.Actinidia chlorotic ringspot-associated virus(AcCRaV)is one of the main viruses that infect kiwifruit.For the rapid detection and monitoring of AcCRaV in kiwifruit,in this study,a reverse transcription loop-mediated isothermal amplification(RT-LAMP)combined with lateral flow dipstick(LFD)method was established,which could achieve rapid and visual detection of AcCRaV in kiwifruit.In this experiment,a set of LAMP specific primers was designed based on the conservative region of the AcCRaV coat protein gene.The optimized reaction system was reacted for 60 min under a constant temperature of 62℃,followed by 5 min test strip color reaction to finish the sample detection.The detection sensitivity of the RT-LAMP assay was 100 times higher than that of RT-PCR.The results of RT-LAMP detection of kiwifruit samples were consistent with those by RT-PCR detection,even more,the RT-LAMP results could be visualized through LFD test strips.In conclusion,this method can provide a time-saving and efficient diagnostic tool for detecting AcCRaV infection in kiwifruit.

关 键 词：猕猴桃 猕猴桃褪绿环斑相关病毒 RT-LAMP 横向流动试纸条 

分 类 号：Q945.8[农业科学—植物病理学]