建立快速检测委内瑞拉马脑炎的RT-LAMP技术  

Detection of Venezuelan equine encephalitis virus by reverse transcription loop-mediated isothermal amplification

作　　者：曹杨 李环 冯龙斐 陈璐萍 胡博然[1] 袁静[5] Cao Yang;Li Huan;Feng Longfei;Chen Luping;Hu Boran;Yuan Jing(School of Tourism and Culinary Science,School of Food Science and Engineering,Yangzhou University,Yangzhou 225009,China;Center of Disease Control and Prevention of the People’s Liberation Army(PLA),Beijing 100071,China;Henan Institute of Science and Technology,Xinxiang 453000,China;Food Institute,Shanghai Ocean University,Shanghai 201306,China;Department of Bacteriology,Capital Institute of Pediatrics,Beijing 100020,China)

机构地区：[1]扬州大学旅游烹饪学院,食品科学与工程学院,225009 [2]中国人民解放军疾病预防控制中心,北京100071 [3]河南科技学院,新乡453000 [4]上海海洋大学食品学院,201306 [5]首都儿科研究所细菌室,北京100020

出　　处：《中华实验和临床病毒学杂志》2020年第1期78-82,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家重大科技专项(2016ZX09J16105-001-004);国家重点研发计划项目(2016YFC1202702)。

摘　　要：目的利用逆转录-环介导等温扩增技术(reverse transcription loop-mediated isothermal amplification,RT-LAMP)建立快速检测委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus,VEEV)的方法。方法针对VEEV nsp1基因序列,设计了5套特异性引物进行筛选,再对反应温度进行优化,最后进行特异性、灵敏性及重复性评价。结果RT-LAMP检测VEEV的最适温度在65℃;同时选取11种病毒进行RT-LAMP,证明了RT-LAMP具有高特异性;运用RT-qPCR与RT-LAMP对比灵敏性,结果表现为RT-LAMP检测的灵敏性超RT-qPCR检测的100倍。针对VEEV进行RT-LAMP的重复性实验,结果为具有较好的重复性。结论RT-LAMP检测VEEV法具有简单快速高效的特点,且装置简单,特异性强、灵敏性高、重复性好。Objective The study aimed to establish a rapid method for detection of Venezuelan equine encephalitis virus(VEEV)by reverse transcription loop-mediated isothermal amplification(RT-LAMP).Methods Firstly,5 sets of specific primers were designed for the nsp1 gene sequence of VEEV.We screened for the best primer from the 5 sets of primers.After the best primer was obtained,the temperature gradient experiment was carried out.Then,11 viruses were selected for specific test of VEEV.Finally,RT-LAMP and RT-qPCR were compared for sensitivity.Results The optimum temperature for VEEV detection by RT-LAMP was 65℃.Amplification result of 10 kinds of virus were negative and VEEV were positive.The result showed that the method has good specificity.It was found that the of RT-LAMP was 100 times higher than that of RT-qPCR.Conclusions The detection method of RT-LAMP is not only simple,rapid and efficient,but also has high specificity and high sensitivity.

关 键 词：委内瑞拉马脑炎病毒 环介导等温扩增 快速检测 nsp1基因 

分 类 号：R4[医药卫生—临床医学] S[农业科学]