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

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.