西藏环状病毒可视化RT-LAMP检测方法的建立与应用

Establishment and application of a visual reverse transcriptase loop-mediated isothermal amplification method for detection of Tibetorbivirus

西藏环状病毒(Tibet orbivirus,TIBOV)为2009年在我国西藏新发现的一种环状病毒属病毒,本研究拟建立TIBOV可视化RT-LAMP检测方法。根据我国分离TIBOV(YNSZ/V290/2019)毒株Seg-9基因的保守序列设计2对特异性引物,并引入1对环引物,通过反应条件优化,建立了可视化RT-LAMP检测方法,其最佳反应条件为:64℃50 min。利用该方法对流行性出血病病毒、蓝舌病病毒、中山病病毒、广西环状病毒、阿卡斑病毒、版纳病毒、芒市病毒等虫媒病毒的核酸样品进行检测,仅TIBOV能被检测出,与其他虫媒病毒均无非特异性扩增;灵敏度试验显示该方法的最低可检测限制为18.2拷贝/μL,敏感性是常规RT-PCR的10倍以上。应用建立的RT-LAMP和RT-PCR方法分别对2596只蠓虫和蚊虫样品核酸进行检测,结果显示,两者阳性检出率分别为3.31%和1.19%,RT-LAMP检出率是RT-PCR的2倍以上。本研究建立的TIBOV可视化RT-LAMP检测方法具有特异性强、敏感性高、快速等优点,为我国开展TIBOV的现场快速检测与流行病学研究提供了有效技术支持。

Tibet orbivirus(TIBOV)is a new member of the genus Orbivirus,which was first isolated from Anopheles maculatus collected in Tibet,China in 2009.To establish a visual detection method for TIBOV,two pairs of specific primers and a pair of loop primers were designed according to the conserved region of the Seg-9 gene of TIBOV,and the RT-LAMP assay was developed by optimization of reaction conditions.The established RT-LAMP reaction was carried out at 64℃for 50 min.The method was used to detect RNA samples of epizootic haemorrhagic disease virus,bluetongue virus,Chuzan virus,Guangxi orbivirus,Akabane virus,Banna virus,Mangshi virus and TIBOV,and the results showed that the method had high specificity which could detect RNA samples of the TIBOV,and had no cross-amplifications with RNA samples of the other viruses.The RT-LAMP assay could detect the limitation of 18.2 copies/μL of the gene recombination plasmid of TIBOV,and was 10-fold more sensitive than RT-PCR method.RT-LAMP and RT-PCR methods were used to detect the nucleic acids of 2596 samples of culicoides and mosquitoes respectively,and the positive detection rates of two methods were 3.31%and 1.19%,and the detection rate of RT-LAMP was more than twice that of RT-PCR.The established visualization RT-LAMP is a specific,sensitive,rapid and high throughput method,which provides effective technical support for the rapid detection and the epidemiological study of TIBOV in China.