版纳病毒和芒市病毒可视化RT-LAMP检测方法的建立及应用  

Establishment and Application of a Visual RT-LAMP Method for Detection of Banna Virus and Mangshi Virus

作　　者：杨振兴[1] 李卓然 何于雯 李占鸿 李乐[1] 廖德芳[1] 杨恒[1] 朱建波[1] YANG Zhen-xing;LI Zhuo-ran;HE Yu-wen;LI Zhan-hong;LI Le;LIAO De-fang;YANG Heng;ZHU Jian-bo(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Yunnan Veterinary and Animal Science Institute,Kunming,Yunnan,650224,China)

机构地区：[1]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,云南昆明650224

出　　处：《动物医学进展》2022年第6期6-12,共7页Progress In Veterinary Medicine

基　　金：2019年云南省“万人计划”产业技术领军人才项目;基础研究计划青年项目(2018FD002);国家重点研发计划项目(2017YFC1200505)。

摘　　要：为建立版纳病毒(BAV)和芒市病毒(MSV)的可视化RT-LAMP检测方法,根据我国分离的BAV(YNSZ043)和MSV(V301/YNJH/2019)毒株Seg-12基因的保守序列,分别设计2对特异性引物和1条环引物,通过反应条件优化,分别建立了2种病毒的可视化RT-LAMP检测方法,其最佳反应条件为64℃50 min,分别利用2种方法检测西藏环状病毒、流行性出血病病毒、蓝舌病病毒、中山病病毒、广西环状病毒和阿卡斑病毒等虫媒病毒的核酸样品,结果2种方法仅能分别检测出BAV和MSV,其他虫媒病毒均无非特异性扩增。灵敏度试验检出BAV和MSV的最低检测限制分别为13.7拷贝/μL和19.0拷贝/μL,敏感性是常规RT-PCR的10倍以上。应用2种病毒的RT-LAMP及RT-PCR方法分别对2596只蠓虫和蚊虫的核酸样品进行检测,结果阳性检出率均是RT-PCR的2倍以上。结果表明,建立的BAV和MSV可视化RT-LAMP检测方法具有特异性强、敏感性高和可视化等优点,为我国开展2种病毒的现场快速检测与流行病学研究提供了有效的技术支撑。To establish a visual detection method for Banna virus(BAV)and Mangshi virus(MSV),two pairs of specific primers and a pair of loop primers were designed respectively according to the conserved region of the Seg-12 gene of BAV(YNSZ043)and MSV(V301/YNJH/2019)strains isolated in China,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.Two methods were used to detect RNA samples of Tibet orbivirus,Epizootic haemorrhagic disease virus,Bluetongue virus,Chuzan virus,Guangxi orbivirus,Akabane virus,BAV and MSV,and the results showed that the two methods had high specificity which could detecte RNA samples of the BAV and MSV respectively,and had no cross-amplifications with RNA samples of the other viruses.In the sensitivity test,the minimum detectable limits for detecting BAV and MSV are 13.7 copies/μL and 19.0 copies/μL,respectively,and the sensitivity is more than 10 times that of conventional RT-PCR.The RT-LAMP and RT-PCR methods of the two viruses were used to detect the RNA of 2596 culicoides and mosquito samples,and the positive detection rate was more than twice that of RT-PCR.The established visualization RT-LAMP for BAV and MSV were a specific,sensitive,rapid and high throughput method,which provides effective technical support for the rapid detection and the epidemiological study of BAV and MSV in China.

关 键 词：版纳病毒 芒市病毒 反转录-环介等温扩增 可视化检测 

分 类 号：S852.659.4[农业科学—基础兽医学] S854.43[农业科学—兽医学]