小反刍兽疫病毒和蓝舌病病毒多重荧光RT-LAMP鉴别检测方法的建立及应用  

Establishment and Apolication of Multiplex Fluorescence-based RT-LAMP Assay for Identification of Peste Des Petits Ruminants Virus and Bluetongue Virus

作　　者：范晴[1] 谢芝勋[1] 谢志勤[1] 谢丽基[1] 黄娇玲 张艳芳[1] 曾婷婷[1] 王盛[1] 罗思思[1] 邓显文[1] 刘加波[1] FAN Qing;XIE Zhi-xun;XIE Zhi-qin;XIE Li-ji;HUANG Jiao-ling;ZHANG Yan-fan;ZENG Ting-ting;WANG Sheng;LUO Si-si;DENG Xian-wen;LIU Jia-bo(Guangxi Veterinary Research Institute,Guangxi Key Laboratory of Veterinary Biotechnology,Nanning,Guangxi,530001,China)

机构地区：[1]广西兽医研究所广西兽医生物技术重点实验室,广西南宁530001

出　　处：《动物医学进展》2020年第4期1-7,共7页Progress In Veterinary Medicine

基　　金：广西重大专项项目(桂科:AA17204057);广西八桂学者专项项目(2019-79);国家万人计划领军人才专项项目(W02060083)。

摘　　要：旨在建立一种可视化多重荧光RT-LAMP方法用于检测小反刍兽疫病毒和蓝舌病病毒的核酸,并初步应用于小反刍兽疫和蓝舌病临床样品的检测。比对GenBank中小反刍兽疫N基因和蓝舌病NS3基因保守区域,设计2套LAMP引物,在每条内引物FIP的5′端标记荧光基团。对反应条件进行优化,验证方法的特异性、灵敏度和干扰性,同时应用该方法检测168份临床样品。结果表明,该方法对小反刍兽疫病毒和蓝舌病病毒高度特异,与口蹄疫病毒、鹿流行性出血热病毒、牛瘟病毒、山羊痘病毒等其他反刍动物病毒均无交叉反应,检测灵敏度为200 copies/μL,干扰性小,可同时检测两个不同浓度的模板。对168份样品的检测结果显示,小反刍兽疫病毒的感染率为3.6%,蓝舌病病毒的感染率为13.1%,无两种病毒混合感染;与荧光RT-PCR方法相比,此多重荧光RT-LAMP方法敏感性为91.7%~100%,特异性为100%。表明建立的多重荧光RT-LAMP可快速、准确地检测小反刍兽疫病毒和蓝舌病病毒,具有较好的临床应用价值。The aim of this study was to develop a multiplex fluorescence-based reverse transcript loop-mediated isothermal amplification assay(RT-LAMP)for detections of peste des petits ruminants virus and bluetongue virus.The established method was used primarily to test 168 clinical samples for PPRV and BTV.The gene sequences of peste des petits ruminants virus and bluetongue virus were download from GenBank and aligned by biologic software,and two sets of LAMP primers were designed according to the conserved region of N gene for PPRV and NS3 gene for BTV.And the inner primers were synthesized with fluorophore at its 5′end.The primers and reaction conditions were optimized to improve the sensitivity,specificity and interference of RT-LAMP.The results showed that the specificity of the RT-LAMP was high without cross reactions with other sheep viruses such as foot and mouth disease virus(FMDV),epizootic hemorrhagic disease virus(EHDV),rinderpest virus(RPV),and goat pox virus(GTPV).Moreover,the sensitivity of RT-LAMP was 200 copies/μL,and no interference between the high concentration and low concentration contemplate.In the clinical detection test for 168 samples,PPRV,BTV and co-infection positive rates were 3.6%,13.1%and 0%,respectively.Further,the multiplex fluorescence-based RT-LAMP was 91.7%-100%sensitive and 100%specific compared to OIE recommended real-time PCR in detection of clinical samples.This multiplex fluorescence-based RT-LAMP is a rapid,sensitive and specific method for the detections of PPRV and BTV,and would be applied clinically.

关 键 词：小反刍兽疫病毒 蓝舌病病毒 多重荧光RT-LAMP 检测 

分 类 号：S852.65[农业科学—基础兽医学]