西藏环状病毒荧光定量RT-PCR检测方法的建立  

Development of the detection method of real-time RT-PCR assay for Tibet orbivirus

作　　者：李楠[1] 何于雯 孟锦昕 王静林[1] LI Nan;HE Yu-wen;MENG Jin-xin;WANG Jing-lin(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory/Yunnan Veterinary and Animal Science Institute,Kunming 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：《中国兽医科学》2020年第11期1360-1364,共5页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2016YFD0500303);云南省科技厅重点基金项目(2019FA015);国家自然科学基金项目(31660714);云南省畜牧兽医科学院基础研究项目(2019RW006,2019RW007,2019RW009);云南省科技人才和平台计划项目(2018HB046)。

摘　　要：为了建立检测西藏环状病毒(Tibet orbivirus,TIBOV)的快速核酸检测方法,根据新分离的西藏环状病毒(DH13C120)Seg-10基因序列,设计了特异性引物及探针。通过优化反应条件,建立了检测西藏环状病毒核酸的荧光定量RT-PCR方法,并对该方法的特异性、敏感性和重复性进行了检测。结果显示,该方法在2.31×10^8~2.31×10^2copies/μL质粒标准品浓度之间具有良好的线性关系,相关系数为0.999;组内与组间的变异系数均小于2%;最低检测下限为2.31×10^1copies/μL;对蓝舌病病毒、鹿流行性出血热病毒、阿卡斑病毒、盖塔病毒和流行性乙型脑炎病毒检测结果均为阴性,无交叉反应;对云南新分离的9株西藏环状病毒进行检测,拷贝数在3.23×10^5~1.54×10^6copies/μL之间,准确率为100%;对2013年采集自云南江城县的48份牛血液样品进行检测,检出西藏环状病毒阳性3份。结果表明,本研究建立的西藏环状病毒荧光定量RT-PCR检测方法具有良好的特异性、敏感性和重复性,适用于西藏环状病毒的快速检测,对西藏环状病毒的检测和流行病学调查提供了技术手段。To develop a rapid method for nucleic acid detection of Tibet orbivirus(TIBOV),the specific primers and probe were designed according to the Seg-10 gene sequence of the newly isolated Tibet orbivirus(DH13C120).The real-time quantitative RT-PCR assay for detecting the nucleic acid of Tibet orbivirus was optimized after modification,and the specificity,sensitivity and repeatability for this optimized assay were evaluated.The results demonstrated that this assay had a good linear relationship between 2.31×10^8 and 2.31×10^2 copies/L of plasmid standard concentration,and the linear correlation coefficient was 0.999.Both the intra-and inter-group coefficients of variation were less than 2%.The minimum detection limit was 2.31×10^1 copies/L.No cross reaction with Bluetongue virus,Epizootic hemorrhagic disease virus,Akabane virus,Getah virus and Japanese encephalitis virus.9 new isolates of Tibet orbivirus isolated in Yunnan Province were detected,and the copy number was 3.23×10^5-1.54×10^6 copies/L,indicating the accuracy of this assay was 100%.48 cattle blood samples collected from Jiangcheng County of Yunnan Province in 2013 were detected,and the results showed that 3 samples were positive for Tibet orbivirus.In conclusion,the new established real-time RT-PCR assay for Tibet orbivirus detection have good specificity,sensitivity and repeatability,and is suitable for the rapid detection of Tibet orbivirus,which provide an potential effective method for detection and epidemiological investigation of Tibet orbivirus.

关 键 词：西藏环状病毒 荧光定量RT-PCR 检测方法 

分 类 号：S882.659.4[农业科学—特种经济动物饲养]