基于TaqMan探针的A型塞尼卡病毒荧光定量RT-PCR方法的建立及应用  

Development and Application of A TaqMan-based RT-qPCR Assay for Detection of Senecavirus A

作　　者：郭振华[1] 阮海宇 乔松林[1] 邓瑞广[1] 张改平[1,2,3] GUO Zhen-hua;RUAN Hai-yu;QIAO Song-lin;DENG Rui-guang;ZHANG Gai-ping(Henan Provincial Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou,Henan,450002,China;College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou,Henan,450002,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou,Jiangsu,225009,China)

机构地区：[1]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [2]河南农业大学牧医工程学院,河南郑州450002 [3]江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《动物医学进展》2020年第5期6-12,共7页Progress In Veterinary Medicine

基　　金：国家重点研发计划项目(2018YFD0501102);河南省生猪产业技术体系项目(S2012-06)。

摘　　要：为建立一种快速、特异、灵敏的A型塞尼卡病毒(SVA)检测方法,通过比对我国不同地区SVA流行毒株的VP1基因序列,在其保守区域设计了1组引物和探针,通过条件优化,建立了基于TaqMan探针的荧光定量RT-PCR检测方法。用该方法检测口蹄疫病毒、猪瘟病毒、日本脑炎病毒和猪繁殖与呼吸综合征病毒等均不发生交叉反应。敏感性分析显示,该方法的最低检出量为278拷贝/μL,比常规RT-PCR的灵敏度高100倍。重复性分析显示,所建立方法的批内变异系数为0.3%~1.0%,批间变异系数为0.8%~2.0%,具有良好的稳定性。进一步对16份疑似SVA感染的临床病料检进行了检测,结果显示8份为SVA核酸阳性。提示建立的检测方法可以用于临床SVA感染的鉴别诊断,为我国SVA的诊断和控制提供了良好的技术支撑。To develop a fast,specific and sensitive detection method for Senecavirus A,we designed a set of primers and probe based on sequence alignment of VP1genes from the SVA prevalent strains in different areas of our country.Finally,aTaqMan-based probe quantitative RT-PCR(RT-qPCR)was established by optimizing the conditions.The detection method was no reactions with cDNA of FMDV,CSFV,JEV and PRRSV etc.The sensitivity assay showed that the lowest detected quantity was 278copies.It is about 100 times more than the conventional RT-PCR.The reproducibility of intra-and inter-assay displayed that the coefficient of variations were 0.3%-1.0%and 0.8%-2.0%,respectively.Furthermore,we detected 16clinical samples and 8were confirmed to be SVA positive.The result suggested that this method can be successfully used in the differential diagnosis of clinical SVA infection.Our study provides a good technical support for the diagnosis and control of SVA in China.

关 键 词：塞尼卡病毒 荧光定量RT-PCR 猪水泡病 诊断 

分 类 号：S852.659.2[农业科学—基础兽医学]