牛结节性皮肤病病毒与山羊痘病毒双重TaqMan实时荧光定量PCR方法的建立  

Establishment and application of a dual TaqMan real-time quantitative PCR method for lumpy skin disease virus and goatpox virus

作　　者：余翰维 冯秋媛 苏洋 张慧 邵晓龙 杨云凤 何小兵[2] 陈国华[2] 房永祥[2] 景志忠[2] 陈轶霞[1] YU Hanwei;FENG Qiuyuan;SU Yang;ZHANG Hui;SHAO Xiaolong;YANG Yunfeng;HE Xiaobing;CHEN Guohua;FANG Yongxiang;JING Zhizhong;CHEN Yixia(College of Life Science and Engineering/Northwest Minzu University,Lanzhou 730030,China;State Key Laboratory for Animal Disease Control and Prevention/Key Laboratory of Veterinary Public Health,Ministry of Agriculture/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州730030 [2]中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室农业部兽医公共卫生重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2024年第9期1188-1194,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32260874);国家动物疫病监测与流行病学调查计划项目(125161031);家畜疫病病原生物学国家重点实验室(应用)基础重大成果培育项目(SKLVEB2021CGQD03);甘肃省科技计划项目(22JR5RA780)。

摘　　要：为建立可快速鉴别牛结节性皮肤病病毒(LSDV)与山羊痘病毒(GTPV)的方法,通过对GenBank中公布的LSDV和GTPV的全基因序列进行比对分析,靶向LSDV基因组的保守区域设计了1对特异性的引物和2条TaqMan探针,经反应条件优化后,建立了一种LSDV和GTPV的双重TaqMan实时荧光定量PCR检测方法,并评估其敏感性、重复性、特异性。结果表明,本方法能从LSDV、GTPV、绵羊痘病毒、羊口疮病毒、牛病毒性腹泻病毒、口蹄疫病毒6种常见的牛源性疾病病原中成功鉴别LSDV和GTPV,具有良好的特异性。LSDV和GTPV基因组的最低检测下限都为3.37×10~1copies/μL。组内、组间变异系数均小于1%。运用本方法对10份临床样品进行检测,符合率达到100%。综上所述,本方法具有良好的特异性、敏感性和重复性,可快速鉴别出样品中的LSDV和GTPV,解决了目前国内容易造成的牛结节性皮肤病漏诊的问题,为全面控制国内牛结节性皮肤病疫情提供了新的技术手段。In order to establish a method to rapidly identify lumpy skin disease virus(LSDV)and goatpox virus(GTPV),the whole gene sequences of LSDV and GTPV published in GenBank were compared and analyzed,and one pair of specific primers and two TaqMan probes were designed based on the conserved sequences of LSDV.After optimization of the reaction conditions,a dual TaqMan real-time quantitative PCR assay for LSDV and GTPV was established and evaluated for sensitivity,reproducibility,and specificity.The results showed that this method can successfully identify LSDV and GTPV from LSDV,GTPV,sheeppox virus,orf virus,bovine viral diarrhea virus and foot-and-mouth disease virus,this result indicates good specificity.The lowest limit of detection for both LSDV and GTPV was 3.37×101 copies/μL.The coefficient of variation was less than 1%for both intra-and inter-group.The method was applied to 10 clinical samples,and the compliance rate was 100%.In conclusion,this method has good specificity,sensitivity and reproducibility,and can rapidly identify LSDV and GTPV in sample.It solves the current situation of overdiagnosis of bovine lumpy skin disease,which is easily caused in China,and provides a new technical means for the comprehensive control of lumpy skin disease in China.

关 键 词：牛结节性皮肤病病毒 山羊痘病毒 双重TaqMan实时定量PCR 

分 类 号：S852.659.1[农业科学—基础兽医学]