牛结节性皮肤病病毒荧光定量PCR检测方法的建立  

Establishment of a real-time PCR for detection of lumpy skin disease virus

作　　者：孔玉方 王慧煜[1] 袁向芬[1] 景志忠[2] 韩雪清[1] KONG Yu-fang;WANG Hui-yu;YUAN Xiang-fen;JING Zhi-zhong;HAN Xue-qing(Chinese Academy of Inspection and Quarantine,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]中国检验检疫科学研究院,北京100176 [2]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《中国兽医科学》2022年第4期439-443,共5页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2016YFD0500907-X)。

摘　　要：根据牛结节性皮肤病病毒(LSDV)的保守基因序列设计了特异性的引物和探针,建立了荧光定量PCR检测方法。对该方法的特异性、敏感性和重复性进行了试验,并用田间临床样品对该方法和OIE推荐的普通PCR进行了验证。结果显示,本研究建立的荧光定量PCR与绵羊痘病毒(SPPV)、山羊痘病毒(GTPV)、牛传染性鼻气管炎病毒(IBRV)和羊口疮病毒(ORFV)均无交叉反应;重组质粒标准品的检测限为1.16×100copies/μL;组间和组内的变异系数均≤2.00%。25份田间临床样品检测结果显示,本研究建立的荧光定量PCR阳性检出率为100%,而OIE推荐的普通PCR方法的阳性检出率为55.6%。上述结果表明,本研究建立的LSDV荧光PCR特异性强、敏感性高、重复性好,为牛结节性皮肤病的监测和防控提供了有效的技术手段。In this study,specific primers and probes were designed based on the conservative gene sequence of LSDV and a fluorescent quantitative PCR detection method was established.The specificity,sensitivity and reproducibility were tested,respectively.Field clinical samples were used to verify real-time PCR and the common PCR recommended by OIE.In result,LSDV could be identified specifically and without any cross reaction with SPPV,GTPV,IBRV and ORFV,respectively.Besides,the real-time PCR detection recombinant plasmid standard sample limited was 1.16×100copies/μL,the coefficients variations were less than or equal to 2%for both intra-assay and inter-assay.Moreover,a clinical test showed that our developed PCR method could accurately detect to field clinical samples of LSDV and detection rate up to 100%.The positive detection rate of the ordinary PCR method recommended by OIE is55.6%.The above-mentioned results showed that the real-time PCR can be used as effective tool for monitoring and prevention and control of LSD.

关 键 词：牛结节性皮肤病病毒 荧光定量PCR 检测方法 

分 类 号：S852.654[农业科学—基础兽医学]