牛结节性皮肤病病毒野毒株TaqMan-MGB荧光定量PCR检测方法的建立  

Development of TaqMan-MGB real-time PCR for rapid detection of wild type lumpy skin disease virus

作　　者：聂福平 孟向阁[2,3] 王昱 杨俊 李贤良 王国民 韩雪清 李应国[1] 侯长军 NIE Fu-ping;MENG Xiang-ge;WANG Yu;YANG Jun;LI Xian-liang;WANG Guo-min;HAN Xue-qing;LI Ying-guo;HOU Chang-jun(Inspection and Quarantine Technology Center,Chongqing Entry-Exit Inspection and Quarantine Bureau of China、Engineering Center for the Research of Imported Terrestrial Animal Disease Prevention and Control in Chongqing、State KeyLaboratory of Bovine Diseases,Chongqing 400020;College of Bioengineering,Chongqing University,Chongqing 400030;College of Animal Science and Technology,Southwest University,Chongqing 400715;China Academy of Inspection and Quarantine,Beijing 100176)

机构地区：[1]重庆出入境检验检疫局检验检疫技术中心/重庆市陆生动物疫病防控技术研究工程中心/牛病检测重点实验室,重庆400020 [2]重庆大学生物工程学院,重庆400030 [3]西南大学动物科技学院,重庆400715 [4]中国检验检疫科学研究院,北京100176

出　　处：《中国兽医科学》2019年第4期403-409,共7页Chinese Veterinary Science

基　　金：国家"十三五"科研项目(2016YFD0500907-X);国家质检公益性项目(201310093);重庆出入境检验检疫局科技自立项目(2017CQIK01)

摘　　要：为鉴别牛结节性皮肤病病毒野毒株(wild type lumpy skin disease virus,WT LSDV),本研究根据GenBank中发表的WT LSDV全基因组序列,设计并合成1对特异性引物和MGB探针,优化反应条件,建立了鉴别检测WT LSDV的TaqMan-MGB荧光定量PCR方法。结果显示,该方法仅对WT LSDV核酸有特异性扩增,对LSDV疫苗株等其他病毒核酸无交叉反应;标准曲线在一定浓度范围内呈现良好的线性关系,标准曲线为y=-3.361 x+34.979,R^2>0.999,扩增效率为98.39%,最低检测限为4.6 copies/μL。组内、组间重复性试验的变异系数均小于3%。对63份模拟临床样品的检测结果显示,本研究建立荧光定量PCR方法的阳性检出率为90.57%,OIE推荐的普通PCR方法的阳性检出率为64.15%,两种方法检测临床样品均为WT LSDV核酸阴性。结果表明,建立的WT LSDV TaqMan-MGB荧光定量PCR方法特异、敏感、快速、重复性好,且能快速准确地检测WT LSDV,适用于临床样品的鉴别检测,为该病的鉴别、监测、防控提供了技术支撑。In order to indent if icate wild type lumpy skin disease virus(WT LSDV),the real-time PCR was established with the specific primers and TaqMan MGB probes designed according to the conserved gene sequences of wi Id LSDV.The reaction conditions,specificity and sensitivity of this method were optimized.The assay was highly specific and sensitive with the detection limit of 4.6 copies/μL for WT LSDV,but no amplication for YM LSDV,SPPV,GTPV,BTV,PPRV,cowpox virus,and swine pox virus.The standard curve exhibited a good 1 inear relationship within a certain concentration range,and the standard curve was y=-3.361x+34.979,R^2>0.999.The amplification efficiency of the method was up to 98.39%.In addition,the coefficient of variation was less than 3.0% for both intra-assay and inter-assay.Moreover,a clinical test showed t hat the met hod could accurately detect the nucleic acids of wi Id LSDV in clinical samples.The above-mentioned results showed that this real-time PCR can be used as an effective tool for rapid detection and epidemic surveillance of LSDV.

关 键 词：牛结节性皮肤病病毒野毒株 荧光定量PCR TaqMan-MGB 

分 类 号：S852.659.1[农业科学—基础兽医学]