绵羊痘病毒和山羊痘病毒实时荧光重组酶聚合酶检测方法的建立  

Development of real-time RPA assay for the detection of SPPV and GTPV

作　　者：杨俊 聂福平 周庆 王昱 史梅梅 李贤良 王国民 吴蕊 张欢 陈冉越 唐昌杰 刘生峰 李应国 YANG Jun;NIE Fu-ping;ZHOU Qing;WANG Yu;SHI Mei-mei;LI Xian-liang;WANG Guo-min;WU Rui;ZHANG Huan;CHEN Ran-yue;TANG Chang-jie;LIU Sheng-feng;LI Ying-guo(Inspection and Quarantine Technology Center,Chongqing Entry-Exit Inspection and Quarantine Bureau of China/Staie Key Laboratory of Bovine Diseases/Engineering Center for Control and Research of Imported Terrestrial Animal Diseases of Chongqing,Chongqing 400020,China;Chongqing Yucheng Product Quality Testing Co.,Ltd.,Chongqing 400020,China)

机构地区：[1]重庆出入境检验检疫局检验检疫技术中心国家牛病重点实验室重庆市进境陆生动物疫病防控研究工程中心,重庆400020 [2]重庆玉成产品质量检测有限公司,重庆400020

出　　处：《中国兽医科学》2019年第5期544-550,共7页Chinese Veterinary Science

基　　金：重庆检验检疫局2017年度自主立项科技计划项目(2017CQIK02);国家质量监督检验检疫总局科技计划项目(2016IK297);重庆市基础研究与前沿探索项目(重庆市自然科学基金)(cstc2018jcyjAX0372)

摘　　要：为建立一种快速、敏感鉴别诊断绵羊痘病毒(SPPV)和山羊痘病毒(G TPV)的方法,本研究根据SPPV和GTPV基因组保守区,分别设计特异性引物和探针,建立了SPPV和GTPV实时荧光RPA (real-time fluorescent recombinase polymerase amplification)检测方法。结果显示,该方法仅对SPPV和GTPV的靶基因扩增呈阳性,而对牛结节性皮肤病病毒(LSDV)、传染性脓疱病毒(O RFV)、蓝舌病病毒(BTV)、小反刍兽疫病毒(PPRV)、水疱性口炎病毒(VSV)、A型口蹄疫病毒(FM DV type A)、O型口蹄疫病毒(FMDV type O)、亚洲1型口蹄疫病毒(FMDV Asia 1)等相关病毒检测结果均为阴性,特异性良好;对SPPV和GTPV检测的灵敏度分别为4.72×10~2copies/μL和4.35×10~2 copies/μL;利用该方法对临床样品进行检测,与OIE推荐的普通PCR检测结果符合率为100%。结果表明,建立的SPPV和GTPV实时荧光RPA方法灵敏、快速、特异性强,为SPPV和GTPV感染的早期诊断提供了技术支持,适用于SPPV和GTPV的检疫、疫情监测及流行病学调查,对疫情暴发后相应控制方案的制定具有重要意义。To establish a sensitive protocol for the detection of sheep pox virus(SPPV) and goat pox virus(GTPV),the real-time fluorescent recombinase polymerase amplification(RPA) was developed with two paires of primers and two probes. The assay was highly specific for amplification from SPPV and GTPV respectively,but no amplification from lumpy skin disease virus,contagious ecthyma virus,bluetongue disease virus,peste des petits ruminants virus,vesicular stomatitis virus,foot-and-mouth disease virus type A,foot-and-mouth disease virus type O,and foot-and-mouth disease virus Asia 1.In addition,the detected limit of the assay was 4.72×10~2 copies/μL for SPPV and 4.35×10~2 copies/μL for GTPV.Using the assay to detect clinical samples,in result,the coincidence between the ordinary PCR assay recommended by OIE and the real-time RPA was 100%. These results showed that the developed real-time RPA assay could be used for the qualitative detection of SPPV and GTPV,providing technical support for the early diagnosis,and was also of great significance for the development of corresponding control measures.

关 键 词：绵羊痘病毒 山羊痘病毒 重组酶聚合酶扩增 

分 类 号：S852.659.6[农业科学—基础兽医学]