4种猪源病毒多重PCR检测方法的建立及初步应用  

Establishment and Preliminary Application of a Multiplex PCR Assay for Four Kinds of Swine-derived Viruses

作　　者：王莹 刘雨田[1] 孙成友[1] 迟田英[1] 宋芳芳 于小静[1] 吴晓东[1] 王志亮[1] Wang Ying;Liu Yutian;Sun Chengyou;Chi Tianying;Song Fangfang;Yu Xiaojing;Wu Xiaodong;Wang Zhiliang(China Animal Health and Epidemiology Center,Qingdao,Shandong 266032,China)

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266032

出　　处：《中国动物检疫》2021年第6期112-117,共6页China Animal Health Inspection

基　　金：国家重点研发计划项目(2017YFD0501801)。

摘　　要：为建立同时检测非洲猪瘟病毒(ASFV)、尼帕病毒(NiV)、猪水疱病病毒(SVDV)以及口蹄疫病毒(FMDV)等4种猪源病毒的多重PCR检测方法,根据相关文献和GenBank中登录的参考病毒株序列,针对保守区域设计特异性引物,在单一PCR基础上对多重PCR条件进行优化。通过各项反应条件优化,确定ASFV、NiV、FMDV、SVDV引物体积比为2:1:1:1,退火温度为57℃以及35个循环时,扩增效果最好。敏感性试验显示,ASFV、NiV、FMDV和SVDV的最低检测限分别为390.0、46.0、20.9以及37.0 copies/μL。特异性试验显示,建立的多重PCR方法不与猪圆环病毒(PCV)、猪细小病毒(PPV)、猪伪狂犬病毒(PRV)、猪流行性腹泻病毒(PEDV)、猪瘟病毒(CSFV)和猪繁殖与呼吸综合征病毒(PRRSV)发生非特异性反应。利用本研究建立的多重PCR方法对48份临床样品进行检测,得到的检测结果与单一PCR一致。结果表明,建立的多重PCR方法具有敏感性高、特异性强、重复性好的优点。本方法的建立为ASFV、NiV、FMDV和SVDV 4种猪病的快速检测、临床诊断和流行病学调查提供了新技术手段。In order to establish a multiplex PCR assay to simultaneously detect African swine fever virus(ASFV),Nipah virus(NiV),swine vesicular disease virus(SVDV)and foot-and-mouth disease virus(FMDV),multiplex PCR conditions were optimized based on single PCR by specific primers designed for conserved domains according to related literature and the reference virus sequence registered in GenBank.The volume ratio of ASFV,NiV,FMDV and SVDV primers was 2:1:1:1 after all reaction conditions were optimized,and the amplification reached the best effect when the annealing temperature was 57℃with 35 cycles.It was shown that,by sensitivity test,the minimum detection limits were 390.0 copies/μL,46.0 copies/μL,20.9 copies/μL and 37.0 copies/μL for ASFV,NiV,FMDV and SVDV,respectively.It was shown that,by specificity test,the established method could not produce nonspecific reaction with porcine circovirus(PCV),porcine parvovirus(PPV),pseudorabies virus(PRV),porcine epidemic diarrhea virus(PEDV),classical swine fever virus(CSFV) and porcine reproductive and respiratory syndrome virus(PRRSV).48 clinical samples were detected by the established method,and the result was consistent with that by single PCR.It was concluded that the established method was characterized by high sensitivity,strong specificity and good repeatability.A new technique was thereby provided for rapid detection,clinical diagnosis and epidemiological investigation of ASFV,NiV,FMDV and SVDV.

关 键 词：非洲猪瘟 尼帕病毒病 口蹄疫 猪水疱病 猪源疫病 多重PCR 

分 类 号：S855.3[农业科学—临床兽医学]