多重PCR方法用于检测猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒诊断方法的建立  

Multiplex PCR for rapid detection of porcine circovirus type Ⅱ,porcine parvovirus,porcine pseudorabies virus and porcine reproductive and respiratory syndrome virus

作　　者：岳丰雄[1,2] 崔尚金[1] 冉多良[2] 戚亭[1] 周盛华[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室猪传染病研究室,黑龙江哈尔滨150001 [2]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《养猪》2010年第3期41-44,共4页Swine Production

摘　　要：本文建立了一种能够同时检测猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)的多重PCR方法(mPCR)。借助于Oligo6.0引物设计软件,设计了4对引物分别用于扩增PCV2ORF2基因353bp片段、PPVNS-1基因271bp片段、PRRSVMN基因美洲型434bp片段、PRVgB基因194bp片段。通过人为混合以上4种病毒进行特异性及敏感性试验,结果表明,该方法具有很好的特异性和敏感性。对猪瘟病毒、大肠杆菌和双蒸水的PCR扩增结果均为阴性;该mPCR方法对PPV的最低检测量为8.64×10-3μg,PRV的最低检测量为2.36×10-3μg,PRRSV的最低检测量为3.68×10-3μg,PCV2的最低检测量为2.90×10-4μg。该方法的建立对临床上PCV2、PPV、PRV、PRRSV的鉴别诊断以及以上这4种病毒的流行病学调查具有十分重要的意义。A multiplex PCR （mPCR） assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect multiple viral infections of swine.Specific primers for each of four common DNA and RNA viruses were designed by Oligo 6.0 software, namely,porcine circovirus type Ⅱ （PCV20RF2）,porcine parvovirus （PPV NS-1）,pseudorabies virus （PRV gB）,porcine reproductive and respiratory syndrome virus（PRRSV MN）, four specific bands,respectively,353 bp （PCV2）,271 bp （PPV）,434 bp （PRRSV） and 194 bp（PRV）,were amplified.The assay was shown to be highly sensitive and specificity when detect composite of all four virus by purpose.It was also effective for detecting one or more of these four same viruses in various combinations in specimens including lymph nodes,lungs,spleens,and tonsils collected from clinically ill pigs,and aborted fetuses. The relative efficiency （compared to performing separate assays for each virus） and apparent sensitivity of mPCR suggest its potential application for routine molecular diagnostic purposes and investigation epidemiology.

关 键 词：多重PCR方法 猪圆环病毒2型 猪细小病毒 猪伪狂犬病病毒 猪繁殖与呼吸综合征病毒 

分 类 号：S858.28[农业科学—临床兽医学]