PCV2 PPV PRV和PRRSV多重PCR检测方法的建立及初步应用  

Establishment and application of a multiplex PCR assay for simultaneous detection of PCV2,PPV,PRV and PRRSV

作　　者：岳丰雄[1] 崔尚金[1] 冉多良[2] 张超范[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：《中国兽医科学》2008年第8期691-696,共6页Chinese Veterinary Science

基　　金：农业科技成果转化项目(04EFN216900362);国家"十一五"高技术研究发展计划(863)项目(2007AA100606)

摘　　要：参照GenBank中的猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)、猪生殖与呼吸综合征病毒(PRRSV)基因序列,设计了4对引物分别用于扩增PCV2 ORF2、PPV NS1、PRV gB、PRRSV N基因的目的片段。通过对反应因素进行优化,建立了检测PCV2、PPV、PRV、PRRSV的多重PCR(mPCR)诊断方法。敏感性和特异性结果显示,该mPCR对4种病毒的最低核酸检测量分别为29.0 pg(PCV2)、17.3 pg(PPV)、36.8 pg(PRRSV)、38.4 pg(PRV),而对猪瘟病毒、猪流感病毒、猪圆环病毒1型、牛病毒性腹泻黏膜病病毒、大肠杆菌的扩增结果均为阴性。119份临床样品的多重PCR检测结果显示,有3份样品同时感染PPV和PCV2,68份样品同时感染PRRSV和PCV2,其余样品均为PCV2阳性。表明,建立的多重PCR方法能够对PCV2、PPV、PRV、PRRSV单个或混合感染的临床样品进行快速鉴别诊断。According to the gene sequences in GenBank of porcine circovirus type 2（PCV2）,porcine parvovirus（PPV） ,porcine pseudorabies virus （PRV） and porcine reproductive and respiratory syndrome vi- rus （PRRSV）,four pairs of specific primers were designed for amplifying the four specific fragments of PCV20RF2,PPV NS1,PRV gB and PRRSV N,respectively. After optimization of annealing temperature and primers concentrations, a multiplex PCR assay（mPCR）was established for simultaneous detection of the four viruses. The sensitivity and specificity tests showed that the mPCR was highly sensitive in that as little as 1 pg of the respective virus,29.0 pg（PCV2）, 17.3 pg（PPV） ,36.8 pg（PRRSV） ,38.4 pg（PRV）. No band was amplified from classical swine fever virus（CSFV）, swine influenza virus（SIV）,porcine circovirus type I（PCV1）, bovine viral diarrhea virus（BVDV）,Escherichia coli by the mPCR. 119 clinical samples were detected by the mPCR. As a result, PCV2 was detected from all the samples, moreover, 3 samples were positive for PPV,68 samples were positive for PRRSV. The multiplex method could be used to effectively detect and differentiate PCV2, PPV, PRV and PRRSV single or co-infection infected in clinical samples.

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

分 类 号：S852.659.2[农业科学—基础兽医学] Q503[农业科学—兽医学]