PCV2、PRV、PPV多重PCR检测方法的建立及其应用

Establishment and Application of a Multiplex PCR for Detecting Porcine Circovirus Type 2,Porcine Pseudorabies Virus and Porcine Parvovirus

根据GenBank中已发表的猪圆环病毒2型(PCV2)、猪伪狂犬病毒(PRV)、猪细小病毒(PPV)有关基因序列,通过序列比对及参考相关文献,设计了3对特异性引物。通过优化,建立了可同时检测PCV2、PRV及PPV 3种病毒的多重PCR方法。结果表明,该方法能同时检测到PCV2、PRV及PPV的最低核酸量分别为40pg、72pg、46pg。对猪圆环病毒3型(PCV3)、猪细环病毒(TTSuV)、猪流行腹泻病毒(PEDV)、猪蓝耳病毒(PRRSV)、猪瘟病毒(CSFV)无扩增。应用该方法对87份临床样品进行检测,PCV2阳性检出率为28.7%(25/87),PRV野毒检出率为11.5%(10/87),PPV检出率为12.6%(11/87)。其中2种以上病毒混合感染阳性率为13.8%(12/87),3种病毒同时感染的阳性率为2.3%(2/87),其结果也与单一PCR结果一致。研究结果提示,该方法可用于PCV2、PRV、PPV三种病原的单一感染或混合感染的鉴别诊断。

A multiplex PCR was established and optimized to simultaneously detect porcine circovirus type 2(PCV2),porcine pseudorabies virus(PRV)and porcine parvovirus(PPV).Three pairs of specific primers were designed for the multiplex PCR based on the available sequences of the three viruses in GenBank.The results of sensitivity show that the detect limitation for PCV2,PRV and PPV is 40 pg,72 pg and 46 pg respectively.Compared to porcine circovirus type 3(PCV3),torque teno sus virus(TTSuVs),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV)and porcine epidemic diarrhea virus(PEDV),the multiplex PCR shows great specificity of PCV2,PRV and PPV.Eighty seven clinical samples were detected using the multiplex PCR.The results show the positive rates of PCV2,PRV and PPV are 28.7%(25/87),11.5%(10/87)and 12.6%(11/87),respectively.Additionally,the positive rate of the co-infection of two or three viruses is 13.8%(12/87),the positive rate of simultaneous infection of the three viruses is 2.3%(2/87).The results are also consistent with the single PCR results.This study provids meaningful information for the differential diagnosis of the single infection or co-infection of PCV2,PRV and PPV in clinical samples.