猪细小病毒、猪伪狂犬病病毒和猪圆环病毒2型多重PCR检测方法的建立与应用

根据GenBank中已发表的猪细小病毒(porcine parvovirus,PPV)、猪伪狂犬病病毒(pseudorabies virus,PRV)和猪圆环病毒2型 (porcine circovirus type 2,PCV2)基因序列,对各病毒基因区进行同源性分析,确定PPV 的VP2、PRV的 gD、和PCV2的ORF2基因为各病毒的诊断靶序列,设计特异性引物,在建立各病毒单项PCR技术的基础上,优化多重PCR反应条件,建立了3种病毒的多重PCR技术,可同时扩增PPV 313 bp、 PRV 217 bp和PCV2 447 bp的特异性片段。用多重PCR技术与单项PCR技术对比检测试验证明两者的符合率为100％,表明建立的多重PCR检测方法,具有特异、快速、准确的特点,可同时鉴别诊断这3种病毒。从10个发病猪场和门诊病例的病猪采集的211份样品,用建立的多重PCR检测方法,检出PPV阳性42份,阳性率为19.91%;PRV阳性26份,阳性率为12.32%;PCV2阳性56份,阳性率为26.54%;2种以上病毒混合感染25份,混合感染阳性率为11.85%。检测结果表明,山西省猪群已感染这3种疫病。

Establishment and Application of Multiplex PCR Assay for Detection of Porcine Parvovirus, Pseudorabies Virus and Porcine Circovirus Type 2

Porcine parvovirus(PPV),pseudorabies virus(PRV) and porcine circovirus type 2(PCV2) were three main viral pathogens of porcine reproduction barrier diseases.To identify and differentiate rapidly the cause(s) of clinical diseases,a multiple PCR/RT-PCR assay was developed.Based on the similarity of the viral sequences deposited in the GenBank database,the VP2 gene of PPV,the gD gene of PRV and the ORF2 gene of PCV2 were selected as the diagnostic targets.By using three pairs of virus-specific primers,three PCR/RT-PCR assay were established to amplify the conservative regions of the three viruses,respectively.Consequently,a multiplex PCR/RT-PCR method to detect the three viruses in one tube was developed.The multiple PCR/RT-PCR system would amplify a 313 bp fragment for PPV,a 217 bp for PRV and a 447 bp for PCV2 simultaneously or separately in the samples,depending on its infection status.By comparison,the test proved that the multiplex PCR method being 100% coincidence with the single PCR,and it could be used for this three virus detection and differential diagnosis.211 samples of pigs collected from 10 nosopoietic pig farms and outpatient cases were detected with the multiplex PCR assay established,the result showed that the positive rate of 42 positive PPV was 19.91%,the positive rate of 26 positive PRV was 12.32% and the positive rate of 56 positive PCV2 was 26.54%,more than,the mixed infection rate of 25 two kinds of mixed infection was 11.85%.It proved that the pigs had been infected by these three epidemics in Shanxi.