猪细小病毒和猪圆环病毒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(PPV)和猪圆环病毒2型(PCV2)基因序列,对各病毒基因区进行同源性分析,确定PPV的VP2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2种病毒的多重PCR技术,可同时扩增PPV的313 bp和PCV2的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二者的总符合率为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2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,用山西省农科院畜牧兽医研究所动物基础医学研究课题组研究的多重PCR检测方法,检出猪圆环病毒2型阳性56份,阳性率为27%;猪细小病毒病阳性42份,阳性率为20%;二者混合感染17份,阳性率为8%。这为掌握山西这2种病毒的感染情况提供了依据。

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

Porcine parvovirus(PPV) and porcine circovirus type 2(PCV2) are two 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 and the ORF2 gene of PCV2 were selected as the diagnostic targets.By using two pairs of virus-specific primers,two PCR/ RT-PCR assay were established to amplify the conservative regions of the two viruses,respectively.Consequently,a multiplex PCR/ RT-PCR method to detect the two viruses in one tube was developed.The multiple PCR/RT-PCR system would amplify a 313 bp fragment for PPV 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 PCRs,and it could be used for this two 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 studied by group,the result showed that the positive rate of 56 positive PCV2 was 27% and the positive rate of 42 positive PPV was 20%,and the positive rate of 17 two diseases mixed infection was 8%.The study provided a basis for the infection situation of these two diseases in Shanxi.