羊痘病毒和羊口疮病毒二重PCR鉴别检测方法的建立

Establishment of duplex PCR assay for quick differentiating capripoxvirus and orf viruses

针对羊痘病毒(capripoxvirus,CaPV)的A29L基因和羊口疮病毒(orf virus,ORFV)的H3L基因,设计、合成了2对特异性引物以进行二重PCR。结果表明,该方法可以在数小时、在同一反应体系中清晰地区分CaPV和ORFV,其PCR产物大小分别为413 bp和708 bp,与预期的片段大小相符,序列分析证实目的基因序列与已发表的序列一致;该检测方法的灵敏度可达1个病毒蚀斑形成单位(PFU)。采用二重PCR对12株CaPV、ORFV和相关病毒、细菌培养物,以及22份田间样品进行检测,与预期结果完全一致,且与相关病毒或健康羊组织样品无交叉反应。结果表明,该方法具有快速、敏感、特异等优点,可作为临床样品中CaPV和ORFV的鉴定和诊断的有效方法。

A simple and fast duplex PCR assay was developed for the simultaneous detecting and differentiating capripoxvirus （CaPV, including goatpox and sheeppox viruses） and orf virus （ORFV）. Two pairs of primers were specifically for CaPV and ORFV （one each） designed and synthesized. Duplex PCR products consisted of fragments of 413 and 708 bp for A29L gene of CaPV and P55 gene of ORFV, respectively. The products were identified by gel electrophoresis. Sequence analysis showed that the amplified gene and those in the literature possessed identical sequences. Sensitivity of the assay was 1 plaque forming unit （PFU） for both CaPV and ORFV. A total of 12 isolates of CaPV, ORFV and other clinically relevant virus and bacterial strains, and 22 field samples were analyzed. The assay was sensitive and highly specific; it had no cross-reactions with those clinically relevant pathogens. The assay may be used for a quick differentiation and diagnosis of CaPV and ORFV in clinical samples.