Ⅰ型鸭病毒性肝炎病毒RT-PCR检测方法的建立

摘要

参考GenBank中Ⅰ型鸭病毒性肝炎病毒(DHV-Ⅰ)的基因序列,应用Primer Premier5.0软件设计了一对引物,建立了适合DHV-Ⅰ快速检测的RT-PCR检测方法.以该方法对实验室分离并保存的DHV强毒株Z10株的RNA为模板进行RT-PCR扩增,得到了预期大小为699 bp的目的片段.将扩增所得的DNA片段进行克隆测序,测序结果与GenBank中的DHV-Ⅰ的相应基因序列比对分析,同源性均达90%以上,表明为DHV-Ⅰ的基因序列,对NDV,IBDV,DPV和GPV等常见病毒扩增结果为阴性.该方法敏感性检测结果表明,最低RNA模板检出量为24 Pg.表明所建立的RT-PCR检测技术具有特异、快速和敏感的特点,可用于Ⅰ型鸭病毒性肝炎病毒的快速诊断.%According to the sequences of duck hepatitis virus type I (DHV-I) published in GenBank,a pair of primers were designed and synthesized. A specific 699 bp fragment was amplified and successfully sequenced from RNA templates of DHV -I Z10 strain. Alignment results showed that the homologies were higher than 90% compared with published sequence of DHV-I in GenBank. The amplified results of newcastle disease virus (NDV) .infectious bursal disease virus (IBDV).duck plague virus (DPV) and goose paramyxo viridae (GPV) were negative. The RT-PCR method could detect the duck hepatitis virus with only 24 pg RNA,which was specific,rapid and sensitive. The results indicated that the method could be used for the rapid diagnosis of DHV-I isolates.