H5亚型和N6亚型禽流感病毒二重RT-PCR检测方法的建立

为了建立一种快速、简便的H5亚型、N6亚型禽流感病毒(AIV)的检测方法,根据GenBank中H5亚型、N6亚型AIV的HA和NA基因保守序列,分别设计了2对特异性引物,通过优化条件,建立了H5亚型和N6亚型AIV二重RT-PCR检测方法。特异性试验结果显示,该方法对H5N6亚型AIV特异性扩增出418bp和251bp目的片段,对H5Ny(y≠6)亚型AIV扩增出418bp目的片段,对HxN6(x≠5)亚型AIV扩增出251bp目的片段,对其他亚型和常见的禽病病原体均未扩增出目的片段;敏感性结果显示,该方法对H5亚型和N6亚型最低检测限为1.59×10-5ng/μL。本研究建立的H5亚型和N6亚型AIV检测方法,具有特异性强,灵敏度高的特点,为H5亚型和N6亚型AIV临床检测以及防控提供了有效方法。

Development of a Duplex RT-PCR for Detecting AIV H5 and N6 Subtypes

In order to establish a rapid,simple H5 and N6 subtypes avian influenza virus (AIV) detection method,according to the sequences of HA and NA genes of H5 and N6 subtypes of AIV,two pairs of specific primers were designed respectively,the conditions were optimized and the duplex RT-PCR assay for detection of AIV H5 and N6 subtypes was established.It was shown that H5N6 AIV could be amplified into two specific bands by this duplex RT-PCR, the lengths of bands were 418 bp(H5 AIV) and 251 bp(N6 AIV),respectively.Samples containing H5 or N6 subtype AIV could be amplified into one specific band,which was 418 bp,251 bp,respectively.No specific band was amplified from other subtypes AIV and avian pathogenic virus.Sensitivity test results showed that as low as 1.59×10-5ng/μL H5N6 AIV could be detected.This duplex RT-PCR assay is a specific,sensitive method for the detection of H5 and N6 subtypes AIV in the same tube,and provided an effective tool to determine the infection of AIV H5 and N6 subtypes.