检测禽流感病毒RT-PCR一步法的建立及H5、H9亚型的鉴定

目的建立禽流感病毒(AIV)通用型RT-PCR一步法,并鉴定AIV H5和H9亚型。方法根据流感病毒高度保守的M1基因序列,设计一套通用型引物,建立AIVRT-PCR一步法。根据H5、H9亚型HA基因序列,分别设计一套特异性引物,其上、下游引物分别位于裂解位点两侧,应用RT-PCR一步法检测AIV H5和H9亚型,并验证所建立方法的敏感性;通过检测NDV、IBV和IBDV等RNA病毒及计算机软件模拟检测,验证其特异性。结果所建立AIV系列RT-PCR检测方法具有特异、敏感、简便和快速的特点。结论该方法可用于AIV的检测,H5、H9亚型的鉴定及致病性分析。

Development of One-step RT-PCR Method for Detection of Avian Influenza Virus and Identification of H5 and H9 Subtypes

Objective To develope one-step RT-PCR method for detection of avian influenza virus(AIV) and H5 and H9 subtyping.Methods Develop an one-step RT-PCR method for detection of AIV by designing a set of general primers according to the highly conserved M1 gene.Design another set of specific primers,of which upstream and downstream primers were located in the two sides of the cleavage site respectively,to develop the one-step RT-PCR method for detecting H5 and H9 subtypes of AIV.Identify the sensitivity of the developed one-step RT-PCR method by detecting 2 AIV strains of H5 subtype and 3 AIV strains of H9 subtype,and the specificity by detecting the RNAs of NDV,IBV and IBDV as well as analogue detection using computer software.Results The developed one-step RT-PCR method was specific,sensitive,simple and rapid.Conclusion The developed one-step RT-PCR method might be used for the detection of AIV,the identification of H5 and H9 subtypes and the analysis of pathogenicity.