禽流感病毒RT-PCR检测方法的建立

参照国内外已发表的禽流感病毒(Avian Influenza Virus，AIV)的基因序列和AIV相关的RT-PCR检测方法，根据AIV高保守、高特异的M蛋白基因设计了一套通用型引物，通过对相关病毒、棉拭子检测，建立了AIV通用型RT-PCR检测方法。根据．AIV HA基因设计了H5、H7、H9亚型的联合RT-PCR引物，其上下游引物分别位于裂解位点两侧，在确定各亚型单项RT-PCR检测方法的反应条件和反应体系的基础上，建立、优化了三联RT-PCR检测方法的反应体系和反应条件，并通过相关病毒、质粒、棉拭子检测，建立了AIV三联RT-PCR检测方法。最终建立了AIV系列RT-PCR检测方法，该方法具有快速、敏感、特异等优点，为AIV检测、流行病学调查、致病性分析及疫苗使用等奠定了基础。

Establishment of rapid test of AIV by RT-PCR

According to avian influenza virus (AIV) gene published and RT-PCR assay for the diagnosis and characterization of AIV reported, the genes of matrix(M) and HA were considered as the target sequences of RT-PCR assay. A set of primers to detect AIV were designed in the conserved M gene of AIV which is conserved and specific. The production of the RT-PCR assay is 567bp. Also H5,H7 and H9 primers which cover the cleavage site of the HA gene respectively was designed. The expected results of the RT-PCR assay would be 290bp, 720bp, 547bp.The optimal condition of single RT-PCR for detection and subtyping(H5,H7 and H9)of avain type A influenza virus by RT-PCR was determined through orthogonal assay,so did the optimal condition about running multiplex RT-PCR. The specificity of two sets of primers were examined by RT-PCR using template extracted from other avian virus. While the sensitivity of related RT-PCR were also determined by a seral dilution of RNA from AIV. This study have paved the way for testing of AIV,defining pathogenic potential, using vaccines against AIV and so on.