为准确区分H5亚型禽流感病毒184.108.40.206分支和220.127.116.11分支的毒株，通过对GenBank和GISAID数据库中发表的以及本实验室保存的H5亚型禽流感病毒HA基因序列进行比对，设计1对特异性引物和2条探针，建立了区分H5亚型禽流感病毒不同分支毒株的实时荧光RT-PCR方法，并对该方法的反应体系和反应参数进行了优化，同时开展了特异性和敏感性试验，以及临床应用检测。结果显示：该方法具有良好的特异性，与其他亚型禽流感病毒及常见禽病病毒无交叉反应；对H5亚型禽流感病毒18.104.22.168分支和22.214.171.124分支毒株的检测灵敏度分别为495.0 copies/μL和22.3 copies/μL；100份临床家禽拭子禽流感病毒检测结果与常规RT-PCR和病毒分离检测结果一致，符合率为100%。结果表明，该方法特异、敏感、准确、耗时少，同时还可区分不同分支，可应用于H5亚型禽流感病毒的准确、快速检测。
In order to accurately distinguish the strains of two evolutionary clades including 126.96.36.199 and 188.8.131.52 of H5 subtype avian influenza virus（AIV），a pair of specific primers and two TaqMan probes were designed by comparing HA gene sequences of H5 subtype AIV registered in Genbank and GISAID with the ones reserved in our laboratory，a duplex real-time RT-PCR assay was established to distinguish different clades of the virus，and its reaction system and parameters were optimized. Meanwhile，the specificity，sensitivity and its clinical application were tested. The results showed that，the specificity of the method was good with no any cross-reaction with other subtypes AIV or common avian viruses；the detection limits of clades 184.108.40.206 and 220.127.116.11 were 495.0 and 22.3 copies/μL，respectively；100 clinical poultry swab samples were tested by the established method，and the results were consistent with those by traditional RT-PCR and virus isolation，with the coincidence rate of 100%. As a conclusion，the established method was specific，sensitive，accurate and convenient，which could be used to distinguish different clades of H5 subtype AIV.