為準(zhǔn)確區(qū)分H5亞型禽流感病毒2.3.2.1分支和2.3.4.4分支的毒株，通過(guò)對(duì)GenBank和GISAID數(shù)據(jù)庫(kù)中發(fā)表的以及本實(shí)驗(yàn)室保存的H5亞型禽流感病毒HA基因序列進(jìn)行比對(duì)，設(shè)計(jì)1對(duì)特異性引物和2條探針，建立了區(qū)分H5亞型禽流感病毒不同分支毒株的實(shí)時(shí)熒光RT-PCR方法，并對(duì)該方法的反應(yīng)體系和反應(yīng)參數(shù)進(jìn)行了優(yōu)化，同時(shí)開(kāi)展了特異性和敏感性試驗(yàn)，以及臨床應(yīng)用檢測(cè)。結(jié)果顯示：該方法具有良好的特異性，與其他亞型禽流感病毒及常見(jiàn)禽病病毒無(wú)交叉反應(yīng)；對(duì)H5亞型禽流感病毒2.3.2.1分支和2.3.4.4分支毒株的檢測(cè)靈敏度分別為495.0 copies/μL和22.3 copies/μL；100份臨床家禽拭子禽流感病毒檢測(cè)結(jié)果與常規(guī)RT-PCR和病毒分離檢測(cè)結(jié)果一致，符合率為100%。結(jié)果表明，該方法特異、敏感、準(zhǔn)確、耗時(shí)少，同時(shí)還可區(qū)分不同分支，可應(yīng)用于H5亞型禽流感病毒的準(zhǔn)確、快速檢測(cè)。
Establishment and Application of a Duplex Real-time RT-PCR Assay for Identifying Two Branches of H5 Subtype Avian Influenza Virus
In order to accurately distinguish the strains of two evolutionary clades including 2.3.2.1 and 2.3.4.4 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 2.3.2.1 and 2.3.4.4 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.
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