H5亚型禽流感病毒一步法RT-PCR检测方法的建立

Development of One Step RTPCR Technique for Detectionof H5 Subtype Avian Influenza

针对家禽中流行较为广泛、危害相对大的H5亚型禽流感病毒的血凝素（HA）基因,通过分析流感数据库221个HA序列,在保守区内用Oligo6.0软件设计并合成了一对引物,建立了用于快速诊断H5亚型禽流感病毒的一步法RT-PCR方法,其扩增的目的片段大小为372bp。通过对H5亚型禽流感病毒尿囊液和棉拭子浸出液进行不同稀释倍数检测,结果表明病毒尿囊液最低检出量为10^-4稀释;阳性棉拭子最低检出量为8倍稀释。用病毒分离和该方法同时检测不同脏器、口咽及泄殖腔棉拭子样品,结果表明该方法检测灵敏度比病毒分离低10～100倍。用该方法检测H1～H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原,仅有H5亚型禽流感病毒扩增出特异性目的条带。该方法具有方便快捷、特异性强、敏感性高等特点,为我国禽流感的快速诊断和分子流行病学调查提供了技术支撑。

A one step RT-PCR was developed to detect H5 subtypes of avian influenza virus （AIV）. A pair of specific oligonucleotide primers was designed for the detection of AIV H5 subtypes according to 221 hemagglutinin （HA） gene sequences. Sensitivity to detection of allantoic fluid by one step RT-PCR reached 10 thousand dilutions and detection of swab samples reached 8 dilutions. The tissue and swab samples infected with H5 subtypes AIV were simultaneity detected by one step RT-PCR and virus isolation method. The results showed that the sensitivity of the assay was lower 10 to 100-fold than virus isolation method and the assay gave an excellent （100 % ） correlation with the conventional virus isolation method. H1 ～ H15 subtypes of avian influenza and other avian diseases were detected by the one step RT-PCR. The results showed the assays were high specific,without cross-reaction to other subtype or other avian diseases. Development of one step RT-PCR will provide effective technical support for the rapid diagnosis and surveillance of molecular epidemiology of H5 subtypes AIV.