H9N2禽流感病毒一步法双重荧光RT-PCR检测方法的建立

目的 建立一种可同时检测禽流感病毒H9N2的HA和NA基因一步法双重荧光RT-PCR方法.方法 针对H9N2禽流感病毒的HA和NA基因保守区,设计相应的特异性引物以及探针,优化检测体系及反应条件,建立一步法双重荧光定量RT-PCR方法.对该方法的灵敏度、特异性、稳定性进行验证与评估,并对家禽粪便标本进行应用检测,以单重实时荧光RT-PCR方法作为参照,检测结果不一致的样本采用测序进行验证.结果 该方法特异性强,与H1、H3、H5、H7亚型禽流感病毒、鸡新城疫及鸡传染性支炎病毒均无交叉反应,对HA和NA基因的最低检出限分别可达50拷贝/μL和25拷贝/μL,组间与组内的变异系数在0.20 ～0.79％之间.对82份粪便标本进行检测,H9N2禽流感病毒的阳性率为8.14％ (7/82),与单重实时荧光RT-PCR法检测结果一致.结论 该方法特异性强、灵敏度高、稳定性好,可应用于临床禽流感样本的检测.

Establishment of one-step duplex real-time RT-PCR method for detection avian influenza virus of H9N2

Objective To develop a duplex real-time RT-PCR assay for rapid and simultaneous detection of avian influenza virus of H9N2.Methods Specific primers and probes were designed for according to the conserved sequences in the HA and NA genes of H9N2 avian influenza virus available in GenBank.The one-step real-time RT-PCR reaction system was established by optimizing the reaction mix and reaction conditions.The specificity, sensitivity and stability of the detection system were evaluated.Results The results showed that HA and NA of H9N2 were detected in different fluorescence channels and there was no cross-reaction with H1, H3, H5, H7 and other common respiratory viruses.The detection limit of duplex real-time RT-PCR assay were 50 copies/μL and 25 copies/μL, respectively.The inner-batch and inter-batch variation coefficient was 0.20％ ～ 0.79％.Conclusions The newly developed duplex real-time RT-PCR assay is a specific, sensitive and rapid method for simultaneous detection HA and NA gene of H9N2 avian influenza virus.