高致病性H7N9流感病毒荧光RT-PCR检测方法的建立

2017年初在中国出现的H7N9流感病毒变异株表现出对禽高致病性的特征,根据高致病性H7N9流感病毒(HP-H7N9)的HA基因序列,设计1组HP-H7N9特异性的引物和探针,在对反应体系进行优化的基础上,建立了HP-H7N9的荧光RT-PCR检测方法并进行了特异性、敏感性和重复性试验,制作了标准曲线。结果显示,本方法只对HP-H7N9特异性扩增,而对低致病性H7N9病毒(LP-H7N9)、H7N3流感病毒(H7N3)、H3N2流感病毒(H3N2)、H5N1禽流感病毒(H5N1)、H9亚型禽流感病毒(H9)、新城疫病毒(NDV)、甲型H1N1流感病毒(H1N1)、传染性法氏囊病病毒(IBDV)、鸡传染性支气管炎病毒(IBV)等病原未见扩增。该方法最低能够检测到35.45 copies的阳性质粒,具有良好的检测灵敏度。本方法的建立将为HP-H7N9的诊断、监测和防控提供快速、特异、敏感的技术手段。

Development of a real-time RT-PCR for detecting highly pathogenic H7N9 influenza virus

Early in 2017,the mutant H7N9 influenza virus appeared in China and it had showed the characteristic of highly pathogenic for poultry.Base on the hemagglutinin(HA)gene sequences of this highly pathogenic H7N9 influenza virus(HP-H7N9),specific primers and probe were designed,a real time one-step RT-PCR method was developed to detect HP-H7N9.It could specifically detect HP-H7N9,non-crossreaction with low pathogenice H7N9 influenza virus(LP-H7N9),H7N3 influenza virus(H7N3),H3N2 influenza virus(H3N2),H5N1 influenza virus(H5N1),H9N2 influenza virus(H9 N2),Newcastle disease virus(NDV),H1N1 influenza virus(H1N1),Infectious bursal disease virus(IBDV),avian infectious bronchitis virus(IBV).The minimum detection limit for positive plasmid was 35.45 copies which showed good sensitivity.It will play an important role in the diagnosis,surveillance,and control of highly pathogenic H7N9 influenza virus.