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试验旨在建立可同时鉴别检测H9和H6亚型禽流感病毒(avian influenza virus,AIV)的二重RT-PCR方法。根据GenBank中H9和H6亚型AIV的HA基因保守序列,分别设计2对特异性引物,优化引物浓度与退火温度等条件,建立了可同时鉴别检测H9和H6亚型AIV的二重RT-PCR检测方法。用该法对H9和H6亚型AIV混合感染样品、H9亚型AIV单一感染样品和H6亚型AIV单一感染样品进行扩增,结果均得到对应的目的条带,而对其余亚型AIV及其他禽病病原体均未扩增出特异性条带。该法对H9和H6亚型AIV的检测下限均为5×104拷贝/μL。本研究建立的二重RT-PCR检测方法特异性强、敏感性高、稳定性和重复性良好,可同时鉴别检测H9与H6两种亚型AIV,为H9与H6亚型AIV的监测提供技术支撑。
The aim of the experiment was to establish a dual RT-PCR method for simultaneous identification of avian influenza virus (AIV) and H9 and H6 subtypes. According to the conserved sequence of HA gene of H9 and H6 subtypes AIV in GenBank, two pairs of specific primers were designed respectively to optimize primer concentration and annealing temperature. A dual RT-PCR method was established to detect H9 and H6 subtypes AIV simultaneously Detection method. This method was used to amplify the H9 and H6 subtype AIV mixed infection samples, the H9 subtype AIV single infection sample and the H6 subtype AIV single infection sample, all of which obtained the corresponding target bands, while the other subtype AIV and other No specific bands were amplified from poultry pathogens. The method for the detection of H9 and H6 subtype AIV lower limit of 5 × 104 copies / μL. The double RT-PCR assay established in this study has high specificity, high sensitivity, good stability and repeatability. It can simultaneously identify and detect AIV subtype H9 and H6, providing technology for the monitoring of H9 and H6 subtypes AIV support.