基于重组酶聚合酶扩增结合侧向流试纸条快速检测H7亚型禽流感病毒的方法研究

Rapid detection of avian influenza virus subtype H7 by recombinase polymerase amplification combined with lateral flow dipstick

目的建立一种快速、灵敏、可视化的H7亚型禽流感病毒等温核酸扩增检测方法。方法针对H7N9禽流感病毒的HA片段保守区序列设计引物、探针,优化反应温度,建立H7亚型禽流感病毒的重组酶聚合酶扩增结合侧流层析试纸条检测技术,评价其特异性和敏感性,并与Real time PCR方法进行比较。结果该方法检测H7亚型禽流感病毒的最低检出限为20拷贝/μL,与其他流感/禽流感病毒无交叉反应,反应可在20~50℃温度范围内进行,临床样本检测结果与Real time PCR法一致性为100.0%。结论该检测方法具有快速、特异及灵敏的特点,为在基层和现场快速检测H7亚型禽流感病毒提供了新的方法。

Objective To establish a rapid,sensitive and visual isothermal amplification assay for the detection of avian influenza virus subtype H7.Methods Primers and probes were designed based on conservative regions of haemagglutinin(HA)gene of H7N9 avian virus,and the reaction temperature was optimized.The detection method based on recombinase polymerase amplification(RPA)combined with lateral flow dipstick(LFD)was established.The sensitivity and specificity of the assay were evaluated,and the performances of the assay were compared with real time PCR.Results The detection limit of avian influenza virus subtype H7 was 20 copies/μL,and no cross reaction with other influenza/avian influenza viruses was observed.The detection could carried out within the temperature range 20-50℃.the detection results of clinical samples by RPA-LFD were 100.0%consistent with those by real time PCR method.Conclusions The RPA-LFD assay is characterized by rapidity,specificity and sensitivity.The method provided a novel tool for rapid detection of the avian influenza virus subtype H7 in primary health care departments and on-site testing.