基于荧光及可视化逆转录环介导等温扩增检测食源性甲型肝炎病毒的方法开发

Development of a method for the detection of foodborne hepatitis A virus based on fluorescence and visualization loop-mediated isothermal amplification

中文关键词:  环介导恒温扩增(LAMP)  甲型肝炎病毒  快速检测  可视化

英文关键词:loop-mediated isothermal amplification  visualization  hepatitis A virus  rapid detection

中文摘要:

      目的 基于荧光及可视化逆转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification, RT-LAMP)开发食源性甲型肝炎病毒(hepatitis A virus, HAV)的检测方法。方法 从美国国家生物技术信息中心中选取世界范围内具有代表性的HAV基因信息, 设计LAMP引物, 开发了一种荧光曲线RT-LAMP和可视化RT-LAMP检测方法。以HAV质粒为模板, 评估方法的扩增效率、灵敏度和稳健性, 并与实时荧光逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)进行检出限及实际样品验证比较。结果 荧光及可视化LAMP方法检测灵敏度为10.76 copies/μL, 与实时荧光RT-PCR一致; 在95%置信水平下经概率回归分析检出限(limit of detection, LOD95%), 荧光曲线RT-LAMP的LOD95%为17.46 copies/μL (7.00~511.29 copies/μL, 95%), 实时荧光RT-PCR的LOD95%为79.30 copies/μL (24.68~3208.71 copies/μL, 95%); 稳健性分析表明方法稳定; 与实时荧光RT-PCR比较, 检测30份实际样品的验证比对符合率为100%。结论 该检测方法经济便捷, 可通过肉眼直接观察结果, 适合于食品安全现场监测, 具有很好的应用前景。

英文摘要:

      Objective To develop a method for the detection of hepatitis A virus (HAV) based on fluorescence and visualization reverse transcription loop-mediated isothermal amplification (RT-LAMP). Methods The representative HAV gene information was selected from National Center for Biotechnology Information, LAMP primers were designed, and a fluorescence curve RT-LAMP and visual RT-LAMP detection method were developed. The amplification efficiency, sensitivity and robustness of the method were evaluated using HAV plasmid as a template, verification and limits of detection for actual samples were compared with real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR). Results The detection sensitivity of fluorescence and visualization LAMP method was 10.76 copies/μL, which was consistent with real-time fluorescence RT-PCR. At a 95% confidence level, by probabilistic regression analysis limit of detection (LOD95%), the LOD95% value of the fluorescence curve RT-LAMP was 17.46 copies/μL (7.00-511.29 copies/μL, 95%), and the LOD95% value of real-time fluorescence RT-PCR was 79.30 copies/μL (24.68-3208.71 copies/μL, 95%); robustness analysis showed that the method was stable; compared with real-time fluorescence RT-PCR, the verification comparison rate of 30 actual samples tested was 100%. Conclusion This detection method is economical and convenient, which can directly observe the results by the naked eye, is suitable for on-site monitoring of food safety, and has a good application prospect.