基于微流控等温扩增技术的甲型H1N1流感病毒快速检测

Rapid detection of influenza A(H1N1)virus using microfluidic isothermal amplification

目的:以甲型H1N1流感病毒检测为例,建立一种简便、快速的流感病毒检测方法,为呼吸道传染病疫情防控提供技术支持。方法:整合环介导等温扩增技术(LAMP)和微流控芯片技术,建立微流控实时荧光逆转录环介导等温扩增(RT-LAMP)快速检测方法;针对甲型H1N1流感病毒的M基因序列中8个不同区段设计LAMP引物,根据扩增效果筛选出1套(6条)引物,并进行临床样本检测验证。结果:所建立的检测方法可实现扩增结果的荧光实时判读,能在恒定温度(65℃)30 min内完成扩增反应,检测限可达10copies/μL,仅对甲型H1N1流感病毒产生扩增曲线,对甲型H3、H5、H7、H9及乙型流感病毒均无扩增;对70份临床样本的检测结果与荧光定量PCR方法一致,特异性和灵敏度均为100%;该方法具有体系封闭、污染小、试剂用量小的优势。结论:所建立的微流控实时荧光RT-LAMP是一种快速、灵敏、特异、简便的检测甲型H1N1流感病毒的方法,便于开展现场检测。

Objective:A facile and rapid on-site detection method for influenza virus was established and validated through the detection of influenza A(H1N1)virus,which was expected to provide technical support for the prevention and control of respiratory infectious diseases.Methods:Through integrating loop-mediated isothermal amplification(LAMP)and microfluidic chip technology,a rapid detection method based on microfluidic real-time fluorescence reverse transcription-loop-mediated isothermal amplification(RT-LAMP)was established.Six primers designed for eight different regions of the M gene sequence of H1N1 virus,were used to complete the specific amplification.Thereafter the clinical sample validation were performed using the real-time fluorescence RT-LAMP.Results:In this study,real-time fluorescence amplification curve could be obtained;meanwhile the rapid amplification of viral nucleic acid could be accomplished at a constant temperature(65℃)within 30 minutes,and the detection limit was as low as 10 copies/μL.The assay could specifically amplify and recognize H1N1 virus,but did not present observed signal to H3,H5,H7,H9 and influenza B virus.The consistent detection results with real-time PCR from 70 clinical sample suggested the good specificity and sensitivity of this assay.Integrated in a closed nucleic acid amplification and detection system,the assay presented the advantage of less pollution and cost effect.Conclusion:The established real-time fluorescence RT-LAMP based on microfluidic chips assay was demonstrated to be a rapid,sensitive,specific and simple method to detect influenza A(H1N1),which was convenient for on-site detection.