Abstract：
The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.
摘要：
COVID-19大流行，除了流感病毒和呼吸道合胞病毒（RSV）的同时发生，强调了对呼吸道感染的有效和可靠的多重诊断方法的要求。现有的多重检测技术基于逆转录定量聚合酶链反应(RT-qPCR)和提取纯化试剂盒,对复杂仪器和高成本的需求限制了它们的可扩展性和可用性。在这项研究中,我们开发了一种基于逆转录环介导等温扩增（RT-LAMP）的即时（POC）设备，可以同时检测四种呼吸道病毒（SARS-CoV-2，流感A，乙型流感,和RSV)，并在不到30分钟的时间内执行两次控制，同时避免使用RNA提取试剂盒。该系统包括一个带有机械组件的一次性微流体盒，该机械组件可自动化样品处理，具有低成本和便携式光学阅读器和智能手机应用程序来记录和分析荧光图像。使用鼻液中掺入病毒颗粒的拭子验证了作为真正的护理点平台的应用。我们的便携式诊断系统准确检测呼吸道病原体特异性病毒RNA，实现共感染信息的反卷积。使用掺加在化学裂解缓冲液中的病毒颗粒确定每种病毒的检测限。我们的POC设备有可能通过修改引物序列来适应新病原体和多种病毒的检测。这项工作强调了一种用于多种呼吸道病毒诊断的替代方法，该方法非常适合资源有限的环境或家庭中的医疗保健系统。
