Multiplexed Airborne Virus Collection and Detection at the Point-of-Care

项目摘要 严重急性呼吸综合征冠状病毒2(SARS-CoV-2)是最新出现的 导致了目前新冠肺炎的大流行。尽管考虑到SARS-CoV-2的主要传播途径 美国疾病控制和预防中心(CDC)正在与呼吸道飞沫密切接触，不断攀升 有证据表明，病毒传播也可能通过气雾剂发生。同样，流感病毒(流感病毒) 通过呼吸道飞沫、气雾剂和接触传播。更重要的是，与流感病毒和 已在患者中观察到SARS-CoV-2。预计SARS-CoV-2和流感病毒将同时传播 未来，对各种空气传播病毒的多路收集和检测是研究传播所必需的 路线，规划和分配资源，减轻对公众健康的影响。 收集空气传播的病毒需要空气采样器，理想情况下需要以下两个功能。其一是拥有 收集的样本在护理点(POC)进行分析，而不是延迟1-2送到实验室 报告检测结果的天数。第二个特征是有效地将病毒气溶胶直接收集到液体中， 而不是放在过滤器上，用于后续的病毒分析。而液体撞击器可以有效地收集 对于微粒(如细菌)来说，它们对病毒等纳米粒就不那么适用了。为了实现这些功能， 我们开发了(1)一种活体病毒气溶胶采样器(VIVAS)，它显示了对病毒的高收集效率 通过水蒸气将纳米大小的病毒气溶胶扩大成微米大小的液滴 缩合；以及(2)阀门启动的裂解、纸基RNA浓缩和RNA扩增设备 (VLEAD)在没有移液的情况下进行病毒裂解和洗涤步骤，随后是逆转录循环- 介导性等温扩增(RT-LAMP)用于准确的遗传鉴定。在这项拟议的研究中，我们 目的将Vivas与VLEAD集成，用于POC的多路传输空气传播病毒收集和检测，评估 SARS-CoV-2和流感病毒的采集和多路检测的集成系统，并在 通过调查卫生诊所和公共场所的空气传播病毒，实现了现实世界的应用。 本研究的意义在于以下几个方面。首先，它将导致一个创新的 在POC收集和检测空气传播的SARS-CoV-2和流感病毒的系统，这将有助于解决主要的 公共卫生问题(即当前的新冠肺炎大流行和预计与流感中的流感病毒同时传播 季节)。其次，综合系统将为研究空中航线的作用提供一个至关重要的工具 在病毒传播中。如果得到确认，将提供适当的个人防护装备和其他补救措施 被用来减少医护人员和普通公众的感染。第三，系统有能力 通过在POC有效收集和及时检测这些病毒来进行环境监测。它 可以作为一种快速大规模筛选方法，及时获得结果，对当地政策制定者有价值 及时向公众和医院发出警告，为局部疫情和临床护理做好准备。

Project Summary Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most recent emerging pathogen that causes the current pandemic of COVID-19. Although the main transmission routes for SARS-CoV-2 considered by the Centers for Disease Control and Prevention (CDC) are respiratory droplets and close contact, mounting evidences have emerged that virus transmission likely also occurs by aerosols. Similarly, influenza viruses (FluV) are transmitted via respiratory droplets, aerosols, and contact. More importantly, co-infection with FluV and SARS-CoV-2 has been observed in patients. With expected concurrent circulation of SARS-CoV-2 and FluV in the future, multiplexed collection and detection of various airborne viruses are desirable for studying transmission routes, planning and allocating resources, and mitigating the impact on public health. The collection of airborne viruses requires air samplers, ideally with two following features. One is to have the collected samples analyzed at the point-of-care (POC), rather than being sent to a lab with a delay of 1-2 days in reporting the test results. The second feature is to efficiently collect virus aerosols directly into a liquid, rather than onto a filter, for subsequent virus analysis. While liquid impingers are effective in collecting microparticles (such as bacteria), they are less so for nanoparticles such as viruses. To realize these features, we have developed (1) a Viable Virus Aerosol Sampler (VIVAS) that shows high collection efficiency of virus particles by enlarging nanometer-sized virus aerosols into micrometer-sized droplets through water vapor condensation; and (2) a Valve-enabled Lysis, paper-based RNA Enrichment, and RNA Amplification Device (VLEAD) that carries out virus lysis and washing steps without pipetting, followed by reverse transcription loop- mediated isothermal amplification (RT-LAMP) for accurate genetic identification. In this proposed research, we aim to integrate VIVAS with VLEAD for multiplexed airborne virus collection and detection at POC, assess the integrated system for collection and multiplexed detection of SARS-CoV-2 and FluV, and validate the system in real-world applications by surveying airborne viruses in a health clinic and public venues. The significance of the proposed research lies in the following aspects. First, it will lead to an innovative system that can collect and detect airborne SARS-CoV-2 and FluV at POC, which will help address a major public health concern (i.e., current COVID-19 pandemic and expected concurrent circulation with FluV in the flu seasons). Second, the integrated system will provide a critically important tool to study the role of airborne route in virus transmission. If confirmed, appropriate personal protection equipment and other remedies will be employed to reduce infections among healthcare workers and the general public. Third, the system is capable of conducting environmental surveillance via efficient collection and timely detection of these viruses at POC. It can function as a rapid mass screening method for timely results that will be valuable for local policymakers to issue prompt warning to the public and for hospitals to prepare for local outbreaks and clinical care.