Rapid Point-of-Care Molecular Test for SARS-CoV-2 and Influenza A/B

项目总结 此第一阶段SBIR建议的目标是开发15分钟多路复用点(POC)分子测试 对于新冠肺炎和甲型流感的定性检测，截至2021年9月，美国已有超过39.5%的病例。 100万例新冠肺炎确诊病例和超过642,000,000人死亡。此外，流感病毒占比超过 在美国，每年有20万人住院，3万至5万人死亡。作为SARS-CoV-2和流感 单凭症状很难区分感染，这是一项可以诊断由 SARS-CoV-2和流感病毒将为公共卫生官员提供他们努力控制的信息 这些重要病毒的传播令人担忧。我们的方法利用快速样品准备(1分钟 上手时间和2.5分钟热失活)和快速金标准实时RT-PCR(12分钟， 40个周期)来扩增靶向病毒基因组。此测试可用于可操作测试的多个环境中 需要结果才能迅速做出知情的治疗决定。我们将证明我们的测试可以提供 快速结果，与使用鼻咽拭子的参考实验室获得的性能水平相似 和人为设计的唾液样本。我们的方法已经使用SARS-CoV-2和流感阳性临床进行了测试 病毒传输介质中的标本。我们计划使用带薄膜的穿梭聚合酶链式反应来实现快速多重RT-PCR 反应堆。利用振动来加强试剂的传质，可以提高聚合酶链式反应的速度和效率 避免需要10到20倍的浓缩试剂(即额外的 费用)以加快反应速度。基于空间多路荧光探针RT-PCR的结果可以 通过机载电子设备进行成像和分析。在完成40个循环后，RT-PCR测试需要大约8分钟 2.5分钟的样品热灭活和3分钟的逆转录步骤，一些高阳性结果可以 最早在6分钟内就能到达。我们的方法将比基于抗原的方法敏感得多 而且比等温扩增方法，如环介导的等温扩增(LAMP)更快。 分子分析的诊断效用将通过比较其稳健性、速度、灵敏度、 与目前的分子检测方法相比，具有较高的特异性。在第一阶段，我们将与我们的合作者一起验证我们的设备 并使用临床标本进行测试。如果成功开发，这种紧凑和最小的工具方法 操作简单，价格低廉，适用于各种规模的初级保健医生办公室、疗养院、 药房、社区卫生所，甚至是居家消费者都采用该平台。

PROJECT SUMMARY The goal of this Phase I SBIR proposal is to develop a 15 minute multiplex point-of-care (POC) molecular test for the qualitative detection of COVID-19 and influenza A/B. As of September 2021, the US has had over 39.5 million confirmed cases of COVID-19 and over 642,000K deaths. Additionally, influenza viruses account for over 200,000 hospitalizations and 30,000–50,000 deaths in the US each year. As SARS-CoV-2 and influenza infections can hardly be differentiated by symptoms alone, a single test that can diagnose illness caused by SARS-CoV-2 and influenza viruses will give public health officials information they need in their efforts to control the spread of these important viruses of concern. Our approach leverages rapid sample preparation (1 min hands-on-time and 2.5 minute of heat inactivation) and the rapid gold-standard real-time RT-PCR (12 minute, 40-cycle) to amplify the targeted viral genome. This test can be utilized in multiple settings where actionable test results are needed to make informed treatment decisions quickly. We will demonstrate that our test can deliver rapid results with a similar level of performance as those obtained in reference labs using nasopharyngeal swabs and contrived saliva samples. Our approach has been tested using SARS-CoV-2 and influenza positive clinical specimens in viral transport media. We plan to achieve rapid multiplex RT-PCR using shuttle PCR with thin-film reactors. We can enhance PCR speed and efficiency by using vibration to enhance reagent mass-transport while avoiding the “extreme PCR” approach that needs 10 to 20 times more concentrated reagents (i.e., additional expense) to speed up the reaction. The result from spatial multiplexed fluorescence probe-based RT-PCR can be imaged and analyzed by on-board electronics. The RT-PCR test takes ~8 minutes to complete 40 cycles after 2.5 min of sample heat inactivation and 3 min of reverse-transcription step, and some high positive results can be obtained in as early as 6 minutes. Our approach will be much more sensitive than antigen-based approaches and faster than isothermal amplification approaches, such as loop mediated isothermal amplification (LAMP). The diagnostic utility of the molecular assay will be demonstrated by comparing its robustness, speed, sensitivity, and specificity with current molecular assays. In Phase I, we will work with our collaborator to validate our device and test using clinical specimens. If successfully developed, this compact and minimal instrumentation approach will be simple to perform and inexpensive enough for all sizes of primary-care physician’s offices, nursing homes, pharmacies, community health clinics, and even at-home consumers to adopt the platform.