EAGER: Rapid Planar PCR for COVID-19 Testing

该项目的目标是开发第一个接触点分子诊断作为新冠肺炎检测的工具。调查人员设想在有人通过门、检查站、机场大门或边境之前进行接触点测试。这项工作的许多关键战略已经制定，包括对各种病毒进行一步化验的可行性，包括导致新冠肺炎的SARS-CoV-2病毒。该项目为扩展该方法/诊断工具的能力提供了基础，目标是建立一种可靠的方法，在不到2分钟的时间内从唾液中检测新冠肺炎阳性，每次测试的成本约为2美元。当该工具起作用时，可以常规用于在机场、医院入口或长期护理设施测试隐藏的疾病传播者。同样，当员工到达医疗机构或其他大型设施工作时，可以使用该方法对他们进行筛查，以保护基本工作人员和患者。最后，该方法可用于工厂、食品加工或配送设施以及大型政府大楼等大型设施的常规筛查，使我们的经济恢复到更正常的状态。该研究还将为博士后和本科生提供培训该项目专注于建立一种新的聚合酶链式反应方法(一种用于快速制作数百万份DNA样本的方法)，将实现对新冠肺炎等包膜病毒进行接触点分子诊断测试的目标。已经为这项工作建立了关键战略：(1)使用光学加热进行快速、均匀的循环；(2)大规模分离，以加速样品制备，而不需要微图案或微流体；以及(3)对包膜病毒进行一步裂解/RP-PCR(快速平面-PCR)分析。研究计划按三个目标组织：(1)通过设计和制造印刷电路，将由于使用单个LED/透镜对而导致的有限区域加热扩展到大区域，该印刷电路将允许每个区域的LED密度更高，允许更多的区域，简化与其余控制电路的连接，并允许使用表面贴装LED驱动器；(2)一旦建立了均匀的大面积加热，则表明扩散限制工作以提供非常大的分区水平，目标是在5×5厘米的样本中实现1,000,000个分区位置；以及(3)通过改变试剂、温度和时间段来证明预期的检测速度和检测极限(~2分钟和~100拷贝/毫升)，以实现三个步骤(裂解、反转录、聚合酶链式反应)的效率和检测速度和检测极限之间的良好折衷。如果成功，项目结果将为推出接触点测试提供基础，该测试可以快速转换为临床应用。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The goal of this project is to develop the first point-of-contact molecular diagnostic as a tool for COVID-19 testing. The investigator envisions a point-of-contact test being performed before someone passes through a door, checkpoint, airport gate, or border. Many of the critical strategies for this work have already been established, including the feasibility of performing a one-step assay on various viruses, including SARS-CoV-2, the virus responsible for COVID-19. This project provides a basis for expanding the capability of the method/diagnostic tool, with the goal of establishing a reliable method for detecting COVID-19 positivity from saliva in less than 2 minutes at a cost of approximately $2/test. When functional, this tool could be used routinely to test for hidden spreaders of disease at airports, entrances to hospitals or long-term care facilities. Similarly, the method could be used to screen employees when they arrive to work at health care facilities or other large facilities to protect essential workers and patients. Finally, the method could be used for routine screening at large facilities such as factories, food processing or distribution facilities, and large government buildings, allowing our economy to return to a more normal state. The research will also provide training for a postdoctoral fellow and an undergraduateThis project focuses on establishing a new method for PCR (Polymerase Chain Reaction, a method used to rapidly make millions of copies of a DNA sample) that will realize the goal of a point-of-contact molecular diagnostic test for envelope viruses such as COVID-19. Already established for this work are critical strategies: (1) for rapid, uniform cycling using optical heating; (2) for large scale partitioning to accelerate sample preparation without micro-patterning or microfluidics; and (3) for performing a one-step lysis/rp-PCR (rapid planar-PCR) assay on envelope viruses. The Research Plan is organized under three objectives: (1) to extend the limited area heating due to use of individual LED/lens pairs to large areas through the design and fabrication of a printed circuit that will allow higher density of LEDs per zone, allow many more zones, simplify connections to the rest of the control circuitry, and allow use of surface mount LED drivers; (2) to show, once uniform, large area heating is established, that the diffusion limitation works to provide a very large level of partitioning, with a goal of achieving 1,000,000 partitioning sites in a 5 x 5 cm sample; and (3) to demonstrate the expected assay speed and detection limit (~2 minutes and ~100 copies/milliliter) by modifying reagents, temperatures, and time periods to achieve a good compromise between efficiency of the three steps (lysis, reverse transcription, PCR) overall assay speed, and limit-of detection. If successful, project results will provide the basis for rolling out a point-of-contact test that can be rapidly translated for clinical applications.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.