RAPID: COVID-19 diagnostics for limited resource settings via improved sample preparation

COVID-19仍然是一个全球性挑战。该疾病的一个关键方面是，多达一半的SARS-CoV-2病毒感染者没有症状，使得仅基于临床表现的筛查和遏制策略成为不可能。除非能够定期筛查有症状感染者和无症状感染者，否则社会无法完全复工复学。因此，迫切需要一种普遍可获得的快速护理点（POC）诊断，其结果准确可靠，可以在前所未有的全球范围内以负担得起的方式部署。然而，几十年来，诊断领域一直在努力使这些复杂的测试与POC设置兼容。在开发超灵敏和快速的核酸扩增测定法方面已经取得了很大进展;然而，自1990年以来，最初RNA提取步骤的核心技术在很大程度上仍然没有改进。RNA提取步骤是开发可全球部署的COVID-19诊断方法的关键瓶颈。该项目将为COVID-19诊断技术带来界面工程的进步，将分子诊断从实验室中分散出来，这是重新开放美国经济和保护社会弱势成员所必需的。该项目的最终影响将是提高SARS-CoV-2 RNA测试的性能和可用性，以便这些测试可以在POC由受过最低限度培训的用户运行。有两个目标：（1）减少与依赖供应链和集中实验室相关的后勤负担，（2）简化RNA提取步骤，消除对复杂设备的需求。标准RNA提取遵循基于固相提取（SPE）的复杂的多步骤方案。该方案需要离心，并且由于抑制性缓冲液的残留而降低了测定性能。该项目将通过整合创新方法直接解决与RNA提取相关的两个瓶颈：两相洗涤（TPW），在提取步骤中减少抑制剂，同时保持RNA产量。TPW技术集成了与水不混溶的清洗缓冲液。TPW通过利用固-液和液-液界面特性和抑制组分的溶解度的组合从萃取柱中去除污染物。 通过TPW获得的额外纯度将提高检测灵敏度并降低成本，并将使冻干试剂和等温扩增的使用成为可能，消除了制冷要求并减少了检测时间（从数小时到数分钟）。 最后，将TPW与基于压力的RNA提取技术结合使用将消除对离心的需要，并将提高RNA提取的速度和可及性。此外，该项目将通过TPW利用界面工程来开发样品制备模块，该模块可用作独立组件，并与其他最先进的放大和读出技术相结合（即插即用），例如工业合作者设计的技术和RAPID计划中目前正在开发的其他传感/检测技术。该项目开发的技术可立即被商业和商业前诊断制造商采用。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

COVID-19 continues to be a global challenge. A critical aspect of the disease is that up to half of all people infected with the SARS-CoV-2 virus are asymptomatic, rendering screening and containment strategies based solely on clinical presentation impossible. Society cannot fully return to work and school unless both symptomatic and asymptomatic infected individuals can be screened regularly. There is therefore an urgent need for a universally accessible, rapid point-of-care (POC) diagnostic with accurate, reliable results that can be deployed in an affordable way on an unprecedented global scale. However, the diagnostics field has struggled for decades to make these complex tests compatible with POC settings. Great progress has been made developing nucleic-acid amplification assays that are ultrasensitive and rapid; yet the core technology for the initial RNA-extraction step remains largely unimproved since 1990. The RNA extraction step is the key bottleneck to developing a globally deployable COVID-19 diagnostic. This project will bring advances in interfacial engineering to COVID-19 diagnostic technology to decentralize molecular diagnostics from the laboratory, which is needed to reopen the US economy and to protect the vulnerable members of society. The ultimate impact of this project will be to improve the performance and availability of SARS-CoV-2 RNA testing so that these tests can be run at the POC by minimally trained users. There are two goals: (1) reduce the logistical burden associated with relying on supply-chains and centralized labs and (2) simplify the RNA extraction step to eliminate the need for complex equipment. Standard RNA extraction follows a complex, multi-step protocol based on solid-phase extraction (SPE). The protocol requires centrifugation, and it suffers from lowered assay performance due to the carryover of inhibitory buffers. This project will directly address both bottlenecks associated with RNA extraction by integrating an innovative approach: a two-phase wash (TPW) that reduces inhibitors while maintaining the RNA yield during the extraction step. The TPW technology integrates a wash buffer immiscible with water. TPW removes contaminants from the extraction column by leveraging the combination of solid-liquid and liquid-liquid interfacial properties and solubility of the inhibitory components. Extra purity obtained via TPW will improve assay sensitivity and reduce cost and will enable the use of lyophilized reagents and isothermal amplification, eliminating refrigeration requirements and reducing testing time (from hours to minutes). Finally, using TPW with a pressure-based RNA-extraction technology will eliminate the need for centrifugation and will improve the speed and accessibility of RNA extraction. Additionally, this project will leverage interfacial engineering via TPW to develop sample-preparation modules that can be used as stand-alone components and combined (plug-and-play) with other state-of-the-art amplification and readout technologies, such as those designed by industrial collaborators and other sensing/detection technologies currently under development in the RAPID program. The technologies developed in this project can be immediately adopted by commercial and pre-commercial diagnostic manufacturers.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.