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

新冠肺炎仍然是一个全球性的挑战。这种疾病的一个关键方面是，多达一半感染SARS-CoV-2病毒的人没有症状，使得仅基于临床表现的筛查和遏制策略是不可能的。除非定期对有症状和无症状的感染者进行筛查，否则社会不可能完全重返工作和学校。因此，迫切需要一种普遍可访问的、具有准确、可靠结果的快速医疗点(POC)诊断，该诊断可以以前所未有的全球规模以负担得起的方式部署。然而，诊断领域几十年来一直在努力使这些复杂的测试与PoC设置兼容。在发展超灵敏、快速的核酸扩增分析方面取得了很大进展，但自1990年以来，最初的RNA提取步骤的核心技术仍然没有得到很大的改进。核糖核酸的提取步骤是开发可全球部署的新冠肺炎诊断程序的关键瓶颈。该项目将为新冠肺炎诊断技术带来界面工程方面的进步，将分子诊断从实验室分散开来，这是重启美国经济和保护社会弱势群体所必需的。该项目的最终影响将是改进SARS-CoV-2 RNA测试的性能和可用性，以便这些测试可以由受过最低培训的用户在POC进行。有两个目标：(1)减少与依赖供应链和中央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要。标准的RNA提取遵循基于固相萃取(SPE)的复杂的多步方案。该方案需要离心法，而且由于抑制缓冲液的携带，它的检测性能降低。该项目将通过整合一种创新的方法直接解决与RNA提取相关的两个瓶颈：两阶段洗涤(TPW)，在提取步骤中减少抑制剂，同时保持RNA产量。TPW技术集成了一种不与水混合的洗涤缓冲液。TPW通过利用抑制组分的固-液和液-液界面性质和溶解性，从萃取柱中去除污染物。通过TPW获得的额外纯度将提高分析灵敏度和降低成本，并将能够使用冷冻干燥试剂和等温放大，消除冷藏要求并减少测试时间(从几小时减少到几分钟)。最后，使用基于压力的RNA提取技术的TPW将不再需要离心，并将提高RNA提取的速度和可获得性。此外，该项目将利用通过TPW的界面工程来开发样品制备模块，这些模块可以作为独立组件使用，并与其他最先进的放大和读出技术相结合(即插即用)，例如由行业合作者设计的那些技术，以及目前正在开发的快速计划中的其他传感/检测技术。该项目开发的技术可以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。

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.