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

RAPID：通过改进样品制备对有限资源环境进行 COVID-19 诊断

• 批准号: 2032467
• 负责人: Rustem Ismagilov
• 金额: $ 14.09万
• 依托单位: California Institute of Technology
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2032467&HistoricalAwards=false
• 关键词: RAPID; COVID; 19; diagnostics; limited

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.

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，这使得仅基于临床表现的筛查和遏制策略变得不可能。除非能够定期筛查有症状和无症状的感染者，否则社会无法完全恢复工作和学校。因此，迫切需要一种普遍可用、快速的即时护理 (POC) 诊断，其结果准确、可靠，并且能够以经济实惠的方式在前所未有的全球范围内部署。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。超灵敏、快速的核酸扩增检测方法的开发取得了巨大进展；然而，自 1990 年以来，初始 RNA 提取步骤的核心技术在很大程度上仍未得到改进。RNA 提取步骤是开发可全球部署的 COVID-19 诊断的关键瓶颈。该项目将为 COVID-19 诊断技术带来界面工程方面的进步，以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，以便这些测试可由经过最低限度培训的用户在 POC 上运行。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 提取遵循基于固相萃取 (SPE) 的复杂多步骤方案。该方案需要离心，并且由于抑制缓冲液的残留而导致测定性能降低。该项目将通过整合创新方法来直接解决与 RNA 提取相关的两个瓶颈：两相清洗 (TPW)，可减少抑制剂，同时在提取步骤中保持 RNA 产量。 TPW 技术集成了与水不混溶的洗涤缓冲液。 TPW 通过利用固-液和液-液界面特性以及抑制成分的溶解度的组合来去除萃取塔中的污染物。 通过 TPW 获得的额外纯度将提高测定灵敏度并降低成本，并将允许使用冻干试剂和等温扩增，消除冷藏要求并缩短测试时间（从几小时缩短到几分钟）。 最后，将 TPW 与基于压力的 RNA 提取技术结合使用将消除离心的需要，并将提高 RNA 提取的速度和可及性。此外，该项目将利用 TPW 的界面工程来开发样品制备模块，这些模块可用作独立组件，并与其他最先进的放大和读出技术组合（即插即用），例如由工业合作者设计的技术以及 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以立即被商业和预商业诊断制造商采用。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。