RAPID: Rapid Assay for RNA Extraction and Concentration for COVID-19 Molecular Diagnostics

获得冠状病毒检测对于遏制和控制2019年冠状病毒病(新冠肺炎)的传播是不可或缺的。新冠肺炎诊断检测目前采用了一种名为逆转录-聚合酶链式反应(RT-PCR)的技术。这项技术需要两个试剂盒。第一种是用来从冠状病毒中提取遗传物质--核糖核酸(RNA)，第二种是用来扩增RNA，以便用RT-PCR进行检测。目前的诊断危机部分是由于RNA提取试剂盒的短缺以及使用试剂盒提取RNA所需的时间。提高这些试剂盒的可用性和时效性对于改善检测的可及性和增强临床诊断的可靠性至关重要。在这个项目中，将使用一种生产多孔吸附材料的新工艺来开发一种替代提取试剂盒，以高效快速地从患者样本中提取核酸。通过研究吸附剂捕获和释放核酸的分子机制并应用这些基本知识，将进一步优化其性能。在开发和临床验证一种新的提取试剂盒之后，将进行大规模生产，解决目前的短缺问题，并改善获得新冠肺炎检测的机会。在该项目的推动下，开发诊断技术的工程师和实施原型的临床医生之间的密切合作，将使科学知识转化为有利于社会健康和福祉的解决方案。该项目还将通过培训研究人员掌握新的冠状病毒诊断技术来提供劳动力发展机会。该项目的目标是开发一种新的、可扩展的核酸分离和浓缩方法，并大规模生产原型试剂盒，以便立即在临床环境中实施。这一概念依赖于使用吸附材料，而不是目前以过滤器和硅胶柱为基础的方法。与使用孔径大小将目标(这里是核酸)与介质物理分离的过滤器不同，吸着剂是一种多孔材料，通过化学亲和力和相互作用捕获目标。因此，使用吸附剂可以提高水流速度、提高核酸捕获效率和更快的样品处理速度，并克服了对多个缓冲液或提取步骤的需要。将使用金属氧化物纳米颗粒和有机硅氧烷聚合物的组合对传统过滤器进行功能化处理，以生产核酸吸附剂。这种功能化将通过一种新的方法来实现，即通过热分解和聚合物偶联的方法来负载纳米粒子。该吸附剂的效率将通过提取和使用定量RT-PCR检测核酸来评估。替代吸附试剂盒的验证将评估快速分离和浓缩新型冠状病毒(SARS-CoV-2)RNA的有效性。该项目还将探索多孔性吸附剂中核酸动力学的基本原理，因为它与核酸大小、吸附剂化学、孔隙率和孔径以及多孔性介质中传输现象的影响有关。提高对影响核酸在多孔介质中的保留、释放和传输的因素的了解，对于生产可靠和高效的核酸提取和检测试剂盒以及开发其他生物分离过程至关重要。对博士后研究人员、研究生和本科生的培训将侧重于如何结合跨学科合作和对基本方面的清楚理解来产生颠覆性技术。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。

Access to coronavirus testing is integral to the effort to curb and control the spread of the 2019 coronavirus disease (COVID-19). COVID-19 diagnostic tests currently implement a technique called reverse transcription-polymerase chain reaction (RT-PCR). This technique requires two reagent kits. The first is used to extract the genetic material, ribonucleic acid (RNA), from the coronavirus, and the second is used to amplify the RNA to enable its detection with RT-PCR. The current diagnostic crisis is due, in part, to shortages of the RNA extraction kits and the time required to extract the RNA using the kits. Increasing both the availability and time-efficiency of these kits is vital to improve testing accessibility and enhance the reliability of clinical diagnostics. In this project, a novel process for producing porous sorbent materials will be used to develop an alternative extraction kit for efficient and rapid extraction of nucleic acids from patient samples. The performance will be further optimized by studying the molecular mechanisms governing nucleic acid capture and release by the sorbents and applying this fundamental knowledge. The development and clinical validation of a novel extraction kit will be followed by mass production, addressing the current shortage and improving access to COVID-19 testing. The close collaboration between engineers developing the diagnostic technologies and clinicians implementing the prototypes, as enabled by this project, will streamline the transition of scientific knowledge into solutions that benefit the health and well-being of society. The project will also provide workforce development opportunities through training researchers in novel diagnostic techniques for coronaviruses.The goal of this project is to develop a novel, scalable approach to nucleic acid separation and concentration and mass-produce prototype kits for immediate implementation in clinical settings. The concept relies on the use of sorbent materials instead of the current filter- and silica column-based approach. Unlike filters that use pore size to physically separate the target (here nucleic acids) from the media, sorbents are a porous material that captures the target by chemical affinity and interactions. As a result, the use of a sorbent enables larger water flow rates, enhanced nucleic acid capture efficiency, and faster sample processing, and overcomes the need for multiple buffers or extraction steps. Functionalization of conventional filters with a combination of metal oxide nanoparticles and organosiloxane polymers will be used to produce the nucleic acid sorbent. The functionalization will be achieved by a new method for supported synthesis of nanoparticles by thermolysis and polymer conjugation. The efficiency of the sorbent will be assessed by the extraction and detection of nucleic acid using quantitative RT-PCR. Validation of the alternative sorbent kit will assess the effectiveness of the rapid separation and concentration of the novel coronavirus (SARS-CoV-2) RNA. The project will also explore the fundamentals of nucleic acid dynamics in porous sorbents as it relates to nucleic acid size, sorbent chemistry, porosity and pore size, and the effect of transport phenomena in porous media. Enhanced understanding of the factors that affect the retention, release, and transport of nucleic acids in porous media is critical to producing reliable and efficient nucleic acid extraction and detection kits and the development of other bio-separation processes. Training of postdoctoral researchers and graduate and undergraduate students will focus on how the combination of transdisciplinary collaboration and a clear understanding of the fundamental aspects can lead to disruptive technologies.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.