RAPID:COVID-19: RT-LAMP-based electrical detection of SARS-CoV-2

自2019年12月导致冠状病毒相关急性呼吸道疾病COVID-19的病毒SARS-CoV-2从动物宿主传播到人类以来，它已迅速蔓延到世界各地，给企业和个人带来死亡、疾病、日常生活中断以及经济损失。COVID-19大流行的快速发展凸显了现有的基于实验室的病毒诊断检测模式的缺陷，其中包括普遍缺乏检测试剂盒，获得检测结果的时间延迟较长，以及假阴性检测率高。现有以实验室为基础的基础设施的缺陷正在造成围绕隔离失败的不确定性，卫生当局之间的混乱以及公众的普遍焦虑。为了解决这一关键和及时的需求，在这项工作中，建议开发一种即时护理设备，用于在10分钟内从鼻液样本中检测SARS-CoV-2的存在。当前病毒病原体检测的基本局限性源于它们对聚合酶链反应（PCR）分析的依赖，这需要复杂的、劳动密集型的、基于实验室的病毒分离、裂解和去除抑制物质的方案。重要的是，PCR需要大量耗时和精确的热循环来酶扩增特定的RNA序列。在这项工作中，开发了一种电子、无标签和无表面修饰的护理点设备，用于在10分钟内从鼻液样本中检测SARS-CoV-2（1-3拷贝/mL）的存在。这项工作将在等温RT-LAMP（逆转录环介导的等温扩增）反应中结合Bst聚合酶，与目标特异性引物和皱巴巴的石墨烯场效应晶体管（gFET）结合，通过感应引物的消耗来电检测扩增事件。这些反应提供了使用简单、廉价和便携的恒电位器快速检测SARS-CoV-2的可能性，并避免了提取RNA的必要性。为了开发建议的检测技术，本提案将首先验证12套SARS-CoV-2特异性RT-LAMP引物。重要的是，没有针对SARS-CoV-2的RT-LAMP引物在商业上可用，在文献中也没有。然后，该提案将论证基于rt - lamp的电气方法检测SARS-CoV-2的可行性。同样，该提案将展示这种方法作为一种全球卫生技术，有助于在世界各地提供低成本诊断，使用便携式和廉价的加热块和恒电位器进行病毒检测和定量。该奖项反映了美国国家科学基金会的法定使命，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。

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the virus that causes the coronavirus-associated acute respiratory disease COVID-19) jumped from an animal reservoir to humans in December 2019, it has rapidly spread across the world, bringing death, illness, disruption to daily life, and economic losses to businesses and individuals. The rapid development of the COVID-19 pandemic highlights the shortcomings in the existing laboratory-based testing paradigm for viral diagnostics that features a widespread lack of test kits, extended time delays to obtain test results, and a high rate of false negative tests. The shortcomings of the existing laboratory-based infrastructure are contributing to uncertainty surrounding quarantine failure, confusion among health authorities, and also to a general public anxiety. To address this critical and timely need, in this work, development of point-of-care device is proposed for detecting the presence of SARS-CoV-2 from nasal fluid samples and in 10 minutes.The fundamental limitations of current assays for viral pathogens stem from their reliance upon polymerase chain reaction (PCR) analysis, which requires complex, labor-intensive, laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. Importantly, PCR requires a large number of time-consuming and precise thermal cycles to enzymatically amplify specific RNA sequences. In this work development of an electrical, label-free and surface modification-free point-of-care device for detecting the presence of SARS-CoV-2 (1-3 copy/mL) from nasal fluid samples and in 10 min. The proposed work will combine Bst polymerase in an isothermal RT-LAMP (reverse transcription loop-mediated isothermal amplification) reaction, with target-specific primers and crumpled graphene field-effect transistors (gFET) to electrically detect the amplification event by sensing the consumption of primers. These reactions offer the possibility of rapidly detecting SARS-CoV-2 using a simple, inexpensive and portable potentiostat and avoiding the necessity of RNA extraction. In order to develop the proposed detection technology, this proposal will first validate twelve sets of RT-LAMP primers specific to SARS-CoV-2. Importantly, No RT-LAMP primers specific to SARS-CoV-2 are available commercially, neither in the literature. Then, the proposal will demonstrate the feasibility of the RT-LAMP-based electrical approach to detect SARS-CoV-2. Likewise, the proposal will demonstrate the approach as a global health technology to contribute to providing low-cost diagnostics around the world using portable and inexpensive heating block and potentiostat for virus detection and quantification.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.