RAPID: Enzyme-free detection of SARS-CoV2 using a PAINT-based single-molecule microscopy assay

引起COVID-19的新型冠状病毒由一个球形包膜组成，包膜包围着由核糖核酸（RNA）组成的基因组。这些RNA控制病毒复制，并且对于每种冠状病毒株具有不同的区域。大多数COVID-19的快速检测测试都会用一种称为酶的蛋白质扩增三个独特的RNA区域。酶的活性大大提高了COVID-19检测的灵敏度，但缺点是需要冷藏、保质期短，以及由于酶活性的丧失而可能出现假阴性结果。这项研究开发了一种无酶COVID-19检测方法，该方法利用了新型冠状病毒基因组沿着锁定到位的稳定脱氧核糖核酸（DNA）分子的结合。当另一个标记DNA分子不断与附着在冠状病毒遗传物质上的DNA对接和脱离时，可以使用显微镜观察到新型冠状病毒。该项目将为快速检测COVID-19提供灵敏和稳定的检测方法。该项目的主要目标是开发一种灵活的无酶，货架稳定的病毒检测，可以很容易地适应当前以及未来的病毒爆发。该项目的目标是开发一种无酶的检测方法，SARS-CoV-2的单分子灵敏度，可以检测突变株的单核苷酸多态性在美国。为了实现这一目标，病毒捕获盖玻片将通过共价连接与70-nt前导序列（LS）反向互补的寡核苷酸（oligo）序列和SARS-CoV-2基因组关键区域的5'序列来工程化。将提取的病毒RNA应用于盖玻片，以拉下与LS捕获锚结合的所有病毒转录物。疾病预防控制中心（CDC）建议的SARS-CoV-2检测的特定区域将被用作靶序列，以设计用于通过基于DNA的纳米级拓扑学点累积（sRNA-PAINT）检测小RNA的探针。通过部署与寡核苷酸“对接链”连接的高度特异性锁核酸（LNA）寡核苷酸探针，使用sRNA-PAINT实现单分子病毒检测和靶区域扩增。 扩增是无酶的，相反，任何单个探针的信号通过寡核苷酸“成像链”与探针的对接链的可预测的结合和解结合来扩增。成像链具有共轭荧光团，并且使用灵敏的相机在显微镜上拍摄超过15分钟的数千张图像中检测它们的结合。检测测定或试剂盒将由寡核苷酸（寡核苷酸）连接的捕获盖玻片、COVID-19特异性寡核苷酸探针、荧光寡核苷酸成像探针、缓冲液、该奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的智力价值和更广泛的评估来支持的。影响审查标准。

The novel coronavirus that causes COVID-19 is composed of a spherical envelope that surrounds a genome made of ribonucleic acids (RNAs). These RNAs control viral replication and have distinct regions for each coronavirus strain. Most rapid detection tests for COVID-19 amplify three unique RNA regions with a protein called an enzyme. The activity of enzymes greatly increases the sensitivity of COVID-19 detection, but have disadvantages of requiring cold storage, short shelf life, and potential for false negative results due to the loss of enzymatic activity. This research develops an enzyme-free COVID-19 test that uses the binding of stabilized deoxyribonucleic acid (DNA) molecules that are locked into place along the novel coronavirus genome. The novel coronavirus can be visualized using a micrscope when another marker DNA molecule constantly docks and undocks from the DNA attached to the coronavirus genetic material. The project will deliver a sensitive and stable test for rapid detection of COVID-19. A major goal of this project is to develop a flexible enzyme-free, shelf-stable viral test that can be readily adapted for the current as well as future viral outbreaks.The goal of this project is to develop an enzyme-free detection method for SARS-CoV-2 with single-molecule sensitivity that can detect single nucleotide polymorphisms of mutated strains in the United States. To achieve this goal, viral capture coverglasses will be engineered by covalently linking oligonucleotide (oligo) sequences that are reverse complementary to the 70-nt leader sequence (LS) and sequences 5’ to key regions of SARS-CoV-2 genome. Extracted viral RNA will be applied to the coverglass to pull down all viral transcripts that bind to the LS capture anchors. The specific regions for SARS-CoV-2 detection suggested by the Center of Disease Control & Prevention (CDC) will be used as target sequences to design probes for small RNA detection via DNA-based points accumulation in nanoscale topography (sRNA-PAINT). Single-molecule viral detection and amplification of target regions will be achieved with sRNA-PAINT by deploying highly specific locked-nucleic acid (LNA) oligonucleotide probes linked to oligo “docking strands”. Amplification is enzyme-free, and instead, the signal for any individual probe is amplified by the predictable binding and unbinding of oligo “imager strands” to the docking strands of the probe. Imager strands have a conjugated fluorophore and their binding is detected in thousands of images taken over 15 minutes on a microscope using a sensitive camera. The detection assay or kit will consist of an oligonucleotide (oligo)-linked capture coverglass, COVID-19 -specific oligo probes, fluorescent oligo imaging probes, buffers, and instructions on how to either deploy the assay on any microscope with single molecule sensitivity or build one.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.