Liposome fusion enabled extracellular vesicle detection for COVID-19

摘要 COVID-19可导致在最初的SARS-CoV-2消退后长期存在的病理变化 感染需要新的、更灵敏的检测方法来更好地了解潜在的机制 参与这一过程，并改善对有症状和无症状COVID-19病例的检测， 包括长期感染，由于金的显著假阴性率， 标准COVID-19检测，逆转录酶定量聚合酶链反应（RT-qPCR）。呼吸 样本似乎是一个穷人的选择，以检测和监测SARS-CoV-2感染超出了相对狭窄的 病毒暴露后的窗口，因为上呼吸道中存在短暂和间歇性的病毒产生 感染后有证据表明，SARS-CoV-2可能通过循环系统传播，这表明 血液是均匀的，常规采集，不适和暴露风险最小， 作为备用诊断和监测样本。然而，RT-qPCR对于以下疾病表现出较差的诊断灵敏度： 分析血液样本时的COVID-19。我们的团队开发了一种快速，超灵敏的COVID-19检测方法 （CRISPR-FDS），其中CRISPR活性与扩增的DNA靶标成比例地切割淬灭的探针，以 使检测灵敏度提高20倍。这种检测方法现在用作研究性检测，可以检测COVID-19病例 鼻咽拭子样本的重复RT-qPCR检测遗漏，并且不需要昂贵的 设备、重要的技术专长或防护设备。我们最近的初步数据表明， 通过分析分离的血浆RNA的CRISPR-FDS测定检测循环无细胞病毒RNA， 诊断COVID-19病例，无论感染部位或持续时间。因此，我们建议调整我们的 CRISPR-FDS方法允许直接定量血浆中的病毒RNA。在该测定中，细胞外囊泡 (EVs)诱导直接从血浆捕获的CRISPR-FDS与装载有CRISPR-FDS的合成脂质体融合 使用标准ELISA，在小体积内检测试剂，以有效扩增和定量靶RNA 工作流我们选择EV用于该测定，因为这些囊泡由感染的细胞大量分泌， 将病毒RNA保存在其管腔内，并可通过抗体从血浆/血清中特异性捕获 靶向它们的表面蛋白以减少来自无细胞核酸的背景。Aim 1将优化CRISPR- FDS测定程序（例如，EV捕获、脂质体包装和融合以及试剂滴定步骤）， 最大化反应灵敏度，并评估如何修饰脂质体表面与靶向特异性 抗体影响测定性能。目的2将评价优化的测定方法的分析性能 与血浆样品的RT-qPCR相比，进行脂质体试验的分析验证，并采用该方法 用于定量分析纵向血浆队列中存在的血浆EV中的SARS-CoV-2 RNA的测定 使用血清或血浆进行EV介导的COVID-19诊断，并定量评估 来自COVID-19患者队列的纵向血液样本中的SARS-CoV-2 RNA。

Abstract COVID-19 can cause pathological changes that persist long after the resolution of the initial SARS-CoV-2 infection. New, more sensitive detection approaches are needed to better understand the potential mechanisms involved in this process and to improve the detection of both symptomatic and asymptomatic COVID-19 cases, including long-term infections, that may be missed by due to the significant false negative rate of the gold- standard COVID-19 test, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Respiratory samples appear to be a poor choice to detect and monitor SARS-CoV-2 infections beyond a relatively narrow window after virus exposure, as there is transient and intermittent viral production in the upper respiratory tract after infection. Evidence indicates that SARS-CoV-2 may spread systemically through the circulation, suggesting that blood, which is homogeneous and routinely collected with minimal discomfort and exposure risk, could serve as an alternate diagnostic and monitoring specimen. RT-qPCR, however, exhibits poor diagnostic sensitivity for COVID-19 when analyzing blood samples. Our team has developed a rapid, ultrasensitive COVID-19 assay (CRISPR-FDS) where CRISPR activity cleaves a quenched probe in proportion to an amplified DNA target to increase assay sensitivity 20-fold. This assay, now used as an investigational test, can detect COVID-19 cases missed by repeated RT-qPCR testing of nasopharyngeal swab samples, and does not require expensive equipment, significant technical expertise, or protective equipment. Our recent preliminary data suggest that detection of circulating cell-free viral RNA by a CRISPR-FDS assay that analyzes isolated plasma RNA can diagnose COVID-19 cases regardless of infection site(s) or duration. We therefore propose to adapt our CRISPR-FDS method to allow direct quantification of viral RNA in plasma. In this assay, extracellular vesicles (EVs) captured directly from plasma are induced to fuse with synthetic liposomes loaded with CRISPR-FDS assay reagents within a small volume to amplify and quantify target RNA efficiently, using a standard ELISA workflow. We selected EVs for this assay, since these vesicles are abundantly secreted by infected cells and preserve viral RNA within their lumen, and can be specifically captured from plasma/serum by antibodies targeting their surface protein to reduce background from cell-free nucleic acid. Aim 1 will optimize CRISPR- FDS assay procedures (e.g., EV capture, liposome packaging and fusion, and reagent titration steps) to maximize reaction sensitivity, and evaluate how modification of the liposome surface with target-specific antibodies influences assay performance. Aim 2 will evaluate the analytical performance of the optimized assay versus RT-qPCR for plasma samples, conduct and analytical validation of the liposome assay, and employ this assay for quantitative analysis of SARS-CoV-2 RNA in plasma EVs present in longitudinal plasma cohort direct EV-mediated COVID-19 diagnosis using serum or plasma, and quantitatively evaluate dynamic changes in SARS-CoV-2 RNA in longitudinal blood samples from a cohort of COVID-19 patients.