Saturday, April 20, 2024
4/20/2024
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.
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