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Global Health Technologies

Global Health Technologies I

SARS-CoV-2 RNA Sub-Categorization in Clinical Specimens with a High-Throughput, Multi-Parametric Approach Based on Single Extracellular Vesicle Characterization

Friday, October 13, 2023
8:15 AM – 8:30 AM PDT
Location: 347 (Center, Level 3)
Introduction:: The global death toll due to the SARS-CoV-2 pandemic has surpassed 6.7 million people. Various bulk analysis techniques have been created to detect the novel coronavirus. However, these methods have limitations, such as low specificity. As a result, a multi-parametric approach has been developed to enhance detection accuracy by correlating antigen and RNA analyses on intact virus particles.



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Materials and Methods:: Our biochip antigen and RNA assay (BARATM) enables the high-throughput screening of 256 samples (Figure A). To prepare clinical specimens for analysis, size exclusion chromatography was utilized for purification. The assay captures non-lysed viral particles and EVs on antibody-decorated biochips, without requiring lysis or amplification steps. The viral RNA was labeled with molecular beacons (MB), generating fluorescence signals that were quantified using total internal reflection fluorescence microscopy (TIRFM). Subsets of EVs (Figure B) and viruses (Figure C) were identified using spike and anti-CD63 detection antibodies.


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Results, Conclusions, and Discussions:: The molecular beacons (MBs) used in our study successfully differentiated between the alpha, beta, gamma, and omicron strains of SARS-CoV-2. BARATM detected SARS-CoV-2 RNA in isolated EVs in saliva and plasma patient specimens through single EVs analysis. In retrospective analyses of saliva specimens from 32 patients and 30 healthy donors, protein detection showed a 94% positive percentage agreement (PPA) and a 100% negative percentage agreement (NPA). Meanwhile, RNA detection had a 91% PPA and a 100% NPA. Multi-parametric detection in saliva using both protein and RNA improved to a 100% PPA. The combined protein and RNA detection in nasal swab specimens resulted in a 95% PPA, whereas individual protein and RNA detection yielded only 88% and 80% PPA, respectively.

This technique would pave the path to sub-categorize viral RNA in clinical specimens and elucidate viral load during different phases of infection. The BARA is currently under review for Emergency Use Authorization from FDA.



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Acknowledgements (Optional): : This work was supported by the U.S. National Institutes of Health (NIH) grant UG3/UH3TR0002884 & U18TR003807 (E.R. & L.J.L.). Additional support was provided by the William G. Lowrie Department of Chemical and Biomolecular Engineering at The Ohio State University.



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