Multiplexed Virus Detection at the Point-of-Care (POC) by a Valve-Enabled Sample Preparation Device with Isothermal Amplification

Carlos Mazanas, Md. Mahbubul Alam, Julia Loeb, John Lednicky, CHANG-YU WU, Z. Hugh Fan, University of Florida

     Abstract Number: 433
     Working Group: Aerosol Science of Infectious Diseases: What We Have Learned and Still Need to Know about Transmission, Prevention, and the One Health Concept

Abstract
With expected co-circulation of SARS-CoV-2 and influenza, their early and accurate detection at point-of-care is crucial for reducing their transmission. To address this need, we developed a valve-enabled lysis, paper-based RNA enrichment, and RNA amplification device (VLEAD) for simultaneous detection of SARS-CoV-2 and influenza A viruses. Each side of the device consists of a buffer unit at the top containing 4 wells (lysis buffer, binding buffer, and two wash buffers) for sample preparation, a mixing unit in the middle, and a detection unit at the bottom. Sample preparation process starts by adding a sample into each mixing well, followed by the release of the lysis buffer into the mixing unit. Then, the binding buffer is released to bind the RNA onto the chromatography paper in the detection unit. The wash buffers in the last two wells are then discharged to purify the collected RNA. Afterwards, the detection units are prepared for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Finally, the detection units are added SYBR Green dye for detection of amplicons by color change.

We have demonstrated the specificity of VLEAD by using SARS-CoV-2 and influenza A H1N1 pdm2009. In one experiment, we used the primer set targeting SARS-CoV-2 to test individual RNA of SARS-CoV-2, influenza A H1N1 pdm2009, and CoV-OC43; in the other experiment we used the primer set targeting Influenza A H1N1 pdm2009 to test the same three viruses. Both results confirmed the specific detection of the target virus with no cross-reaction with any of the other two viruses. VLEAD also showed great sensitivity with limit of detection at 10 genome equivalents for SARS-CoV-2 and 0.06 TCID50 for Influenza A H1N1 pdm2009.

Acknowledgements: This work is supported by National Science Foundation (CBET-2030844), National Institutes of Health (R01AI158868), and the University of Florida.