Optimization of\nReverse Transcription Loop-Mediated\nIsothermal Amplification for In Situ Detection of SARS-CoV‑2\nin a Micro-Air-Filtration Device Format

The Coronavirus disease 2019 (COVID-19) pandemic has\nsupercharged\ninnovation in the field of molecular diagnostics and led to the exploration\nof systems that permit the autonomous identification of airborne infectious\nagents. Airborne virus detection is an emerging approach for determining\nexposure risk, although current methods limit intervention timeliness.\nHere, we explore reverse transcription loop-mediated isothermal amplification\n(RT-LAMP) assays for one-pot detection of Severe acute respiratory\nsyndrome Coronavirus 2 (SARS-CoV-2) (SCV2) run on membrane filters\nsuitable for micro-air-filtration of airborne viruses. We use a design\nof experiments statistical framework to establish the optimal additive\ncomposition for running RT-LAMP on membrane filters. Using SCV2 liquid\nspike-in experiments and fluorescence detection, we show that single-pot\nRT-LAMP on glass fiber filters reliably detected 0.10 50% tissue culture\ninfectious dose (TCID<sub>50</sub>) SCV2 per reaction (3600 E-gene\ncopies) and is an order of magnitude more sensitive than conventional\nRT-LAMP.