Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification for In Situ Detection of SARS-CoV-2 in a Micro-Air-Filtration Device Format

Abstract

The Coronavirus disease 2019 (COVID-19) pandemic has supercharged innovation in the field of molecular diagnostics and led to the exploration of systems that permit the autonomous identification of airborne infectious agents. Airborne virus detection is an emerging approach for determining exposure risk, although current methods limit intervention timeliness. Here, we explore reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for one-pot detection of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) (SCV2) run on membrane filters suitable for micro-air-filtration of airborne viruses. We use a design of experiments statistical framework to establish the optimal additive composition for running RT-LAMP on membrane filters. Using SCV2 liquid spike-in experiments and fluorescence detection, we show that single-pot RT-LAMP on glass fiber filters reliably detected 0.10 50% tissue culture infectious dose (TCID₅₀) SCV2 per reaction (3600 E-gene copies) and is an order of magnitude more sensitive than conventional RT-LAMP.

Figure 1

(a) Effect of reaction volume on reaction times for nonoptimized RT-LAMP reactions on-filter, showing individual values and 90% confidence intervals. (b) Plot showing the effect of different additives on RT-LAMP Tp from the first stage DOE. Error bars depict 90% confidence intervals. Confidence intervals that pass over the zero mark are not considered statistically significant. A negative value indicates that the additive reduced the Tp. (c, d) Pareto charts of standard effects of different additives on their effect on Tp and time to false positive (Tfp), respectively, at 95% confidence. Bars indicate the size but not the direction of the standard effect for the coded additives. The red horizontal line at 1.96 indicates the 95% significant level. Pairs of letters indicate interactions between terms and second-order coefficients.