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

Jacob Fry; Jean Y H Lee; Julie L McAuley; Jessica L Porter; Ian R Monk; Samuel T Martin; David J Collins; Gregory J Barbante; Nicholas J Fitzgerald; Timothy P Stinear

doi:10.1021/acsomega.4c05784

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

ACS Omega. 2024 Sep 20;9(39):40832-40840. doi: 10.1021/acsomega.4c05784. eCollection 2024 Oct 1.

Authors

Affiliations

¹ ARC Centre of Excellence in Exciton Science, The School of Chemistry, The University of Melbourne, Masson Rd, Parkville, Victoria 3010, Australia.
² Department of Microbiology and Immunology, The Doherty Institute for Infection and Immunity, The University of Melbourne, 792 Elizabeth Street, Melbourne, Victoria 3000, Australia.
³ Department of Biomedical Engineering, The University of Melbourne, Building 261/203 Bouverie St, Carlton, Victoria 3053, Australia.
⁴ Graeme Clarke Institute, The University of Melbourne, Chemical Engineering 2 Building 167, Parkville, Victoria 3010, Australia.
⁵ Defence Science and Technology Group, Australian Department of Defence, 506 Lorimer Street, Fishermans Bend, Victoria 3207, Australia.

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.