Since the COVID-19 pandemic is expected to become endemic, quantification of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in ambient waters is critical for environmental surveillance and for early detection of outbreaks. Herein, we report the development of a membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system that is designed for the rapid point-of-use quantification of SARS-CoV-2 particles in environmental waters. The mgLAMP system integrates the viral concentration, in-assay viral lysis, and on-membrane hydrogel-based RT-LAMP quantification using enhanced fluorescence detection with a target-specific probe. With a sample-to-result time of less than 1 h, mgLAMP successfully detected SARS-CoV-2 below 0.96 copies/mL in Milli-Q water. In surface water, the lowest detected SARS-CoV-2 concentration was 93 copies/mL for mgLAMP, while the reverse transcription quantitative polymerase chain reaction (RT-qPCR) with optimal pretreatment was inhibited at 930 copies/mL. A 3D-printed portable device is designed to integrate heated incubation and fluorescence illumination for the simultaneous analysis of nine mgLAMP assays. Smartphone-based imaging and machine learning-based image processing are used for the interpretation of results. In this report, we demonstrate that mgLAMP is a promising method for large-scale environmental surveillance of SARS-CoV-2 without the need for specialized equipment, highly trained personnel, and labor-intensive procedures.
Keywords: RT-LAMP; SARS-CoV-2; environmental quantification of SARS-CoV-2 in milliliters of environmental water samples; hydrogel; membrane.