Isothermal amplification as a water safety tool: rapid detection of viruses in surface water and wastewater

Abstract

This study introduces a simple and rapid multi-wavelength, semi-quantitative detection strategy for monitoring SARS-CoV-2 and MS2 bacteriophage in water and wastewater using reverse transcription loop-mediated isothermal amplification (RT-LAMP). By integrating microplate-based spectrophotometry, we enabled higher throughput monitoring through simple optical measurements, thereby reducing the complexity of sample processing. Our findings demonstrate that RT-LAMP can be performed at lower temperatures, such as 45 °C, with incubation times of ≤60 minutes, while maintaining assay accuracy. The RT-LAMP yielded a conservative positivity threshold of ≥0.25 ΔOD_434–560nm for both SARS-CoV-2 and MS2, with limits of detection (LOD) of ∼180 copies per μL and 1000 PFU mL⁻¹ for SARS-CoV-2 and MS2, respectively. Statistically significant agreement with RT-qPCR was observed above 100 copies per μL (p < 0.001), with strong inverse correlations between Cq values and ΔOD_434–560nm readings for both targets (p < 0.001). Variability was primarily confined to low-template samples (<100 copies per μL), where stochastic primer dynamics and matrix inhibitors likely broadened coefficient of variation percentages; however, precision tightened to <10% once targets exceeded 500 copies per μL. To assess real-world applicability, RT-LAMP was applied to raw wastewater and eluates from granular activated carbon (GAC)-based passive samplers in surface waters. In wastewater, RT-LAMP detected endogenous SARS-CoV-2 and MS2 with 100% and 85% positive predictive values, respectively, aligning with RT-qPCR benchmarks. In surface waters, SARS-CoV-2 was detected in 10% of RT-LAMP replicates, while MS2 remained undetected. These results support the use of isothermal amplification with spectrophotometry and scalable sampling for rapid, field-deployable viral detection.