Long-term monitoring of SARS-CoV-2 load and variant composition at a large metropolitan wastewater treatment plant using a simple two-step direct capture RNA extraction, droplet digital PCR, and targeted mutation assays

Wastewater surveillance offers an objective, comprehensive, and cost-effective means of monitoring the prevalence and genomic heterogeneity of pathogens circulating in a community. Here, a novel two-step extraction procedure for the direct capture of SARS-CoV-2 RNA from raw wastewater is presented. Combined with reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR) detection, the method provides a fast and sensitive method for measuring viral RNA concentrations in wastewater. The method was used to measure the concentration of SARS-CoV-2 RNA in daily samples of wastewater entering a major metropolitan wastewater treatment plant over the course of 32 months, from November 2020 through June 2023. In addition, targeted mutation assays were used with RT-ddPCR to characterize the evolving presence and prevalence of specific SARS-CoV-2 variant sub-lineages in the wastewater stream over time. The results demonstrate the utility of these methods to accurately measure the total load of SARS-CoV-2 RNA, and chronicle its evolving variant composition, in wastewater treatment plant influent, providing near-real-time characterization of COVID-19 disease prevalence and trends in the served community.