Monitoring SARS-CoV-2 RNA in Wastewater with RT-qPCR and Chip-Based RT-dPCR: Sewershed-Level Trends and Relationships to COVID-19

We evaluated the performance of reverse transcription quantitative PCR (uniplex and duplex RT-qPCR) and chip-based digital PCR (duplex RT-dPCR) using CDC N1 and CDC N2 assays for longitudinal monitoring of SARS-CoV-2 RNA in influent wastewater samples (n = 281) from three wastewater plants in Ohio from January 2021 to January 2022. Human fecal virus (PMMoV) and wastewater flow rate were used to normalize SARS-CoV-2 concentrations. SARS-CoV-2 measurements and COVID-19 cases were strongly correlated, but normalization effects on correlations varied between sewersheds. SARS-CoV-2 measurements by RT-qPCR were strongly correlated with 7-day moving average COVID-19 cases (average Spearman's ρ = 0.58, p < 0.05). SARS-CoV-2 was detected more frequently in samples with duplex RT-dPCR than with duplex RT-qPCR during periods of low COVID-19 cases. Duplex and uniplex RT-qPCR N1 concentrations were more strongly correlated with cases (ρ = 0.62) than N2 (ρ = 0.52). RT-dPCR correlations (average ρ = 0.21) were weaker than those of RT-qPCR (average ρ = 0.58). We also share practical experience from establishing wastewater surveillance. Per sample, RT-qPCR had a lower cost ($6 vs $18) and sample turnaround time (3-4 h vs 7-9 h) than RT-dPCR. These findings reinforce selection and use of PCR-based wastewater surveillance tools.