Comparative analyses of SARS-CoV-2 RNA concentrations in Detroit wastewater quantified with CDC N1, N2, and SC2 assays reveal optimal target for predicting COVID-19 cases.

To monitor COVID-19 through wastewater surveillance, global researchers dedicated significant endeavors and resources to develop and implement diverse RT-qPCR or RT-ddPCR assays targeting different genes of SARS-CoV-2. Effective wastewater surveillance hinges on the appropriate selection of the most suitable assay, especially for resource-constrained regions where scant technical and socioeconomic resources restrict the options for testing with multiple assays. Further research is imperative to evaluate the existing assays through comprehensive comparative analyses. Such analyses are crucial for health agencies and wastewater surveillance practitioners in the selection of appropriate methods for monitoring COVID-19. In this study, untreated wastewater samples were collected weekly from the Detroit wastewater treatment plant, Michigan, USA, between January and December 2023. Polyethylene glycol precipitation (PEG) was applied to concentrate the samples followed by RNA extraction and RT-ddPCR. Three assays including N1, N2 (US CDC Real-Time Reverse Transcription PCR Panel for Detection of SARS-CoV-2), and SC2 assay (US CDC Influenza SARS-CoV-2 Multiplex Assay) were implemented to detect SARS-CoV-2 in wastewater. The limit of blank and limit of detection for the three assays were experimentally determined. SARS-CoV-2 RNA concentrations were evaluated and compared through three statistical approaches, including Pearson and Spearman's rank correlations, Dynamic Time Warping, and vector autoregressive models. N1 and N2 demonstrated the highest correlation and most similar time series patterns. Conversely, N2 and SC2 assay demonstrated the lowest correlation and least similar time series patterns. N2 was identified as the optimal target to predict COVID-19 cases. This study presents a rigorous effort in evaluating and comparing SARS-CoV-2 RNA concentrations quantified with N1, N2, and SC2 assays and their interrelations and correlations with clinical cases. This study provides valuable insights into identifying the optimal target for monitoring COVID-19 through wastewater surveillance.