Szlag, DavidHunawill, EmilyWu, ColinAvery, AdamWendell, DougWestrick, Randal2024-09-252024-09-252023-01-01https://hdl.handle.net/10323/18163Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Our study focuses on identifying and quantifying the presence of SARS-CoV-2 in the sewage based on identifying three gene targets which target the nucleocapsid gene (N1 and N2) and the envelope gene (E). The first set of experiments looked at the degradation of these targets at 4C, 25C, and 35C. As temperature increased so did the rate constants. Based on the half-life data, N1 degraded faster than N2 at 4C, all targets degraded at the same rate at 25C, and E degraded faster than N1 and N2 at 35C. Frequency of the gene targets was then assessed. E occurred less frequently than N1 and N2 adding 2.3 more SARS-CoV-2 detections leading to its removal from testing. The N1 and N2 gene targets were both necessary with removal of one resulting in minimally 9.6 loss of SARS-CoV-2 detections. We investigated normalization of the N1 and N2 gene targets with four different fecal indicators (pepper mottled mild virus, HF183, lachno3, and crAssphage) to improve the correlation between the sewage signal and clinical cases. Normalizing the data did not result in an increased correlation between the clinical and sewage data. Bovine coronavirus (BCoV) and Phi6 viruses were evaluated as surrogates to estimate SARS-CoV-2 inhibition in a single duplex reaction. Duplexing these targets was successful without significant signal loss, and these targets were used to estimate SARS-CoV-2 inhibition. Compared to BCoV, Phi6 suggested over 10 more fully inhibited samples. Several workflow modifications including bovine serum albumin (BSA), dilution, polyvinylpyrrolidone, and pasteurization were applied to the sewage samples in an attempt to reduce sample inhibition. BSA was able to reduce inhibition for both N1 and N2. Lastly, BCoV, Phi6, and murine hepatitis virus (MHV) retrieval were compared to the retrieval of the SARS-CoV-2 gene targets in sewage to determine if they accurately depicted the amount of inhibition the SARS-CoV-2 targets exhibited. BCoV and MHV were better surrogates to assess SARS-CoV-2 inhibition. SARS-CoV-2 RNA concentration in sewage were shown to correlate with COVID-19 cases.BCoVCOVID-19Phi6SARS-CoV-2