Pasteurization, storage conditions and viral concentration methods influence RT-qPCR detection of SARS-CoV-2 RNA in wastewater

The COVID-19 pandemic presents many public health challenges including the tracking of infected individuals from local to regional scales. Wastewater surveillance of viral RNA has emerged as a complementary approach to track and monitor the presence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in a variety of communities of different land use and population size. In the present study, we investigate how five different parameters (pasteurization, storage temperature, storage time, polyethylene glycol (PEG) concentration, and pellet mass) affect the detection of the SARS-CoV-2 N gene and fecal abundance indicator pepper mild mottle virus (PMMoV) gene. Pre-treatment of 24-h composite wastewater samples (n = 14) by pasteurization at 60 degrees C resulted in a significant reduction of total RNA concentration and copies of the SARS-CoV-2 N gene copies/L (paired Student's t-test, P < 0.05). Comparing the wastewater samples collected from 6 wastewater treatment plants (WWTPs) for a storage period of 7 and 14 days at 4 degrees C, -20 degrees C and -80 degrees C, demonstrated a decrease in SARS-CoV-2 N gene copies/L when samples were stored for 14 days at -20 degrees C. Polyethylene glycol-NaCl for purification and concentration of viral particles from the wastewater samples demonstrated that a short PEG incubation of 2 h during centrifugation at 4 degrees C was suffi-cient for the consistent detection of the SARS-CoV-2 N gene from a 30 mL sample volume. Combined, this paper presents method recommendations for developing a reliable, accurate, sensitive, and reproducible estimation of the SARSCoV-2 virus in diverse domestic wastewater samples.