Detection of SARS-CoV-2 Proteins in Wastewater Samples by Mass Spectrometry.

The recent COVID-19 pandemic overwhelmed the health system worldwide, and there was a need to track outbreaks and try to use this information as an early warning system. Wastewater-based epidemiology (WBE) enabled detection of the SARS-CoV-2 virus in wastewater treatment plant influents. Until now, the most used technique for this detection has been the quantitative polymerase chain reaction (qPCR)-based quantification of SARS-CoV-2 RNA. This study proposes a mass spectrometry (MS)-based method that detected specific SARS-CoV-2 proteins in wastewater, 5 and 6 days ahead of the case data for two municipalities. We identified unique peptides of eight proteins related to the SARS-CoV-2 virus and COVID-19 infection. We detected the nonstructural protein (NSP) pp1ab (transcribed after host cell infection) most frequently in all of the samples. As a result, we suspect that in the active cases of COVID-19, the pp1ab protein is present in high abundance in the urine and feces and that this protein could be used as an alternative biomarker. These data were collected before mass vaccination occurred in the population.

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 °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 °C, -20 °C and -80 °C, demonstrated a decrease in SARS-CoV-2 N gene copies/L when samples were stored for 14 days at -20 °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 °C was sufficient 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 SARS-CoV-2 virus in diverse domestic wastewater samples.