Recovery of SARS-CoV-2 from Wastewater Using Centrifugal Ultrafiltration

The COVID-19 pandemic is a global crisis and continues to impact communities as the disease spreads. Clinical testing alone provides a snapshot of infected individuals but is costly and difficult to perform logistically across whole populations. The virus which causes COVID-19, SARS-CoV-2, is shed in human feces and urine and can be detected in human waste. SARS-CoV-2 can be shed in high concentrations (>10(7) genomic copies/mL) due to its ability to replicate in the gastrointestinal tract of humans through attachment to the angiotensin-converting enzyme 2 (ACE-2) receptors there. Monitoring wastewater for SARS-CoV-2, alongside clinical testing, can more accurately represent the spread of disease within a community. This protocol describes a reliable and efficacious method to recover SARS-CoV-2 in wastewater, quantify genomic RNA levels, and evaluate concentration fluctuations over time. Using this protocol, viral levels as low as 10 genomic copies/mL were successfully detected from 30 mL of wastewater in more than seven-hundred samples collected between August 2020 and March 2021. Through the adaptation of traditional enteric virus methods used in food safety research, targets have been reliably detected with no inhibition of detection (RT-qPCR) observed in any sample processed. This protocol is currently used for surveillance of wastewater systems across New Castle County, Delaware.