In this study, 14 virus concentration protocols based on centrifugation, filtration, polyethylene glycol (PEG) precipitation and ultrafiltration were tested for their efficacy for the quantification of SARS-CoV-2 in wastewater samples. These protocols were paired with four RNA extraction procedures resulting in a combination of 50 unique approaches. Bovine respiratory syncytial virus (BRSV) was used as a process control and seeded in each wastewater sample subjected to all 50 protocols. The recovery of BRSV obtained through the application of 50 unique approaches ranged from <0.03 to 64.7% (±1.6%). Combination of centrifugation as the solid removal step, ultrafiltration (Amicon-UF-15; 100 kDa cut-off; protocol 9) as the primary virus concentration method, and Zymo Quick-RNA extraction kit provided the highest BRSV recovery (64.7 ± 1.6%). To determine the impact of prolonged storage of large wastewater sample volume (900 mL) at −20 °C on enveloped virus decay, the BRSV seeded wastewaters samples were stored at −20 °C up to 110 days and analyzed using the most efficient concentration (protocol 9) and extraction (Zymo Quick-RNA kit) methods. BRSV RNA followed a first-order decay rate ( k = 0.04/h with r 2 = 0.99) in wastewater. Finally, 21 wastewater influent samples from five wastewater treatment plants (WWTPs) in southern Maryland, USA were analyzed between May to August 2020 to determine SARS-CoV-2 RNA concentrations. SARS-CoV-2 RNA was quantifiable in 17/21 (81%) of the influent wastewater samples with concentration ranging from 1.10 (±0.10) × 10 4 to 2.38 (±0.16) × 10 6 gene copies/L. Among the RT-qPCR assays tested, US CDC N1 assay was the most sensitive followed by US CDC N2, E_Sarbeco, and RdRp assays. Data presented in this study may enhance our understanding on the effective concentration and extraction of SARS-CoV-2 from wastewater.

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