Journal
SCIENCE OF THE TOTAL ENVIRONMENT
Volume 808, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.scitotenv.2021.152033
Keywords
Wastewater; Surveillance; SARS-CoV-2; COVID-19; Virus concentration; RT-qPCR
Categories
Funding
- University of Maryland
- Maryland Transportation Institute
- Department of Civil and Environmental Engineering
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This study evaluated 14 virus concentration protocols for quantification of SARS-CoV-2 in wastewater samples, finding the most effective combination for virus recovery. The impact of prolonged storage on enveloped virus decay was also assessed, along with the quantification of SARS-CoV-2 RNA in influent wastewater samples from wastewater treatment plants. The study may enhance understanding of the effective concentration and extraction of SARS-CoV-2 from wastewater.
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 degrees C on enveloped virus decay, the BRSV seeded wastewaters samples were stored at -20 degrees 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) x 10(4) to 2.38 (+/- 0.16) x 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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