Evaluation of multiple analytical methods for SARS-CoV-2 surveillance in wastewater samples

Abstract

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 r² = 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) × 10⁴ to 2.38 (±0.16) × 10⁶ 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.

Keywords: COVID-19; RT-qPCR; SARS-CoV-2; Surveillance; Virus concentration; Wastewater.

Graphical abstract

Fig. 1

Average natural log (ln) reduction of BRSV RNA (average ln (C_t/C₀)) where C is BRSV RNA concentrations in wastewater samples stored at −20 °C measured on day 0, 47, 77, and 110 and C₀ is the seed BRSV RNA concentration (before freezing).

Fig. 2

SARS-CoV-2 measurements (mean values of N1 and N2 assays, blue bars) and recovery of surrogate virus (BRSV, red triangles) RNA seeded into wastewater samples from the five WWTPs. Mean of triplicate analysis for each sampling date for each assay were reported with error bars indicating standard deviations of triplicate analysis. SARS-CoV-2 concentrations for samples below assay limit of detection (ALOD) (gray bars) were calculated using 3 GC/reaction. * indicates samples that were not seeded with BRSV for background BRSV analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)