Comparison of five polyethylene glycol precipitation procedures for the RT-qPCR based recovery of murine hepatitis virus, bacteriophage phi6, and pepper mild mottle virus as a surrogate for SARS-CoV-2 from wastewater

Abstract

Polyethylene glycol (PEG) precipitation is one of the conventional methods for virus concentration. This technique has been used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. The procedures and seeded surrogate viruses were different among implementers; thus, the reported whole process recovery efficiencies considerably varied among studies. The present study compared five PEG precipitation procedures, with different operational parameters, for the RT-qPCR-based whole process recovery of murine hepatitis virus (MHV), bacteriophage phi6, and pepper mild mottle virus (PMMoV), and molecular process recovery of murine norovirus using 34 raw wastewater samples collected in Japan. The five procedures yielded significantly different whole process recovery of MHV (0.070%–2.6%) and phi6 (0.078%–0.51%). The observed concentration of indigenous PMMoV ranged from 8.9 to 9.7 log (7.9 × 10 8 to 5.5 × 10 9 ) copies/L. Interestingly, PEG precipitation with 2-h incubation outperformed that with overnight incubation partially due to the difference in molecular process recovery efficiency. The recovery load of MHV exhibited a positive correlation ( r = 0.70) with that of PMMoV, suggesting that PMMoV is the potential indicator of the recovery efficiency of SARS-CoV-2. In addition, we reviewed 13 published studies and found considerable variability between different studies in the whole process recovery efficiency of enveloped viruses by PEG precipitation. This was due to the differences in operational parameters and surrogate viruses as well as the differences in wastewater quality and bias in the measurement of the seeded load of surrogate viruses, resulting from the use of different analytes and RNA extraction methods. Overall, the operational parameters (e.g., incubation time and pretreatment) should be optimized for PEG precipitation. Co-quantification of PMMoV may allow for the normalization of SARS-CoV-2 RNA concentration by correcting for the differences in whole process recovery efficiency and fecal load among samples.

Relationships between SARS-CoV-2 in Wastewater and COVID-19 Clinical Cases and Hospitalizations, with and without Normalization against Indicators of Human Waste

“…In brief, samples were pasteurized to inactivate the ambient SARS-CoV-2 virus (90 °C for 60 min) . Raw wastewater samples were then concentrated using the polyethylene glycol precipitation (PEG) method, , which required centrifugation of samples (5 min) before PEG8000 (Sigma-Aldrich, Burlington, MA) and sodium chloride were added. The treated wastewater samples were incubated overnight at 4 °C on a shaker .…”

COVID-19 case prediction via wastewater surveillance in a low-prevalence urban community: a modeling approach

“…PMMoV is detected throughout the year and abundant in wastewater (10 6 -10 10 copies/L) (Kitajima et al 2014;Symonds et al 2018; Ahmed et al 2020b), which may allow for using it as an internal control of RT-qPCR for a wastewater sample (Haramoto et al 2020). A previous study reported that the recovered load of PMMoV correlated with that of murine hepatitis virus, suggesting that PMMoV is the potential indicator of the efficiency of SARS-CoV-2 (Torii et al 2022). We concluded that there was no significant loss throughout the analysis because the PMMoV concentration was consistent in both influent and concentrated samples.…”

Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy

“…In particular, this method allowed for a better recovery efficiency of SARS-CoV-2 when compared to Dextran and polyethylene glycol-6000 (PEG) two-phase separation, in contrast to other Italian preliminary results [15]. PEG-8000 precipitation without chloroform has also shown a good performance in concentrating SARS-CoV-2 from wastewater matrices in other published studies [11,29,30]. In the study from Ahmed, W. et al, the mean ± SD of the recovery of murine hepatitis virus (as a proxy of SARS-CoV-2) was shown to be 44.0% ± 27.7, similar to that observed in our study, where the recovery of spiked SARS-CoV-2 ranged from 31.4% to 76% by using PEG-8000 precipitation [30].…”