Selection of surrogate viruses for process control in detection of SARS-CoV-2 in wastewater

Abstract

Since SARS-CoV-2 RNA in wastewater is often present at low concentration or under detection limit, ensuring the reliability of detection processes using appropriate process controls is essential. The objective of this study was to evaluate applicability and limitations of candidate surrogate viruses as process controls under combinations of different virus concentration and RNA extraction methods. Detection efficiency of SARS-CoV-2 spiked in wastewater was compared with those of candidate surrogate viruses of bacteriophage ϕ6, pepper mild mottle virus (PMMoV), F-specific coliphage (F-phage), and murine norovirus (MNV). After inactivated SARS-CoV-2 and ϕ6 were spiked in two different wastewaters, the viruses in solid and liquid fractions of wastewater were concentrated by centrifuge and polyethylene glycol (PEG) precipitation, respectively. Viral RNA was extracted by using QIAamp Viral RNA Mini Kit and 3 other commercially available extraction kits, then quantified by reverse transcription-quantitative PCR using CDCN1 assay. Regardless of extraction kits, SARS-CoV-2 was consistently detected with good efficiency from both liquid (11–200%) and solid fractions (7.1–93%). Among the candidate process controls, PMMoV was widely detected at good efficiencies from both liquid and solid fractions regardless of selection of RNA extraction kits. F-phage and MNV also showed good detection efficiencies in most combinations of wastewater fractions and RNA extraction kits. An enveloped virus ɸ6 was found often undetected or to have very low detection efficiency (0.1–4.2%) even when SARS-CoV-2 spiked in wastewater was detected with good efficiency. Consequently, PMMoV is widely applicable as process control for detection of SARS-CoV-2 either in liquid fractions concentrated by PEG precipitation, or in solid fractions concentrated by centrifuge.

SARS-CoV-2 RNA in Wastewater Was Highly Correlated With the Number of COVID-19 Cases During the Fourth and Fifth Pandemic Wave in Kobe City, Japan

“…Recovery rate using the PMMoV copy number have been conducted for concentration efficiency of wastewater. Previous studies that compared the liquid and solid fraction showed that the detection efficiency of PMMoV ranged 8.0%–30% in the PEG-precipitated liquid fraction and 6.0%–17% in the solid fraction ( Graham et al, 2021 ), and 12%–102% from liquid fractions and 9.4%–62% from solid fractions ( Alamin et al, 2022 ). Our reported recovery values tended to be lower than the range reported in previous studies.…”

Wastewater Surveillance of SARS-CoV-2 at a Canadian University Campus and the Impact of Wastewater Characteristics on Viral RNA Detection

“… 32 SARS-CoV-2 and MHV are, for instance, abundant in the solids fraction of wastewater unlike nonenveloped enteric viruses. 33 For concentration methods including the solid phase, enveloped viruses might be better surrogates. 33 This study started processing with the ultrafiltration method, and therefore MS2 was spiked into the samples as it is known to partition more into the liquid phase.…”

“… 33 For concentration methods including the solid phase, enveloped viruses might be better surrogates. 33 This study started processing with the ultrafiltration method, and therefore MS2 was spiked into the samples as it is known to partition more into the liquid phase. For consistency reasons, the use of MS2 for all samples in this study was continued, both for ultrafiltration and centrifugation processed samples.…”

Detection of SARS-CoV-2 and Other Viruses in Wastewater: Optimization and Automation of an Aluminum Hydroxide Adsorption–Precipitation Method for Virus Concentration

“…For example, the recovery rate for PPV in tap water was 5.9% while it was 22.3% for PEDV, which might be explained by the differences in the size and outer layer structure of the virus particles. 55 It is also interesting that the recovery rates in wastewater were not so different from those in tap water ( Figure 2 ). In some cases, the recovery rates of some viruses in wastewater were even higher than those in tap water.…”