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. 2022 Jun 1:823:153737.
doi: 10.1016/j.scitotenv.2022.153737. Epub 2022 Feb 9.

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

Md Alamin  1 , Shohei Tsuji  2 , Akihiko Hata  3 , Hiroe Hara-Yamamura  4 , Ryo Honda  5
Affiliations

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

Md Alamin et al. Sci Total Environ. .

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.

Keywords: Murine norovirus (MNV); Pepper mild mottle virus (PMMoV); Polyethylene glycol (PEG) precipitation; Pseudomonas phage ϕ6; Solid fraction; Wastewater-based epidemiology (WBE).

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Conflict of interest statement

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: this study was supported by Japan Science and Technology Agency (JST) MIRAI Program (Grant No. JPMJMI18DC), JST CREST (Grant No. JPMJCR20H1) and partly sponsored by the joint research project with Shimadzu Corporation. Maxwell RNA extraction kits were provided by Promega K.K., Japan.

Figures

Unlabelled Image
Graphical abstract
Fig. 1
Fig. 1
Flow diagram of concentration, RNA extraction and Detection for the sample processing. QIAamp = QIAamp Viral RNA Mini Kit, Maxwell GMO = Maxwell RSC PureFood GMO and Authentication Kit, Maxwell viral TNA = Maxwell RSC Viral Total Nucleic Acid (TNA) Purification Kit.
Fig. 2
Fig. 2
(a) Total RNA concentration (μg/mL), (b) RNA quality by A260/A280, and (c) A260/A230 by concentrated liquid and solids. The concentrates from wastewaters A and B were extracted by QIAamp Viral RNA Mini Kit (QIAamp), Maxwell RSC PureFood GMO and Authentication Kit (GMO), Maxwell RSC Viral Total Nucleic Acid Purification Kit (Viral). Dotted lines are the quality values of the pure RNA.
Fig. 3
Fig. 3
Detection efficiency of SARS-CoV-2 spiked in wastewater. Liquid fractions of wastewaters were concentrated by PEG precipitation; solid fractions by centrifuge. Extraction from each concentrate was triplicated. QIAamp = QIAamp Viral RNA Mini Kit, Maxwell GMO = Maxwell RSC PureFood GMO and Authentication Kit, Maxwell viral TNA = Maxwell RSC Viral Total Nucleic Acid (TNA) Purification Kit.
Fig. 4
Fig. 4
Detection efficiency of CDCN1 and the surrogate viruses of ɸ6, MNV and PMMoV by different combination of concentrated fraction and extraction kits. Dotted line shows limit of detection (LOD).
See this image and copyright information in PMC

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