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Title: Development of an efficient wastewater testing protocol for high-throughput country-wide SARS-CoV-2 monitoring
Authors: Mailepessov, Diyar
Arivalan, Sathish
Kong, Marcella
Griffiths, Jane
Low, Swee Ling
Chen, Hongjie
Hapuarachchi, Hapuarachchige Chanditha
Gu, Xiaoqiong
Lee, Wei Lin
Alm, Eric J.
Thompson, Janelle
Wuertz, Stefan
Gin, Karina
Ng, Lee Ching
Wong, Judith Chui Ching
Keywords: Engineering::Environmental engineering
Issue Date: 2022
Source: Mailepessov, D., Arivalan, S., Kong, M., Griffiths, J., Low, S. L., Chen, H., Hapuarachchi, H. C., Gu, X., Lee, W. L., Alm, E. J., Thompson, J., Wuertz, S., Gin, K., Ng, L. C. & Wong, J. C. C. (2022). Development of an efficient wastewater testing protocol for high-throughput country-wide SARS-CoV-2 monitoring. Science of the Total Environment, 826, 154024-.
Journal: Science of the Total Environment 
Abstract: Wastewater-based surveillance has been widely used as a non-intrusive tool to monitor population-level transmission of COVID-19. Although various approaches are available to concentrate viruses from wastewater samples, scalable methods remain limited. Here, we sought to identify and evaluate SARS-CoV-2 virus concentration protocols for high-throughput wastewater testing. A total of twelve protocols for polyethylene glycol (PEG) precipitation and four protocols for ultrafiltration-based approaches were evaluated across two phases. The first phase entailed an initial evaluation using a small sample set, while the second phase further evaluated five protocols using wastewater samples of varying SARS-CoV-2 concentrations. Permutations in the pre-concentration, virus concentration and RNA extraction steps were evaluated. Among PEG-based methods, SARS-CoV-2 virus recovery was optimal with 1) the removal of debris prior to processing, 2) 2 h to 24 h incubation with 8% PEG at 4 °C, 3) 4000 xg or 14,000 xg centrifugation, and 4) a column-based RNA extraction method, yielding virus recovery of 42.4-52.5%. Similarly, the optimal protocol for ultrafiltration included 1) the removal of debris prior to processing, 2) ultrafiltration, and 3) a column-based RNA extraction method, yielding a recovery of 38.2%. This study also revealed that SARS-CoV-2 RNA recovery for samples with higher virus concentration were less sensitive to changes in the PEG method, but permutations in the PEG protocol could significantly impact virus yields when wastewater samples with lower SARS-CoV-2 RNA were used. Although both PEG precipitation and ultrafiltration methods resulted in similar SARS-CoV-2 RNA recoveries, the former method is more cost-effective while the latter method provided operational efficiency as it required a shorter turn-around-time (PEG precipitation, 9-23 h; Ultrafiltration, 5 h). The decision on which method to adopt will thus depend on the use-case for wastewater testing, and the need for cost-effectiveness, sensitivity, operational feasibility and scalability.
ISSN: 0048-9697
DOI: 10.1016/j.scitotenv.2022.154024
Rights: © 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:ASE Journal Articles
CEE Journal Articles
SBS Journal Articles
SCELSE Journal Articles

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