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Validating and optimizing the method for molecular detection and quantification of SARS-CoV-2 in wastewater
Journal article   Open access   Peer reviewed

Validating and optimizing the method for molecular detection and quantification of SARS-CoV-2 in wastewater

Yuanyuan Qiu, Kanti Pabbaraju, Bonita E Lee, Tiejun Gao, Nicholas J Ashbolt, Steve E Hrudey, Mathew Diggle, Grahma Tipples, Rasha Maal-Bared and Xiaoli Pang
Science of the Total Environment, Vol.812, 151434
15/03/2022
DOI: https://doi.org/10.1016/j.scitotenv.2021.151434
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Published (Version of record)CC BY V4.0,  Open Access
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Abstract

SARS-CoV-2 Wastewater RT-qPCR hCoV-229E PMMoV
Wastewater surveillance of SARS-CoV-2 has become a promising tool to estimate population-level changes in community infections and the prevalence of COVID-19 disease. Although many studies have reported the detection and quantification of SARS-CoV-2 in wastewater, remarkable variation remains in the methodology. In this study, we validated a molecular testing method by concentrating viruses from wastewater using ultrafiltration and detecting SARS-CoV-2 using one-step RT-qPCR assay. The following parameters were optimized including sample storage condition, wastewater pH, RNA extraction and RT-qPCR assay by quantification of SARS-CoV-2 or spiked human coronavirus strain 229E (hCoV-229E). Wastewater samples stored at 4 °C after collection showed significantly enhanced detection of SARS-CoV-2 with approximately 2–3 PCR-cycle threshold (Ct) values less when compared to samples stored at −20 °C. Pre-adjustment of the wastewater pH to 9.6 to aid virus desorption followed by pH readjustment to neutral after solid removal significantly increased the recovery of spiked hCoV-229E. Of the five commercially available RNA isolation kits evaluated, the MagMAX-96 viral RNA isolation kit showed the best recovery of hCoV-229E (50.1 ± 20.1%). Compared with two-step RT-qPCR, one-step RT-qPCR improved sensitivity for SARS-CoV-2 detection. Salmon DNA was included for monitoring PCR inhibition and pepper mild mottle virus (PMMoV), a fecal indicator indigenous to wastewater, was used to normalize SARS-CoV-2 levels in wastewater. Our method for molecular detection of SARS-CoV-2 in wastewater provides a useful tool for public health surveillance of COVID-19.

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