Comparing methods for recovering genomic RNA from SARS-CoV-2 in municipal wastewater for molecular analysis
Date
2023-12-04
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Abstract
SARS-CoV-2, an enveloped virus with a single stranded positive sense RNA genome (Baltimore class 4), is shed in the excreta (e.g., feces, sputum, urine) of people infected with the virus, resulting in the presence of viral genetic fragments in municipal wastewater. This RNA is quantifiable using wastewater-based surveillance (WBS) molecular biology and genomic methods. RNA extracted from wastewater can be assessed using RT-qPCR to track disease trends within populations of various sizes (e.g., whole city, neighbourhoods, hospitals, etc) in a non-invasive, unbiased, and highly inclusive way. Despite accelerated development and utilisation of WBS during the COVID-19 pandemic, many unknowns and challenges still confront this burgeoning field. Fundamental structural differences between enveloped SARS-CoV-2 and the majority of viruses in wastewater (e.g., non-enveloped enteric viruses) raise important questions about how previously established methods perform for this novel coronavirus. Establishing a reliable workflow for wastewater-based monitoring of SARS-CoV-2 requires a series of connected methods, including viral recovery from wastewater and downstream molecular and/or genomic analyses. Selecting the best possible components of an overall workflow requires method comparison and optimisation. In this study, several wastewater processing methods were assessed for the recovery of a bovine coronavirus surrogate, which has a similar structure to that of SARS-CoV-2, to establish an effective method for viral recovery from wastewater. This comparison revealed that ultrafiltration and direct extraction performed better than electronegative membrane filtration, which exhibited RT-qPCR gene abundance estimates an order of magnitude lower than the other two methods. Consequently, two validated workflows implementing ultrafiltration and direct extraction were then compared during a 29-month longitudinal analysis of SARS-CoV-2 quantification using RT-qPCR. Despite each workflow representing a distinct method for viral recovery, trends were similar with both methods indicating that consistent application of a given workflow, and not necessarily which workflow is used, is paramount in WBS. RNA extraction methods were furthermore compared using an amplicon-based sequencing strategy to assess the SARS-CoV-2 variant profiles in municipal wastewater samples. This comparison did not reveal significant differences in sequencing results between the two extraction strategies, but rather underscored the importance of wastewater sample handling and storage prior to genomic assessment.
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Keywords
Wastewater, RT-qPCR, SARS-CoV-2, Wastewater-based surveillance
Citation
McCalder, J. A. (2023). Comparing methods for recovering genomic RNA from SARS-CoV-2 in municipal wastewater for molecular analysis (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.