Evaluation of low-cost SARS-CoV-2 RNA purification methods for viral quantification by RT-qPCR and next-generation sequencing analysis: Implications for wider wastewater-based epidemiology adoption

Wastewater: Based Epidemiology (WBE) consists of quantifying biomarkers in sewerage systems to derive real-time information on the health and/or lifestyle of the contributing population. WBE usefulness was vastly demonstrated in the context of the COVID-19 pandemic. Many methods for SARS-CoV-2 RNA determination in wastewater were devised, which vary in cost, infrastructure requirements and sensitivity. For most developing countries, implementing WBE for viral outbreaks, such as that of SARS-CoV-2, proved challenging due to budget, reagent availability and infrastructure constraints. In this study, we assessed low-cost methods for SARS-CoV-2 RNA quantification by RT-qPCR, and performed variant identification by NGS in wastewater samples. Results showed that the effect of adjusting pH to 4 and/or adding MgCl₂ (25 mM) was negligible when using the adsorption-elution method, as well as basal physicochemical parameters in the sample. In addition, results supported the standardized use of linear rather than plasmid DNA for a more accurate viral RT-qPCR estimation. The modified TRIzol-based purification method in this study yielded comparable RT-qPCR estimation to a column-based approach, but provided better NGS results, suggesting that column-based purification for viral analysis should be revised. Overall, this work provides evaluation of a robust, sensitive and cost-effective method for SARS-CoV-2 RNA analysis that could be implemented for other viruses, for a wider WEB adoption.