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.

Autor: Reyes-Calderón A; Centro de Investigación y Tecnología del Agua - CITA, Universidad de Ingenieria y Tecnologia - UTEC, Jr. Medrano Silva 165, Lima, 15063, Peru., Mindreau-Ganoza E; Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Av. Germán Amézaga s/n, Lima, 15081, Peru., Pardo-Figueroa B; Centro de Investigación y Tecnología del Agua - CITA, Universidad de Ingenieria y Tecnologia - UTEC, Jr. Medrano Silva 165, Lima, 15063, Peru., Garcia-Luquillas KR; Centro de Investigación y Tecnología del Agua - CITA, Universidad de Ingenieria y Tecnologia - UTEC, Jr. Medrano Silva 165, Lima, 15063, Peru., Yufra SP; Departamento de Ingeniería Metalúrgica e Ingeniería Ambiental, Universidad Nacional de San Agustín, Av. Independencia s/n, Arequipa, 04001, Peru., Romero PE; Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Av. Germán Amézaga s/n, Lima, 15081, Peru., Antonini C; Departamento de Ingeniería Industrial, Universidad de Ingenieria y Tecnologia - UTEC, Jr. Medrano Silva 165, Lima, 15063, Peru., Renom JM; Departamento de Ciencias, Universidad de Ingenieria y Tecnologia - UTEC, Jr. Medrano Silva 165, Lima, 15063, Peru., Mota CR; Departamento de Engenharia Sanitária e Ambiental, Escola de Engenharia, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos, Belo Horizonte, 6.627, 31270-901, Brazil., Santa-Maria MC; Centro de Investigación y Tecnología del Agua - CITA, Universidad de Ingenieria y Tecnologia - UTEC, Jr. Medrano Silva 165, Lima, 15063, Peru.
Jazyk: angličtina
Zdroj: Heliyon [Heliyon] 2023 Jun; Vol. 9 (6), pp. e16130. Date of Electronic Publication: 2023 May 16.
DOI: 10.1016/j.heliyon.2023.e16130
Abstrakt: 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 MgCl 2 (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.
Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(© 2023 The Authors.)
Databáze: MEDLINE
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