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Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater

Citation:

Avgeris M, Adamopoulos PG, Galani A, Xagorari M, Gourgiotis D, Trougakos IP, Voulgaris N, Dimopoulos MA, Thomaidis NS, Scorilas A. Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater. Int J Mol Sci 2021;22

Abstract:

Considering the lack of effective treatments against COVID-19, wastewater-based epidemiology (WBE) is emerging as a cost-effective approach for real-time population-wide SARS-CoV-2 monitoring. Here, we report novel molecular assays for sensitive detection and mutational/variant analysis of SARS-CoV-2 in wastewater. Highly stable regions of SARS-CoV-2 RNA were identified by RNA stability analysis and targeted for the development of novel nested PCR assays. Targeted DNA sequencing (DNA-seq) was applied for the analysis and quantification of SARS-CoV-2 mutations/variants, following hexamers-based reverse transcription and nested PCR-based amplification of targeted regions. Three-dimensional (3D) structure models were generated to examine the predicted structural modification caused by genomic variants. WBE of SARS-CoV-2 revealed to be assay dependent, and significantly improved sensitivity achieved by assay combination (94%) vs. single-assay screening (30%-60%). Targeted DNA-seq allowed the quantification of SARS-CoV-2 mutations/variants in wastewater, which agreed with COVID-19 patients' sequencing data. A mutational analysis indicated the prevalence of D614G (S) and P323L (RdRP) variants, as well as of the Beta.1.1.7/alpha variant of concern, in agreement with the frequency of Beta.1.1.7/alpha variant in clinical samples of the same period of the third pandemic wave at the national level. Our assays provide an innovative cost-effective platform for real-time monitoring and early-identification of SARS-CoV-2 variants at community/population levels.

Notes:

Avgeris, MargaritisAdamopoulos, Panagiotis GGalani, AikateriniXagorari, MarietaGourgiotis, DimitriosTrougakos, Ioannis PVoulgaris, NikolaosDimopoulos, Meletios-AthanasiosThomaidis, Nikolaos SScorilas, Andreaseng16689/Special Account for Research Grants of the National and Kapodistrian University of AthensSwitzerland2021/08/28 06:00Int J Mol Sci. 2021 Aug 7;22(16). pii: ijms22168498. doi: 10.3390/ijms22168498.