Cargando…

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

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-...

Descripción completa

Detalles Bibliográficos
Autores principales: Avgeris, Margaritis, Adamopoulos, Panagiotis G., Galani, Aikaterini, Xagorari, Marieta, Gourgiotis, Dimitrios, Trougakos, Ioannis P., Voulgaris, Nikolaos, Dimopoulos, Meletios-Athanasios, Thomaidis, Nikolaos S., Scorilas, Andreas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395163/
https://www.ncbi.nlm.nih.gov/pubmed/34445204
http://dx.doi.org/10.3390/ijms22168498
_version_ 1783744110632370176
author Avgeris, Margaritis
Adamopoulos, Panagiotis G.
Galani, Aikaterini
Xagorari, Marieta
Gourgiotis, Dimitrios
Trougakos, Ioannis P.
Voulgaris, Nikolaos
Dimopoulos, Meletios-Athanasios
Thomaidis, Nikolaos S.
Scorilas, Andreas
author_facet Avgeris, Margaritis
Adamopoulos, Panagiotis G.
Galani, Aikaterini
Xagorari, Marieta
Gourgiotis, Dimitrios
Trougakos, Ioannis P.
Voulgaris, Nikolaos
Dimopoulos, Meletios-Athanasios
Thomaidis, Nikolaos S.
Scorilas, Andreas
author_sort Avgeris, Margaritis
collection PubMed
description 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 Β.1.1.7/alpha variant of concern, in agreement with the frequency of Β.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.
format Online
Article
Text
id pubmed-8395163
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-83951632021-08-28 Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater Avgeris, Margaritis Adamopoulos, Panagiotis G. Galani, Aikaterini Xagorari, Marieta Gourgiotis, Dimitrios Trougakos, Ioannis P. Voulgaris, Nikolaos Dimopoulos, Meletios-Athanasios Thomaidis, Nikolaos S. Scorilas, Andreas Int J Mol Sci Article 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 Β.1.1.7/alpha variant of concern, in agreement with the frequency of Β.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. MDPI 2021-08-07 /pmc/articles/PMC8395163/ /pubmed/34445204 http://dx.doi.org/10.3390/ijms22168498 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Avgeris, Margaritis
Adamopoulos, Panagiotis G.
Galani, Aikaterini
Xagorari, Marieta
Gourgiotis, Dimitrios
Trougakos, Ioannis P.
Voulgaris, Nikolaos
Dimopoulos, Meletios-Athanasios
Thomaidis, Nikolaos S.
Scorilas, Andreas
Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater
title Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater
title_full Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater
title_fullStr Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater
title_full_unstemmed Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater
title_short Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater
title_sort novel nested-seq approach for sars-cov-2 real-time epidemiology and in-depth mutational profiling in wastewater
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395163/
https://www.ncbi.nlm.nih.gov/pubmed/34445204
http://dx.doi.org/10.3390/ijms22168498
work_keys_str_mv AT avgerismargaritis novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater
AT adamopoulospanagiotisg novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater
AT galaniaikaterini novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater
AT xagorarimarieta novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater
AT gourgiotisdimitrios novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater
AT trougakosioannisp novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater
AT voulgarisnikolaos novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater
AT dimopoulosmeletiosathanasios novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater
AT thomaidisnikolaoss novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater
AT scorilasandreas novelnestedseqapproachforsarscov2realtimeepidemiologyandindepthmutationalprofilinginwastewater