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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-...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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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 |
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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 |
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