Next Article in Journal
The Role of the Second Extracellular Loop of Norepinephrine Transporter, Neurotrophin-3 and Tropomyosin Receptor Kinase C in T Cells: A Peripheral Biomarker in the Etiology of Schizophrenia
Previous Article in Journal
Major Facilitator Superfamily Transporter Gene FgMFS1 Is Essential for Fusarium graminearum to Deal with Salicylic Acid Stress and for Its Pathogenicity towards Wheat
Article

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

1
Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, 15771 Athens, Greece
2
Laboratory of Clinical Biochemistry and Molecular Diagnostics, Second Department of Pediatrics, School of Medicine, National and Kapodistrian University of Athens, “P. & A. Kyriakou” Children’s Hospital, 11527 Athens, Greece
3
Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771 Athens, Greece
4
Department of Cell Biology and Biophysics, Faculty of Biology, National and Kapodistrian University of Athens, 15771 Athens, Greece
5
Division of Geophysics & Geothermics, Department of Geology & Geoenvironment, National and Kapodistrian University of Athens, 15771 Athens, Greece
6
Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens, 11528 Athens, Greece
*
Author to whom correspondence should be addressed.
Academic Editor: James K. Bashkin
Int. J. Mol. Sci. 2021, 22(16), 8498; https://doi.org/10.3390/ijms22168498
Received: 21 June 2021 / Revised: 30 July 2021 / Accepted: 4 August 2021 / Published: 7 August 2021
(This article belongs to the Section Molecular Biology)
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. View Full-Text
Keywords: COVID-19; 2019-nCov; SARS-CoV-2 mutations; SARS-CoV-2 sequencing; sewage; nested PCR; SARS-CoV-2 NGS; wastewater-based epidemiology; D614G; spike protein COVID-19; 2019-nCov; SARS-CoV-2 mutations; SARS-CoV-2 sequencing; sewage; nested PCR; SARS-CoV-2 NGS; wastewater-based epidemiology; D614G; spike protein
Show Figures

Figure 1

MDPI and ACS Style

Avgeris, M.; Adamopoulos, P.G.; Galani, A.; Xagorari, M.; Gourgiotis, D.; Trougakos, I.P.; Voulgaris, N.; Dimopoulos, M.-A.; Thomaidis, N.S.; 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, 8498. https://doi.org/10.3390/ijms22168498

AMA Style

Avgeris M, Adamopoulos PG, Galani A, Xagorari M, Gourgiotis D, Trougakos IP, Voulgaris N, Dimopoulos M-A, Thomaidis NS, Scorilas A. Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater. International Journal of Molecular Sciences. 2021; 22(16):8498. https://doi.org/10.3390/ijms22168498

Chicago/Turabian Style

Avgeris, Margaritis, Panagiotis G. Adamopoulos, Aikaterini Galani, Marieta Xagorari, Dimitrios Gourgiotis, Ioannis P. Trougakos, Nikolaos Voulgaris, Meletios-Athanasios Dimopoulos, Nikolaos S. Thomaidis, and Andreas Scorilas. 2021. "Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater" International Journal of Molecular Sciences 22, no. 16: 8498. https://doi.org/10.3390/ijms22168498

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop