@article{2980709,
    title = "Novel nested-seq approach for SARS-CoV-2 real-time epidemiology and in-depth mutational profiling in wastewater",
    author = "Avgeris, M. and Adamopoulos, P.G. and Galani, A. and Xagorari, M. and Gourgiotis, D. and Trougakos, I.P. and Voulgaris, N. and Dimopoulos, M.-A. and Thomaidis, N.S. and Scorilas, A.",
    journal = "International Journal of Molecular Sciences",
    year = "2021",
    volume = "22",
    number = "16",
    publisher = "MDPI AG",
    issn = "1422-0067",
    doi = "10.3390/ijms22168498",
    keywords = "virus RNA, environmental monitoring;  epidemiology;  human;  isolation and purification;  pandemic;  procedures;  virology;  wastewater, COVID-19;  Environmental Monitoring;  Humans;  Pandemics;  RNA, Viral;  SARS-CoV-2;  Waste Water",
    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 B.1.1.7/alpha variant of concern, in agreement with the frequency of B.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. © 2021 by the authors. Licensee MDPI, Basel, Switzerland."
}