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.
【저자키워드】 COVID-19, 2019-nCoV, Wastewater-based epidemiology, Spike protein, D614G, SARS-CoV-2 mutations, SARS-CoV-2 sequencing, sewage, nested PCR, SARS-CoV-2 NGS, 【초록키워드】 Treatment, SARS-CoV-2, pandemic, Epidemiology, variant, SARS-CoV-2 variant, variants, Prevalence, amplification, clinical samples, sensitivity, wastewater, Region, SARS-CoV-2 variants, RdRP, reverse transcription, SARS-CoV-2 RNA, DNA sequencing, SARS-CoV-2 mutations, nested PCR, Alpha variant, genomic variants, PCR assays, quantification, Molecular assay, platform, RNA stability, Combination, Frequency, Analysis, (RdRp, Real-time monitoring, P323L, sequencing data, National, Modification, regions, approach, effective, clinical sample, predicted, lack, caused, significantly, indicated, applied, with COVID-19, 【제목키워드】 SARS-CoV-2, novel, Profiling,