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

Margaritis Avgeris,Ioannis P Trougakos,Nikolaos Voulgaris,Andreas Scorilas,Marieta Xagorari,Panagiotis G Adamopoulos,Nikolaos S Thomaidis,Dimitrios Gourgiotis,Aikaterini Galani,Meletios-Athanasios Dimopoulos

doi:10.3390/ijms22168498

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 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.

Highlights

A cluster of severe pneumonia cases of unknown origin, linked to the Huanan seafood wholesale market in Wuhan, Hubei Providence, China, were reported by the Chinese Health Authorities on 31 December 2019
The analyses led to the identification of 300 nt sequences bearing negative z-scores, which were predicted to possess low minimum free energy (MFE) native values, being more stable
Here, that severe acute respiratory syndrome (SARS)-CoV-2 detection in wastewater is assay/genomic region dependent, and that significantly improved detection specificity was achieved by the combination of novel nested PCR/real-time PCR assays against highly stable SARS-CoV-2 genomic regions

Summary

Introduction

A cluster of severe pneumonia cases of unknown origin, linked to the Huanan seafood wholesale market in Wuhan, Hubei Providence, China, were reported by the Chinese Health Authorities on 31 December 2019. CoVs constitute a family of enveloped positive-strand RNA viruses of 26–32 kilobases (kb) [3] This seventh member of the CoV family that causes disease in humans, was further characterized showing sequence homology of approximately 79% and 50% with the SARS-CoV ( referred to as SARS-CoV-1) of the 2002 outbreak in China and the Middle East respiratory syndrome (MERS)-CoV of the 2012 outbreak in the Middle East (mainly in Saudi Arabia), respectively [4]. Thereafter, this novel CoV was named SARS-CoV-2 [5], and was rapidly spread to most countries worldwide, leading to the announcement of the COVID-19 pandemic by the World Health Organization (WHO) on 12 March 2020. Risk-based community/population-wide PCR screening strategies have clearly contributed to the control of epidemics, lessening of death rates, and reopening of economic activities (see Wuhan and Singapore examples [9,10]), with the current methods/resources, only symptomatic and suspicious cases are being tested and only a fraction of them is being sequenced so far in most countries, highlighting the need for population-wide screening approaches for SARS-CoV-2 spread and mutational/variant monitoring

Methods

Results

Conclusion