Abstract
Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.
Keywords: BA.4; BA.5; COVID-19 surveillance; Omicron; SARS-CoV-2 monitoring; variant of concern; wastewater-based epidemiology (WBE).
【저자키워드】 omicron, SARS-CoV-2 monitoring, variant of concern, COVID-19 surveillance, BA.4, BA.5, Wastewater-based epidemiology (WBE)., 【초록키워드】 COVID-19, Treatment, SARS-CoV-2, Epidemiology, variant, polymorphism, Delta, variants, RNA, Spread, Prevalence, digital PCR, RT-qPCR, PCR, Transmissibility, Immune escape, L452R, Germany, mutant, plant, single nucleotide, inactivated, Analysis, SARS-CoV-2 epidemiology, Support, fraction, North, MONITOR, NGS sequencing, state, Occurrence, was used, virus, evaluated, required, majority, Initially, RNA purified, 【제목키워드】 SARS-CoV-2, detection, wastewater,