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Application of dPCR and RT-qPCR assays for SARS-CoV-2 Variant Detection in Wastewater

Poster: Wastewater-based epidemiology has been widely used to monitor COVID-19 trends in communities, the spread of SARS-CoV-2 variants in the population, and the effectiveness of public health prevention measures. Since the beginning of the pandemic, there have been five lineages classified as variants of concerns (VOCs) of SASR-CoV-2 (Alpha, Beta, Gamma, Delta, and Omicron) described by the WHO. Since November 2021, significant changes in circulating strains have been primarily Omicron sub-lineages. Variants have been successfully identified and quantified in wastewater using next-generation sequencing (NGS) methods that are time-consuming and expensive. We applied allele-specific dPCR and RT-qPCR assays with sequence-specific primers and fluorescently-labeled oligonucleotide probes developed for genotyping clinical samples to estimate the proportion of four Omicron sub-lineages (BA1, BA2, BQ1, and XBB) in archived wastewater samples. Samples were collected weekly from the influent of several wastewater treatment facilities, public schools, and a county jail in 2022 and 2023 in Atlanta, GA, USA. The assays were applied in proportionally mixed Omicron sub-lineage standards, which indicated that they were sensitive, specific, and quantitatively-reliable. Omicron sub-lineage detection using the assays was aligned well with the NGS results from the same wastewater samples. Wastewater samples collected between mid-February 2022 and mid-April 2022 showed a rapid displacement of BA1 with BA2, and samples collected between mid-December 2022 and early-March 2023 demonstrated a clear transition from BQ1 to XBB. These sub-lineage displacements detected by PCR assays were concordant with these sub-lineage dynamics in clinical samples reported in the literature. These results suggest that dPCR and RT-qPCR based assays can be used for specific and timely detection and monitoring of SARS-CoV-2 variants in wastewater.


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