Real-Time Monitoring of SARS-CoV-2 Variants in Oklahoma Wastewater through Allele-Specific RT-qPCR

Abstract

During the COVID-19 pandemic, wastewater surveillance was used to monitor community transmission of SARS-CoV-2. As new genetic variants emerged, the need for timely identification of these variants in wastewater became an important focus. In response to increased reports of Omicron transmission across the United States, the Oklahoma Wastewater Surveillance team utilized allele-specific RT-qPCR assays to detect and differentiate variants, such as Omicron, from other variants found in wastewater in Oklahoma. The PCR assays showed presence of the Omicron variant in Oklahoma on average two weeks before official reports, which was confirmed through genomic sequencing of selected wastewater samples. Through continued surveillance from November 2021 to January 2022, we also demonstrated the transition from prevalence of the Delta variant to prevalence of the Omicron variant in local communities. We further assessed how this transition correlated with certain demographic factors characterizing each community. Our results highlight RT-qPCR assays as a rapid, simple, and cost-effective method for monitoring the community spread of SARS-CoV-2 genetic variants in wastewater. Additionally, they demonstrate that specific demographic factors such as ethnic composition and household income can correlate with the timing of SARS-CoV-2 variant introduction and spread.

Keywords: Omicron; RT-qPCR; SARS-CoV-2; early warning; sequencing; variant; wastewater surveillance.

Figure 1

Average concentrations (10-day moving averages) of SARS-CoV, Delta and Omicron variants in (a) wastewater treatment plants and (b) sewersheds in Oklahoma, November 2021–January 2022.

Figure 2

Average proportion (%) of Omicron and Delta variants in Oklahoma wastewater treatment plants (WWTP) and sewersheds, Oklahoma 2021–2022.

Figure 3

Detection of the Omicron strain through variant-specific genome deletion analysis. (A) Genome browser snapshot depicting the 69–70del mutation within the Spike protein coding region of SARS-CoV-2 and showing mixed presence and absence of the Omicron-specific 69–70del mutation (dotted red rectangle). (B) Regional prevalence estimates for daily occurrences of SARS-CoV-2 variants illustrates circulating variants at the regional level, with the "other" category consolidating lineages below 1%, excluding Delta and Omicron.

Figure 4

Representative genomic region of SARS-CoV-2 highlighting mutations associated with variants: Delta (green), Omicron (purple), shared by Delta and Omicron (blue), and unassigned (grey). The Omicron-specific deletion at 60-71del is indicated in red, with mutation frequency shown on the Y-axis.

Figure 5

Association between Omicron wastewater indicators and demographic factors, Oklahoma 2021–2022.