An allele-specific primer extension assay to quantify the proportion of B.1.1.7-specific SARS-CoV-2 RNA in wastewater

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed millions of lives globally to date. Rapid accumulation of co-occurring mutations has led to the emergence of viral variants which appear to be more transmissible, virulent, or both. Variants of concern (VOC) now include those belonging to the B.1.1.7, B.1.351, and P.1 lineages. Early detection of VOC and the ability to retrospectively follow their respective, longitudinal prevalence in communities is wanting. Wastewater-based epidemiology (WBE) allows tracking of disease prevalence in the general population using RT-qPCR to detect viral fragments, but ongoing longitudinal studies have yet to differentiate between these variants. Here, we describe and validate a primer extension strategy to amplify and distinguish B.1.1.7-specific, from non-B.1.1.7 alleles by combining new forward, and existing CDC 2019-nCoV_N1 qPCR probes and reverse primers. This assay can be quickly implemented within a current SARS- CoV-2 WBE framework with minimal cost with the goal of providing early detection of increasing B.1.1.7 transmission in a community prior to identification through clinical testing and confirmation via secondary screening strategies. As such, this assay can provide public health units with an additional and much needed metric to be able to rapidly triangulate B.1.1.7 prevalence.