Context. The COVID-19 pandemic has caused unprecedented disruption in all aspects of life globally. The emergence of new variants of concern (VOC) that have increased transmissibility and tout vaccine immune evasion suggests that variant tracking is required to manage the COVID-19 pandemic.
Gap. There is a critical need for monitoring of SARS-CoV-2 VOCs in real time via wastewater-based surveillance. However, wastewater presents unique methodological challenges, as assays need to be variant specific yet accurately quantitative for meaningful interpretation, and this poses a major hurdle for adaptation of methods developed for variant detection in clinical samples for use in wastewater.
Aim. To develop an assay that can quantitatively track SARS-CoV-2 VOCs in wastewater, that is easily implemented using existing SARS-CoV-2 wastewater diagnostics.
Methods. We develop and present an open-source method based on allele specific RT-qPCR (AS RT-qPCR) that detects and quantifies VOCs by targeting spike protein mutations at loci predictive of specific VOCs. We explore the specificity and quantitative accuracy of such assays at SARS-CoV-2 variant tracking in wastewater.
Findings. We show that the AS RT-qPCR assays we developed for B.1.1.7, can reliably detect and quantify low levels of B.1.1.7 with low cross-reactivity, and at variant proportions down to 1% in a background of mixed SARS-CoV-2.
Utilization. We applied our method to wastewater samples from the United States collected between Oct 2020 to Mar 2021 to track B.1.1.7 occurrence in 19 communities. AS RT-qPCR results align with clinical trends, and summation of B.1.1.7 and wild-type sequences quantified by our assays match SARS-CoV-2 levels indicated by the US CDC N1/N2 assay. This work paves the path for AS RT-qPCR as a method for rapid inexpensive surveillance of SARS-CoV-2 variants in wastewater.
