Characterization and Resistance to Mutation of a Single-Channel Multiplex PCR Assay for SARS-CoV-2

Throughout the COVID-19 pandemic, wastewater surveillance has been used worldwide to provide valuable public health data. RT-qPCR is frequently used as a quantitative methodology for wastewater surveillance but is susceptible to mutations in target regions. These mutations may lead to misinterpretation of surveillance data; a drop in signal could be concluded to be a result of lower viral load, when in fact it is caused by reduced detection efficiency. We describe a novel approach to mitigating the impacts of such mutations: monitoring the cumulative signal from two targets (N1 and N2) via independent amplification reactions using identically labeled probes; a "single-channel multiplex" approach. Using the IDEXX Water SARS-CoV-2 RT-qPCR test, we demonstrate equivalent intra-assay repeatability and quantitative results from the combined N1N2 test when compared to individual N1 and N2 assays. Furthermore, we show that while mutations in B.1.1.529, BA.5.2, and BA.5.2.1 significantly impact the performance of the N1 assay, the impact on the N1N2 assay was negligible, and nearly within acceptable margin of error for technical replicates. These findings demonstrate that a single-channel multiplex approach can be used to improve the robustness of wastewater surveillance and minimize the risk of future mutations leading to unreliable public health data.