Hyperplex PCR enables highly multiplexed analysis of point mutations in wastewater: Long-term SARS-CoV-2 variant...

Wastewater-based surveillance (WBS) allows the analysis of pathogens, chemicals or other biomarkers in wastewater to derive unbiased epidemiological information at population scale. After re-gaining attention during the SARS-CoV-2 pandemic, the field holds promise as a surveillance and early warning system by tracking emerging pathogens with pandemic potential. Expanding the current toolbox of analytical techniques for wastewater analysis, we explored the use of Hyperplex PCR (hpPCR) to analyse SARS-CoV-2 mutations in wastewater samples collected weekly in up to 22 sites across Sweden between October 2022 and December 2023. The samples were tested using a probe panel ranging from 10- to 18-plex, continuously adapted within 1-2 weeks to quantify relevant mutations of concern over time. For cross-validation, the samples were simultaneously analysed with commonly used methods including quantitative PCR (qPCR) and next-generation sequencing (NGS). hpPCR is demonstrated herein to provide (1) systematic single nucleotide specificity with a straightforward probe design, (2) high multiplexity with minimal panel re-optimization requirements and (3) 4-5-week earlier mutation detection relative to NGS with comparable performance of mutation frequency quantification (Pearson r = 0.88, n = 50). Hence, hpPCR is shown to be a powerful complementary tool to the current workflow involving NGS and qPCR by facilitating the assembly of dynamic high-plex panels compatible with high-frequency monitoring of multiple key pathogens and/or variants in WBS.