Underestimation of SARS-CoV-2 in wastewater due to single or double mutations in the N1 qPCR probe binding region

Wastewater surveillance using RT-qPCR has now been widely adopted to track circulating levels of SARS-CoV-2 virus in many sewersheds. The CDC qPCR assays targeting two regions (N1 and N2) within the N gene are commonly used, but a discrepancy between the two biomarkers has been noticed by independent studies using these methods since late 2021. The reason is presumed to be due to mutations in regions targeted by the N1 qPCR probe. In this study, we systematically investigated and unequivocally confirmed that the underlying reason for this discrepancy was mutations in the N1 probe target, and that a single mutation could cause a significant drop in signal. We first confirmed the proportion of related mutations in wastewater samples (Jan 2021-Dec 2022) using nested PCR and LC-MS. Based on relative proportions of N1 alleles, we separated the wastewater data into four time periods corresponding to different variant waves: Period I (Alpha and Delta waves with 0 mutation), Period II (BA.1/BA.2 waves with a single mutation found in all Omicron strains), Period III (BA.5.2* wave with two mutations), and Period IV (BQ.1* wave with two mutations). Significantly lower N1 copies relative to N2 copies in samples from Periods II-IV compared to those from Period I was observed in wastewater. To further pinpoint the extent to which each mutation impacted N1 quantification, we compared the qPCR response among different synthetic oligomers with corresponding mutations. This study highlighted the impact of even just one or two mutations on qPCR-based wastewater surveillance of SARS-CoV-2.