Wastewater surveillance has rapidly emerged as an early warning tool to track COVID-19.
However, the early warning measurement of new SARS-CoV-2 variants of concern (VOCs) in
wastewaters remains a major challenge. We herein report a rapid analytical strategy for
quantitative measurement of VOCs, which couples nested polymerase chain reaction and
liquid chromatography–mass spectrometry (nPCR-LC-MS). This method showed a
greater selectivity than the current allele-specific quantitative PCR (AS-qPCR) for
tracking new VOC and allowed the detection of multiple signature mutations in a single
measurement. By measuring the Omicron variant in wastewaters across nine Ontario
wastewater treatment plants serving over a three million population, the nPCR-LC-MS
method demonstrated a better quantification accuracy than next-generation sequencing
(NGS), particularly at the early stage of community spreading of Omicron. This work
addresses a major challenge for current SARS-CoV-2 wastewater surveillance by rapidly
and accurately measuring VOCs in wastewaters for early warning.