A One-Step Quadruplex RT-ddPCR Assay for Simultaneous Detection and Absolute Quantification of Respiratory Viruses in Wastewater from Multiple Settings

Abstract

Wastewater-based surveillance of respiratory viruses increasingly requires multiplex assays capable of detecting low-abundance targets in complex environmental matrices without standard curves. In this study, a highly sensitive and specific one-step quadruplex reverse transcription droplet digital PCR assay was developed for the simultaneous quantification of SARS-CoV-2, respiratory syncytial virus, influenza A virus, and influenza B virus in wastewater. The assay achieved equivalent limits of detection of 0.32–0.44 copies/μL, excellent linearity (R2 > 0.999), and satisfactory precision, with all intra- and interassay coefficients of variation below 25%. No significant quantitative differences were observed between the quadruplex assay and the corresponding singleplex assays (Wilcoxon signed-rank test, P > 0.05). When applied to 38 wastewater samples collected across multiple settings, the assay detected all four viruses at rates of 65.79%–81.58%, with 89.47% of samples positive for two or more viruses, underscoring the value of multiplex monitoring for cocirculating respiratory viruses. Compared with reverse transcription quantitative PCR, the assay showed good qualitative agreement with the evaluated commercial quadruplex RT-qPCR kit (κ = 0.625–0.855, P < 0.001), although discordant results were target-dependent. These findings support the feasibility of the developed assay for multiplex detection of respiratory viruses in wastewater under the conditions tested.