Clade-specific multiplex digital PCR assay for monkeypox virus detection in spike-in wastewater samples

Monkeypox virus (MPXV) is a zoonotic pathogen that has recently caused outbreaks in non-endemic areas. Wastewater-based surveillance is independent of individual testing and offers substantial potential for monitoring population-level disease dynamics of MPXV at the clade level, as clades differ in transmissibility and mortality rate. Here, we report the validation of a four-plex digital PCR assay to detect and quantify the major clades and subclades of MPXV. The assay demonstrated strong linearity, analytical sensitivity (limits of detection ranged from 6.7 to 9.6 copies per reaction across targets) and specificity in distinguishing and quantifying MPXV by clade, which is advantageous for both clinical and wastewater applications.