Development of a dual-target qPCR assay for enhanced MPXV detection in wastewater and environmental matrices

Abstract The emergence of mpox as a global public health concern demands accurate and efficient molecular diagnostic tools for surveillance and outbreak management. The single-plex mpox virus (MPXV)-specific assays targeting B7R and TNF receptor (G2R) genes are widely used for this purpose. However, single-target approaches are vulnerable to viral mutations and may exhibit variable sensitivity across viral lineages and sample matrices, necessitating complementary assays for confirmation. Hence, we developed a duplex qPCR assay, combining B7R and G2R assay targets, enhancing laboratory efficiency and detection reliability. The analytical performance of the novel duplex assay was evaluated against its single-plex formats using synthetic DNA controls comprising mpox clades I and II sequences. The duplex assay’s utility for detecting MPXV variants was further evaluated using isolates belonging to recent outbreak lineages (Clade IIb and Clade Ib) and 42 environmental samples. The B7R and G2R Generic (G2RG) duplex assay demonstrated comparable performance to their respective single-plex assays when tested against synthetic controls, isolates, and MPXV-positive environmental samples. The newly developed duplex assay potentially serves as a robust molecular tool for MPXV detection in various sample matrices, including challenging environmental matrices such as wastewater. Besides enhancing screening efficiency and reducing cost, the simultaneous detection of two targets ensures detection reliability and thereby reduces the risk of false-negative results even if mutations occur in one of the target regions.