Development and Application of a Quadruplex TaqMan‐MGB qPCR Assay for Simultaneous Detection of Important Mosquito‐Borne Orthoflaviviruses

Mosquito-borne orthoflaviviruses, such as Japanese encephalitis virus (JEV), West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV), pose considerable public health challenges due to their zoonotic potential and the absence of specific therapeutics. Conventional detection methods exhibit critical limitations in multiplexing capability, prolonged turnaround time, and procedural complexity. These factors hinder rapid outbreak response and underscore the necessity for improved detection platforms. This study developed a quadruplex quantitative real-time PCR (qRT-PCR) assay utilizing Minor Groove Binder (MGB) probes for the simultaneous detection of JEV, WNV, DENV, and ZIKV. The MGB probe-based qPCR assay demonstrated high specificity in distinguishing these four orthoflaviviruses and exhibited significantly enhanced sensitivity compared to conventional TaqMan probes, achieving detection limit of 2 copies/reaction. This method was successfully applied to detect virus-positive samples in cell cultures, laboratory mosquitoes, and field-collected mosquitoes, thereby validating the practical utility of the MGB probe-based qPCR assay. Compared to conventional multiplex detection methods, our MGB-based approach provides a promising detection platform, thereby demonstrating its potential utility in the high-throughput surveillance of mosquito-borne orthoflaviviruses.