A combinatorial probe-coding multiplex RT-qPCR assay for simultaneous detection and differentiation of dengue, Zika, and chikungunya viruses in human and mosquito samples

Arboviruses such as Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV) remain significant public health threats in tropical and subtropical regions. These viruses are transmitted primarily by Aedes mosquitoes and often co-circulate in the same geographic areas, leading to outbreaks with overlapping clinical presentations and cross-reactivity in serological testing, particularly between ZIKV and DENV. These aspects create diagnostic challenges, and the development of molecular diagnostic methods is critically necessary. Here, we developed a diagnostic kit based on the Multicolor Combinational Probe Coding (MCPC) concept to simultaneously detect six viral targets (ZIKV, DENV1–4, and CHIKV) and an internal control (IC), by encoding targets with four spectrally distinct dyes. Using a panel of 660 characterized human serum or plasma samples, and 9 artificial DENV-4 samples, we obtained diagnostic sensitivity and specificity above 94% and 99%, respectively, for all targets. The assay enables the simultaneous analysis of up to 91 samples, substantially increasing testing throughput compared to commercially available products. The test was also applied to mosquito specimens of Aedes spp., Anopheles spp., Haemagogus spp., and Culex spp., demonstrating its utility as a robust tool not only for emergency response but also for arbovirus surveillance in public health settings.