Development of a TaqMan-Based Multiplex Real-Time RTPCR Assay for Detection of Mosquito-Borne Viral Diseases

Abstract

Background: Mosquito-borne viral diseases, including Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), Yellow fever (YFV), and West Nile (WNV), represent a significant global health burden. Existing singleplex molecular assays are often time-consuming and resource-intensive. We aimed to develop and analytically validate a one-step multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of these major arboviruses. Methods: Primers and hydrolysis probes were designed against conserved genomic regions. In vitro transcribed RNA standards were used to establish standard curves, determine PCR efficiency, and calculate limits of detection (LOD). A clinical validation panel comprising 250 samples with pre-determined infection statuses was used to assess diagnostic sensitivity and specificity against singleplex reference assays. Cross-reactivity was evaluated against a panel of related and unrelated pathogens. Results: The multiplex assay demonstrated linear detection across eight orders of magnitude (10¹ to 10⁸ copies/μL) with PCR efficiencies ranging from 94.2% to 102.1% and coefficient of determination (R²) values ≥ 0.992 for all targets. The 95% LOD ranged from 10 to 50 copies/μL. No cross-reactivity was detected with any of the non-target organisms tested. In the clinical cohort, the assay achieved an overall diagnostic sensitivity of 98.0% (95% CI: 93.8–99.5%) and specificity of 100% (95% CI: 97.5–100%), with complete concordance observed between multiplex and singleplex assays for all targets except one low-titer ZIKV sample. Conclusion: The developed TaqMan multiplex qRT-PCR assay provides a rapid, sensitive, and highly specific platform for syndromic screening of major mosquito-borne viruses. The analytical and clinical validation framework supports its potential utility in both clinical diagnostics and vector surveillance programs.