Integrated validation of internally controlled one-step multiplex real-time RT-PCR for dengue virus serotyping and monoplex assay for quantification

Dengue is a major global health challenge, yet no commercially validated molecular kits currently provide both serotyping and viral load quantification in a single integrated format. To address this gap, we developed and internally validated a one-step multiplex real-time RT-PCR assay that simultaneously detects all four dengue virus (DENV) serotypes in a single reaction (5-PLEX), together with a complementary monoplex system for quantitative viral load measurement. The 5-PLEX assay demonstrated high specificity and reliable serotyping performance during acute and early critical phases of dengue infection (day of illness ≤4). Monoplex assays for each serotype showed strong linearity, precision, and low analytical limits of detection (3–10 copies/reaction), enabling accurate quantification. Based on these findings, we propose a diagnostic framework: 5-PLEX for samples collected within the first four days of illness, the previously validated 3-PLEX assay for samples collected from day 5 of illness onward, and monoplex assays for serotype-specific viral load measurement. Internal and external process controls were incorporated to support accuracy and reproducibility within the validated framework and to facilitate future multi-laboratory evaluation. This harmonized is designed to facilitate standardized implementation across laboratories and supports improved inter-laboratory consistency in clinical diagnostics, genomic surveillance, therapeutic trials, and pathogenesis research. • Validated a multiplex real-time RT-PCR assay for detecting and serotyping DENV • Developed a framework integrating multiplex serotyping and monoplex quantification • Monoplex assays provide precise viremia measurement • Supports reliable diagnostics, enhances surveillance, and enables multi-site trials