Comprehensive conservation profiling enables a robust multiplex RT-qPCR assay for simultaneous pan-detection and serotype discrimination of dengue virus.

Dengue virus (DENV) remains a critical global health threat, necessitating rapid pan-detection and precise serotyping. In this study, we developed a multiplex real-time RT-qPCR assay for simultaneous detection and identification of DENV1-4. Guided by a systematic genomic conservation analysis of 4,754 sequences, we targeted the highly conserved 5' untranslated region (5' UTR) to design one pan-DENV and four serotype-specific primer-probe sets. The assay demonstrated robust analytical performance, with the limit of detection (LOD) ranging from 15.8 to 1.58 × 103 copies/mL and the lowest LOD (15.8 copies/mL) observed for serotype I with specific primer-probe sets. There was no cross-reactivity with other common pathogens of the Orthoflavivirus or Alphavirus genus pathogens, and clinical specificity was further validated as 100% using a negative cohort of non-dengue febrile samples. Validation using 53 clinical DENV-positive samples showed 100% concordance with a reference diagnostic kit for serotype identification. While the pan-DENV set served as a high-specificity screening tool with broad reactivity, it exhibited a clinical sensitivity of 88.7%, particularly missing low-titer samples (Ct > 30). This discrepancy, coupled with the superior sensitivity of the serotype-specific sets, suggests the presence of incomplete genomic fragments in acute-phase sera, highlighting the advantage of our 5' UTR-targeted approach in assessing viral genomic integrity. This dual-layered multiplex assay provides a robust, rapid, and cost-effective tool for clinical diagnosis and epidemiological surveillance.IMPORTANCEDengue fever is a leading cause of systemic viral disease worldwide. Co-circulation of four distinct serotypes complicates clinical management and increases severe disease risk. Here, we presented a novel multiplex RT-qPCR assay enabling simultaneous pan-DENV detection and serotype identification in two tubes, comprising six reactions including an internal control (IC), thereby streamlining dengue diagnosis. A key innovation is the systematic selection of the 5' UTR as the diagnostic target, guided by big-data genomic analysis. Unlike assays targeting the 3' UTR, which may overrepresent viral loads by capturing redundant subgenomic orthoflavivirus RNA (sfRNA), our 5' UTR-targeted design provides a more stringent measure of genomic integrity. Furthermore, the modular performance of our assay combining broad screening via a pan-DENV set with high-sensitivity subtyping which offers a dual-layer diagnostic framework. This work provides a high-performance tool for timely clinical intervention and enhances our molecular understanding of DENV fragments in clinical specimens.