Orthoflavivirus detection by quantitative polymerase chain reaction coupled with the high-resolution melting analysis as a versatile discrimination tool

Orthoflaviviruses represent a genetically diverse group of RNA viruses, posing a major challenge for broad and reliable molecular detection. In this study, we developed a robust qPCR-based assay targeting the highly conserved coding region of the non-structural protein 5 (NS5), followed by high-resolution melting (HRM) analysis to enhance discriminatory capacity. Initial classification based on melting temperature profiles enabled separation of orthoflaviviruses into eight groups. Incorporation of HRM analysis further increased resolution, allowing differentiation of up to fifteen distinct orthoflaviviruses. The specificity of the assay was confirmed using a panel of diverse virus species and validated on a large set of orthoflaviviral samples. The combined qPCR-HRM strategy demonstrates strong potential for sensitive detection and classification of both known and previously uncharacterized orthoflaviviruses, providing a valuable tool for surveillance and diagnostic applications.