Advancing multiplex diagnostics: A novel framework for simultaneous detection of diverse pathogens using respiratory RNA viruses as a model system

Numerous infectious pathologies share clinical presentation but require different management. The search for multiple pathogens in a single test could improve the resolution of these cases. For example respiratory infections, caused either by viral, bacterial or fungal agents. Rapid differential detection of common respiratory RNA viruses could be benefit by such a strategy. We present a diagnostic method based on real-time quantitative PCR (qPCR) coupled with high-resolution melt (HRM) analysis. While applicable to diverse pathogens, this study focuses on detecting three respiratory RNA viruses: influenza A (H1N1), respiratory syncytial virus (RSV), and SARS-CoV-2. To develop a rapid, specific, and cost-effective system, we designed a multiplex assay using SYTO 9 fluorescent dye and pathogen-specific primers. Post-amplification HRM analysis generated melt curves, with first-derivative plots enabling discrimination of the three viruses. Samples tested in quintuplicate (10¹-10³ copies/μL) yielded distinct melt peaks. Mean melting temperatures (average Tm ± SD) were 83.64 ± 0.08 °C (SARS-CoV-2), 80.45 ± 0.07 °C (influenza H1N1), and 75.99 ± 0.07 °C (RSV). The system robustly differentiated all three viruses (100 % accuracy). Preliminary validation with clinical samples showed strong agreement with a commercial kit: κ = 0.87 (95 % CI: 0.61-1.00) for SARS-CoV-2 and κ = 1.00 (95 % CI: 0.74-1.00) for RSV. Therefore, the designed method represents a rapid and cost-effective alternative for differential diagnosis in a single reaction tube.