Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 17 2 2025 04 05 204 210 EN Nafiseh Fotros Department of Biotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Reihaneh Bashiri Department of Biotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Samira Mohammadi-Yeganeh Medical Nanotechnology and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Mahdi Paryan Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran 2024 09 23 2025 02 09 Background and Objectives: Viral infections of the respiratory system are a major public problem due to their ease of spread, pandemic potential, and significant rate of death. Diagnosing these infections requires laboratory testing, as clinical symptoms alone are often insufficient. Influenza A, Influenza B, and COVID-19 are common infections that burden the population, especially during winter. We developed a multiplex real-time PCR method to simultaneously detect Influenza A and B, as well as COVID-19. Compared to existing detection kits, it offers higher accuracy, lower costs, and faster results, making it an efficient diagnostic tool. Materials and Methods: We designed primer/TaqMan probes for the M2 gene of Influenza A, N gene of SARS-CoV-2, and NS1 gene of Influenza B. Reaction components were optimized and functional parameters were tested using standard samples with known viral copy numbers. Results: The method’s detection limit is 10 copies for Influenza A and B, and 60 for SARS-CoV-2. Sensitivity and specificity for Influenza A are 88% and 100%, for Influenza B, 95.6% and 100%, and for SARS-CoV-2, 90.4% and 100%. Conclusion: This multiplex real-time PCR method can accurately detect and distinguish SARS-CoV-2, Influenza B, and Influenza A infections. https://ijm.tums.ac.ir/index.php/ijm/article/view/5047 https://ijm.tums.ac.ir/index.php/ijm/article/download/5047/1758