Development and validation of a multiplex RT-qPCR method for the simultaneous detection of influenza type A, B and SARS-COV-2 viruses

Q3 Medicine

Medicine in Novel Technology and Devices Pub Date : 2025-01-22 DOI:10.1016/j.medntd.2025.100350

Samira Karimkhani , Ehsan Lotfi , Fatemeh Karamali , Mahsa DarestaniFarahani , Reza Keikha , Mahmood Barati

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Abstract

The study aimed to evaluate the diagnosis between SARS-COV-2 and influenza viruses using the Multiplex qPCR molecular method, highlighting the importance of these methods in disease management.

In this study, primers and probes were designed for the hemagglutinin region (HA) of influenza A, the M region of influenza B virus, and the RdRp region of SARS-COV-2. Optimization was performed using qPCR methods, and the method's analytical sensitivity and specificity were assessed. Finally, the process was compared to the commercial kit, Generi-Biotech Company (GB SARS-CoV-2 Influenza A/B).

The best annealing temperature for this method was determined to be 58 °C. Analytical sensitivity showed detection limits of 500 copies of the virus genome for SARS-CoV-2, 250 copies for influenza A, and 500 copies for influenza B. Clinical evaluations confirmed that the designed kit exhibited 100 % sensitivity and specificity, identical to the GB commercial kit, establishing its comparable diagnostic performance.

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