SIMULTANEOUS DETECTION OF THREE RESPIRATORY VIRUSES BY A SINGLE QPCR AMPLIFICATION FOLLOWED BY HIGH RESOLUTION MELTING ANALYSIS

M. CAPUTO; S. GINART; D. CORACH; L. GARRIGÓS; F. REMES LENICOV; A. SALA

Mar del Plata

Congreso; Reunión Conjunta SAIC 2023; 2023

SOCIEDAD ARGENTINA DE INVESTIGACION CLINICA

After pandemic confinement, other respiratory viruses, such as Influenza and RSV, have started to circulate again. Since acute symptoms, overlap with those of SARS-CoV-2, molecular methods are required to identify the infecting virus. Accordingly, many research groups worked on the development of specific, rapid and cost/efficient methods to detect these viral infections. Most methods were developed on probe based qPCR techniques, strategies that requires expensive reagents. The aim of this work was to develop a diagnostic method based on a multiplex qPCR-HRM to identify specific sequences of RSV, H1N1 and SARS-CoV-2. M&M: synthetic targets of DNA sequences were designed to detect SARS-CoV-2 (103bp); Influenza (H1N1) (98 bp) and RSV (111bp). Online tools were employed for primer design and melting temperature (Tm) simulations. Quantitative PCR was carried out in multiplex reaction, mixing RSV, H1N1 and SARS-CoV-2 primers and SYTO9TM intercalating dye to visualize the progress of the qPCR/HRM melting. Results: Based on the GC content and amplicons length it was possible to detect the tested viruses. Specific viral sequences displayed differential Tm: RSV 76.1°C, H1N1 80.7°C and SARS-CoV-2 83.8°C. The sensitivity was assessed with serial dilutions of synthetic targets. Detection limit was up to 10 copies/ul for SARS-CoV-2 and H1N1, and 100 copies/ul for RSV. Simulated co-infections were efficiently detected. A preliminary assay with cDNA from nasopharyngeal swabs of patients previously diagnosed were concordant. Conclusion: This technique was able to discriminate three respiratory viruses, with high sensitivity and reproducibility. Preliminary tests involving positive samples showed result consistent with our approach. The next steps will include testing an extended number of diagnosed patients and optimize the reaction by coupling with a previous RT-PCR step. This development constitutes a simple and cost-effective strategy for viral diagnosis.