Validation of a multiplex qRT-PCR assay for the detection of RSV, influenza A/B virus and SARS-CoV-2

The significant burden of viral respiratory diseases necessitates rapid detection of key pathogens. Simultaneous testing for SARS-CoV-2, RSV, and influenza A/B as an initial step, followed by broader panels as needed, offers a cost-effective diagnostic strategy. This study aimed to validate a new commercial multiplex qRT-PCR assay (Diagnovital® RTA Laboratories, Turkey) for the simultaneous detection of these viruses.Analytical sensitivity was determined using Probit regression analysis on serial dilutions (10⁵–10¹ copies/ml) for each target virus. Specificity was evaluated with 120 negative samples and 32 positive samples for non-target respiratory viruses. External quality control panels and clinical specimens positive for RSV (n = 39), influenza A/B (n = 71), SARS-CoV-2 (n = 64) were used for accuracy testing. Intra- and inter-assay precision were analyzed using samples near the limit of detection. The performance was compared to routine diagnostic tests.The assay’s analytical sensitivity was 420.7, 296.7, 368.6, 1362.6, and 1459.7 copies/ml for SARS-CoV-2 Alpha and Omicron variants, influenza A, influenza B, and RSV, respectively. Analytical specificity was 100%, and precision showed CV% < 5. Detection rates for SARS-CoV-2, influenza A, influenza B, and RSV were 100%, 95.1%, 97.5%, and 94.8%, respectively, with false negatives occurring in samples with Ct > 33. Comparative analysis showed high correlations between assays, with strong agreement (Cohen’s κ ranging from 0.861 to 1).These findings demonstrate the clinical applicability of the Diagnovital® assay, though false negatives may occur in low-concentration samples.