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.