Development and comparison of a rapid RT-qPCR assay with qiastat-Dx respiratory panel for the co-detection of SARS-CoV-2, influenza A virus and respiratory syncytial virus

Year 2025, Volume: 42 Issue: 4, 404 - 410, 31.12.2025

Buket Baddal , Gokce Akan , Gülten Tuncel , Melis Kalaycı , Aysegul Bostancı , Tamer Sanlidag

Abstract

Molecular multiplex assays for simultaneous detection of respiratory viral pathogens are central for the efficient identification of multiple pathogens with overlapping clinical presentation. Following the COVID-19 pandemic, influenza viruses, respiratory syncytial virus (RSV) and SARS-CoV-2 began to co-circulate and health authorities issued a warning against a triple epidemic. In this study, a rapid and low-cost multiplex RT-qPCR assay was developed for the detection of influenza A virus, SARS-CoV-2 and RSV A/B. The assay was designed to include two mastermixes (MMs), MM-1 containing primer-probe mix to target SARS-CoV-2 and influenza A virus, and MM-2 containing primer-probe mix for RSV A and B. The assay was validated using 80 clinical oro-nasopharyngeal specimens and the results were compared to Qiastat-Dx respiratory panel. The four-target RT-qPCR assay was able to detect all of the SARS-CoV-2 positive (n=33) specimens and RSV A/B positive specimens (n=22), while the assay was unable to detect 2 of the influenza A positive samples (n=25) in comparison to Qiastat-Dx Analyzer, which had Ct > 35. All of the negative samples were detected correctly (n=60). Overall, PPA (95% CI) in SARS-CoV-2 samples was 100 % (87-100 %), 92 % (72.5-98.6 %) in influenza A samples and 100 % (81.5-100 %) for RSV A/B samples. In conclusion, a rapid, reliable and cost-effective multiplex RT-qPCR assay was developed for the simultaneous detection of four pathogens from nasopharyngeal swabs. This assay has the potential to enhance laboratory diagnostic capabilities and improve public health responses during future epidemics, enabling timely interventions.

Keywords

Respiratory syncytial virus , influenza A virus , SARS-CoV-2 , triple epidemic , molecular diagnosis

Ethical Statement

The study protocol was approved by the Institutional Review Board at Near East University (YDU/2023/116-1762). Informed consent has been taken from the individuals who voluntarily participated for the optimization of the in-house assay.

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