Clinical and analytical validation of a novel switching RT-qPCR assay for the simultaneous detection of seven...

Rapid and accurate identification of respiratory viruses is essential for effective clinical management and infection control. However, conventional RT-qPCR methods are time-consuming and require complex workflows. To address these limitations, we developed a novel Switching RT-qPCR assay designed for multiplex detection of respiratory viruses with enhanced efficiency and sensitivity. This study aimed to evaluate the analytical and clinical performance of the switching RT-qPCR assay in the detection of seven major respiratory viruses: Influenza A and B, respiratory syncytial virus (RSV) A and B, adenovirus (AdV), human metapneumovirus (hMPV), and SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). Nasopharyngeal swab specimens (n = 808) were collected from patients with respiratory symptoms. RNA was extracted and tested using both a Switching RT-qPCR assay and commercial multiplex RT-qPCR method (Allplex Respiratory Panels and Allplex SARS-CoV-2 Assay). The concordance, sensitivity, specificity, and analytical performances of the two methods were compared. The Switching RT-qPCR assay demonstrated a clinical sensitivity of 93.6% and a specificity of 100%, with an overall concordance rate exceeding 96%, compared with the conventional assay. Analytical sensitivity was comparable or superior across most viral targets, particularly RSV A, AdV, and hMPV. The duplex assay format enabled the efficient simultaneous detection of two targets per reaction, reducing the overall testing time while maintaining analytical accuracy. This Switching RT-qPCR assay offers a reliable, rapid, and efficient alternative for respiratory virus detection. Its simplified duplex format and high concordance with standard molecular diagnostics indicate its potential utility in routine clinical laboratories.