Development and Application of a Quadruplex Real‐Time RT‐PCR Assay for Simultaneous Detection and Subtyping of Respiratory Syncytial Virus A and B

Purpose To establish a quadruplex real‐time fluorescent quantitative RT‐PCR method for the simultaneous detection of respiratory syncytial virus (RSV) and its Subtypes A and B (RSV‐A and RSV‐B) and to conduct a preliminary analysis of the genetic variation characteristics of RSV strains isolated from our hospital. Methods Highly specific primers and fluorescently labeled probes were designed using Primer Express 3.0 software. Following optimization of the reaction system, standard curves were constructed, and the sensitivity, reproducibility, and specificity of the quadruplex fluorescent quantitative RT‐PCR method were evaluated. The established assay was applied to test 407 sputum specimens collected from pediatric patients with suspected infections. Positive results were confirmed by sequencing, followed by sequence identity comparison and phylogenetic tree construction. Results The selected primers and probes specifically detected RSV and effectively distinguished between RSV‐A and RSV‐B subtypes. The method demonstrated a detection limit of 103 copies/mL, with an intra‐assay coefficient of variation (CV) of < 2.00%. No cross‐reactivity was observed with nucleic acids from other common respiratory viruses. Among the 407 clinical specimens, 47 tested positive for RSV, including 39 RSV‐A (positivity rate: 9.6%) and 8 RSV‐B (positivity rate: 2.0%). These positive findings were consistent with sequencing results. The four tested RSV‐A Subtype G genes showed the highest nucleotide sequence identity (98.9%–99.3%) with the 2013 Beijing strain (NA1/AB470478). Among the four tested RSV‐B Subtype G genes, one exhibited the highest nucleotide sequence identity (98.8%) with the 2013 Beijing strain (BA8/HM459873), while the other three showed the highest nucleotide sequence identity (97.7%–99.4%) with the 2013 Hangzhou strain (BA10/KP336526). Conclusion A quadruplex real‐time fluorescent quantitative RT‐PCR method was successfully developed for the simultaneous detection of RSV and its A and B subtypes in a single tube. The assay exhibits high specificity, sensitivity, and reproducibility, providing a novel and efficient detection tool for clinical diagnostics and epidemiological studies. RSV infections in our hospital involved cocirculation of Subtypes A and B, with Subtype A being predominant. Furthermore, the G gene of Subtype B may exhibit greater variability than that of Subtype A.