Abstract

Novel extraction-free RPCR-MC assay realizes single-tube simultaneous detection of six respiratory pathogens. • Single-copy sensitivity, high specificity, strong anti-interference, and 12-month stability at -20°C confirmed. • Clinical validation with 150 samples shows 100% consistency with Sanger sequencing; Class III Medical Device certified. • Four fluorescence channels + internal control enable extension to other pathogens for infectious disease control. Objectives Acute respiratory infections commonly result from a spectrum of pathogens, including SARS-CoV-2, influenza A (IAV) and B viruses (IBV), respiratory syncytial virus (RSV), human adenovirus (HAdV), and Mycoplasma pneumoniae (MP). This study aimed to develop and validate a rapid, extraction-free multiplex RT-PCR assay coupled with polychromatic melting curve analysis (RPCR-MC). This assay enables simultaneous detection of all six pathogens in a single tube. Methods Specific primers and TaqMan probes were designed to target conserved regions of each pathogen. An extraction-free workflow was established by direct lysis of throat swab samples. The assay integrated four fluorescence channels (FAM, HEX, ROX, and CY5) with melting curve analysis to differentiate amplicons based on their melting temperatures (Tm). We evaluated analytical performance (sensitivity, specificity, repeatability, and stability) using synthetic plasmids, viral culture supernatants, and clinical samples. The results were further validated by Sanger sequencing. Results The RPCR-MC assay achieved a limit of detection of 1 copy/μL for all six pathogens. No cross-reactivity was observed with 30 potentially interfering substances. In the clinical evaluation of 249 samples, the assay identified 62 SARS-CoV-2, 33 IAV, 62 IBV, 19 RSV, 61 HAdV, and 28 MP cases, including 34 co-infections. The assay achieved an overall accuracy of 99.6% and demonstrated excellent agreement with commercial reference kits. The entire workflow was completed with