Development of a one-pot RPA–CRISPR/Cas12a assay for rapid multiplex detection of respiratory pathogens

Respiratory tract infections impose a substantial clinical and public health burden, and timely etiological identification remains challenging. Rapid panel testing is therefore needed for common respiratory pathogens. In this study, we developed a closed-tube, one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay using a multichamber 8-strip tube format to enable parallel fluorescence readout while reducing the risk of aerosol contamination. The assay targets 9 pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae. Analytical performance, including limit of detection, specificity, and repeatability, was evaluated using viral recombinant plasmids and bacterial genomic DNA. Clinical evaluation was performed using available respiratory specimens and bacterial isolates, with reverse-transcription quantitative polymerase chain reaction for viral targets and conventional bacterial identification for bacterial targets as reference methods. The assay provided results within approximately 50 to 60 minutes. The limits of detection for viral targets were 1 copy/μL for influenza A virus and adenovirus and 2 copies/μL for influenza B virus and respiratory syncytial virus. No cross-reactivity was observed under the tested conditions. Repeatability was satisfactory, with coefficients of variation below 15% across targets. In a clinical evaluation of 34 specimens or isolates, the one-pot assay showed 100% overall agreement with the reference methods. These findings indicate that the closed-tube, one-pot RPA-CRISPR/Cas12a platform enables rapid and parallel detection of common respiratory pathogens and may support point-of-care testing and deployment in resource-limited settings.