Simultaneous detection of 15 respiratory pathogens with a fluorescence probe melting curve analysis-based multiplex real-time PCR assay

Acute respiratory tract infections are common worldwide and caused by a great diversity of pathogens. A rapid and accurate diagnosis method of respiratory infection is crucial for timely clinical intervention. Here, by combining fluorescence melting curve analysis and multiplex real-time assay, we developed a novel method which can simultaneously detect 15 respiratory viruses. The specificity for target genes was 100%, as assessed with a panel of 47 respiratory pathogens, which indicated no cross-reactions. The assay’s limits of detection at the nucleic acid level ranged from 5 copies/μL to 500 copies/μL nucleic acids. Compared with conventional culture method, our assay showed more than 75% sensitivity and 100% specificity for each respiratory pathogen in 384 clinical samples. Even more, the kappa correlation for all the pathogens ranged from 0.86 to 1.00. Overall, this method has the characteristics of high throughput, low cost and high sensitivity and precision, which demonstrated our method is well suited for routine clinical testing in respiratory infection.