Development of a multiplex real-time PCR assay with fluorescence probe-melting-curve analysis for one-tube detection of respiratory pathogens

Pathogens causing respiratory infections have reemerged globally since the COVID-19 pandemic. Rapid and accurate identification of respiratory pathogens is critical for diagnosis and treatment. In this study, we developed and evaluated a multiplex real-time PCR assay with a fluorescence probe melting curve (mqPCR-PMC) assay to simultaneously detect six common pathogens, namely, influenza virus A and B (IFV-A and -B), respiratory syncytial virus (RSV), human rhinovirus (HRV), human adenovirus (HAdV) and Mycoplasma pneumoniae (MP). The primers and probes optimized in this assay exhibited a specificity of 100% with a panel of 57 pathogens. The detection limits of the assay ranged from 248 copies/ml to 394 copies/ml. Moreover, the coefficients of variation ranged from 0.11% to 0.30% for Tm and from 1.59% to 4.10% for Rm. The clinical accuracy of the assay was validated using 760 pharyngeal samples. Compared with a commercial kit based on conventional fluorescence RT-PCR, the assay showed more than 87% sensitivity and 99% specificity for each pathogen. The kappa values ranged from 0.927 to 1.000. Moreover, the assay was more precise for HRV and more sensitive on IFV-A, HAdV, MP and RSV. In summary, the mqPCR-PMC assay was demonstrated to be highly sensitive, highly specific and high throughput, and has potential for clinical use in respiratory infectious disease diagnosis.