AI Insight
Researchers developed a multiplex real-time PCR assay that can simultaneously detect six common respiratory pathogens (influenza A and B, RSV, rhinovirus, adenovirus, and Mycoplasma pneumoniae) in a single test tube using fluorescence probe melting curve analysis. The assay demonstrated 100% specificity, high sensitivity (detection limits of 248-394 copies/ml), and was validated against 760 clinical pharyngeal samples, showing over 87% sensitivity and 99% specificity compared to commercial testing kits. The test proved particularly effective for detecting rhinovirus and showed improved sensitivity for influenza A, adenovirus, Mycoplasma pneumoniae, and RSV.
Why it matters
This diagnostic tool enables faster, more cost-effective identification of multiple respiratory pathogens simultaneously, which is crucial for appropriate treatment decisions and infection control, especially given the resurgence of respiratory infections since the COVID-19 pandemic. The high-throughput capability could significantly improve clinical laboratory efficiency during respiratory disease outbreaks.
Understand the Science
by Mingyu Ji, Hongjiao Dong, Yihui Xu, Yunshan Wang, Qianqian Zhao
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.