Multiplex PCR with probe melting curve detects six respiratory pathogens with high clinical accuracy
The study reports the development and clinical validation of a multiplex real-time PCR assay using fluorescence probe melting curve analysis for simultaneous detection of influenza A and B, RSV, rhinovirus, adenovirus, and Mycoplasma pneumoniae. Evaluated against 760 pharyngeal samples, the assay demonstrated greater than 87% sensitivity and 99% specificity per pathogen compared with a commercial RT-PCR kit, with kappa agreement ranging from 0.927 to 1.000. Analytical performance showed 100% specificity against a 57-pathogen panel and detection limits between 248 and 394 copies per milliliter. The platform offers a high-throughput, single-tube alternative for respiratory pathogen screening, supporting routine molecular diagnostics workflows.
The original study
Development of a multiplex real-time PCR assay with fluorescence probe-melting-curve analysis for one-tube detection of respiratory pathogens.
- Authors
- Ji M, Dong H, Xu Y, Wang Y, Zhao Q
- Journal
- PloS one
- Type
- Journal Article
- PMID
- 42715193
Original abstract
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.