Development and performance evaluation of a multiplex fluorescent PCR method for the detection of fastidious pathogens causing respiratory infections in children.

Introduction: This study was performed to develop a multiplex fluorescent PCR method for the concurrent detection of six fastidious bacteria associated with respiratory tract infections. These bacteria include Streptococcus pneumoniae（SPN）, Bordetella pertussis (BP), Neisseria meningitidis (NM), Legionella pneumophila (LP), Moraxella catarrhalis (MC), and Haemophilus influenzae (HI).

Methodology: A multiplex fluorescent PCR test using SYBR Green as a DNA dye was developed and optimised. Clinical samples from 296 children with respiratory tract infections were then tested using the proposed method to assess its applicability for detecting the six pathogens.

Results: The SYBR Green-based multiplex fluorescent PCR method was successfully employed for the simultaneous identification of five pathogens through the analysis of melting curves and determination of the melting temperatures (Tm) values. However, the method exhibited limitations in distinguishing HI, necessitating separate detection using singleplex fluorescent PCR for this pathogen. In the methodological evaluation involving 296 clinical specimens, the multiplex fluorescent PCR successfully detected SPN, MC, NM, BP, and LP with sensitivities of 97.3%, 96.3%, 85.7%, 95.7%, and 80%, and areas under the ROC curve (AUC) of 0.977, 0.978, 0.927, 0.976 and 0.900, respectively.

Conclusions: Compared to conventional bacterial culture methods, the multiplex fluorescent PCR method with SYBR Green as the DNA dye is a sensitive and cost-effective approach for the simultaneous and rapid identification of five fastidious bacteria, making it a valuable alternative for clinical diagnostic laboratories.