Methodological establishment and diagnostic value of a multiplex fluorescent PCR assay for the detection of three fastidious respiratory pathogens

作者：Jingchao Shi, Yijun Zhu, Shuyun Chen, Xiaoyun Shan, Bo Liu, Kai Shen, Keqiang Chen · 发表于：PLoS ONE · 年份：2025 · DOI：10.1371/journal.pone.0328651 · 被引用次数：1 · 研究领域：Pneumonia and Respiratory Infections、Bacterial Infections and Vaccines、Respiratory viral infections research

BACKGROUND: Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are common fastidious bacteria responsible for respiratory tract infections, particularly in children and immunocompromised individuals. Due to their demanding growth requirements, traditional culture methods often yield low sensitivity and delayed results, posing challenges for early and accurate diagnosis. OBJECTIVE: To establish a TaqMan probe-based multiplex fluorescent PCR method for the simultaneous rapid detection and identification of three important respiratory fastidious pathogens: Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. METHODS: By designing and optimizing TaqMan probes and primers targeting Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, the sensitivity, specificity, and reproducibility of the TaqMan probe-based multiplex fluorescent PCR assay were examined. A total of 173 clinical samples which are sputum and alveolar lavage fluid were tested simultaneously using traditional culture methods and multiplex fluorescent PCR assays, and the results were compared for consistency. For samples with inconsistent detection results, targeted high-throughput sequencing (tNGS) was used for confirmation. RESULTS: The limit of detection of the TaqMan probe-based multiplex fluorescent PCR method was 100 Copies/ml for Streptococcus pneumoniae, 20 Copies/ml for Haemophilus influenzae, and 50 Copies/ml for Moraxella catarrhalis. T...