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Background and Objective. Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis are primary respiratory bacterial pathogens contributing to morbidity and mortality in developing countries. This study evaluated the diagnostic performance of multiplex real-time PCR with fluorescence melting curve analysis (MCA) assay, which was used to detect eight respiratory bacterial pathogens simultaneously. Methods. A total of 157 sputum specimens were examined by multiplex real-time with fluorescence MCA, and the results were compared with the conventional culture method. Results. Multiplex real-time PCR with fluorescence MCA specifically detected and differentiated eight respiratory bacterial pathogens by different melting curve peaks for each amplification product within 2 hours and exhibited high repeatability. The limit of detection ranged from 64 to 102 CFU/mL in the multiplex PCR system. Multiplex real-time PCR with fluorescence MCA showed a sensitivity greater than 80% and a 100% specificity for each pathogen. The kappa correlation of eight bacteria ranged from 0.89 to 1.00, and the coefficient of variation ranged from 0.05% to 0.80%. Conclusions. Multiplex real-time PCR with fluorescence MCA assay is a sensitive, specific, high-throughput, and cost-effective method to detect multiple bacterial pathogens simultaneously.

Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis