Abstract
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.
Highlights
Lower respiratory tract infections are among the leading causes of hospitalization and death around the world [1,2,3,4]. e causative agents of lower respiratory tract infections are diverse [5], while respiratory bacterial pathogens remain to be the primary cause [6,7,8,9]
The performance of MCA using SYBR Green I is problematic as the melting temperature of the dye can be affected by the dye concentration [26] and DNA concentration [27]. ese shortcomings limit the application of SYBR Green I for melting curve analysis based on multiplex PCR [28]
In order to realize the simultaneous detection of respiratory bacterial coinfections and improve the accuracy of detecting respiratory bacterial pathogens, the current study aims to establish an assay that combines multiplex real-time PCR techniques with EvaGreen MCA to detect eight common respiratory bacteria simultaneously
Summary
Lower respiratory tract infections are among the leading causes of hospitalization and death around the world [1,2,3,4]. e causative agents of lower respiratory tract infections are diverse [5], while respiratory bacterial pathogens remain to be the primary cause [6,7,8,9]. Fluorescence melting curve analysis (MCA) based on dyes was developed to overcome these limitations [24]. Ese shortcomings limit the application of SYBR Green I for melting curve analysis based on multiplex PCR [28]. Another dye, EvaGreen, has been found to be more sensitive and stable than SYBR Green I, owing to its ability to insert into each single-nucleotide saturated DNA helix [29]. In order to realize the simultaneous detection of respiratory bacterial coinfections and improve the accuracy of detecting respiratory bacterial pathogens, the current study aims to establish an assay that combines multiplex real-time PCR techniques with EvaGreen MCA to detect eight common respiratory bacteria simultaneously
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
