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

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.

Figure 1

The specificity of EvaGreen multiplex real-time PCR protocol for the eight bacteria. (a) Results obtained for each bacterium when tested by multiplex real-time PCR protocol. (b) Each distinct peak corresponded to a specific bacterial standard strain DNA amplified by multiplex real-time PCR protocol, where nontemplate negative controls (NTC) did not show any peaks.

Figure 2

Sensitivity of multiplex real-time PCR protocol for detection of single bacterium.

Figure 3

The sensitivity of simultaneous detection of S. aureus, S. pneumoniae, E. coli, and M. tuberculosis; the specific T_m peak of M. tuberculosis disappears when each bacterial concentration drops to 3.2 × 10³ CFU/mL.

Figure 4

(a)–(h) Standard curves were obtained by plotting the fluorescence intensity (y-axis) values against the log₁₀ of the bacterial concentration (x-axis) of the eight respective standard bacterial strains. The curve equations and R² are as follows: y = 6.53x − 5.52, R² = 0.988 for A. baumannii (a); y = 7.15x − 22.87, R² = 0.987 for E. coli (b); y = 5.92x − 14.08, R² = 0.980 for K. pneumoniae (c); y = 3.66x + 1.02, R² = 0.965 for H. influenzae (d); y = 1.42x + 4.19, R² = 0.947 for S. pneumoniae (e); y = 5.81x − 6.37, R² = 0.998 for S. aureus (f); y = 4.15x − 13.39, R² = 0.995 for P. aeruginosa (g); y = 1.08x + 1.34, R² = 0.943 for M. tuberculosis (h).