BACKGROUND: To develop a real-time PCR assay for simultaneous detection and identification of clinically common bacteria and fungi causing meningitis from cerebrospinal fluid (CSF) samples. METHODS AND RESULTS: A total of 11 Gram-positive bacteria, 9 Gram-negative bacteria, and 7 fungi were identified correctly with a universal primer and probe designed based on ribosomal RNA conserved sequences. The detection limit was defined as 10(2) copies of plasmid DNA as C(T) value < 35 for boundaries. Among 137 CSF samples from patients with suspicion of meningitis, 23 samples were positive with a rate of 16.8%, and significantly higher than that of conventional methods, such as microbial culture and India ink stain (chi2 = 5.82, p < 0.05). CONCLUSIONS: TaqMan probe-based real-time PCR was proven to be a more rapid, sensitive, and specific method for the simultaneous detection of common bacteria and fungi. The method we established was not only applied to detect pathogens of CSF specimens, but also can also be applied to diagnose other biological fluids; it may be a supplementary assay and screening method to diagnose microorganisms.
BACKGROUND: To develop a real-time PCR assay for simultaneous detection and identification of clinically common bacteria and fungi causing meningitis from cerebrospinal fluid (CSF) samples. METHODS AND RESULTS: A total of 11 Gram-positive bacteria, 9 Gram-negative bacteria, and 7 fungi were identified correctly with a universal primer and probe designed based on ribosomal RNA conserved sequences. The detection limit was defined as 10(2) copies of plasmid DNA as C(T) value < 35 for boundaries. Among 137 CSF samples from patients with suspicion of meningitis, 23 samples were positive with a rate of 16.8%, and significantly higher than that of conventional methods, such as microbial culture and India ink stain (chi2 = 5.82, p < 0.05). CONCLUSIONS: TaqMan probe-based real-time PCR was proven to be a more rapid, sensitive, and specific method for the simultaneous detection of common bacteria and fungi. The method we established was not only applied to detect pathogens of CSF specimens, but also can also be applied to diagnose other biological fluids; it may be a supplementary assay and screening method to diagnose microorganisms.