Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR.
A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.
Gullsby
Karolina
K
Centre for Research & Development, Uppsala University/County Council of Gävleborg, 801 87 Gävle, Sweden.
Storm
Martin
M
Bondeson
Kåre
K
D018829:Adhesins, Bacterial / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D023521:Chlamydophila Infections / Q000175:diagnosis*; D016993:Chlamydophila pneumoniae / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009177:Mycoplasma pneumoniae / Q000302:isolation & purification*; D010614:Pharynx / Q000382:microbiology; D011019:Pneumonia, Mycoplasma / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C035471:adhesin, Mycoplasma pneumoniae; C078167:OMPA outer membrane proteins