Preparation of Bordetella pertussis DNA from respiratory samples for real-time PCR by commercial kits.
Bordetella pertussis is increasingly detected by real-time PCR, but most kits for extracting Bordetella-DNA from respiratory samples are not validated for this material. Respiratory clinical materials were spiked with Bordetella pertussis cells. Four ion-exchange chromatography methods from one manufacturer were used for DNA preparation. Two real-time PCRs detecting the IS481 of Bordetella pertussis and based either on a hybridisation probes format (LightCycler) or on a TaqMan format were used. All kits effectively prepared DNA for Bordetella pertussis real-time PCR. The procedures were linear over a broad range, and the lower level of sensitivity was similar. Sensitivities measured as CT values were different. Inter-assay CVs were between 6.8% and 17.3%. Two kits did not effectively remove inhibitory substances from the respiratory samples. Commercial kits are useful for preparing Bordetella pertussis DNA from respiratory samples, but even kits from one manufacturer show significant differences in effectiveness and removal of inhibitory substances.
Riffelmann
M
M
Institut für Infektiologie Krefeld GmbH, Lutherplatz 40, 47800 Krefeld, Germany. riffelmann_hyg@Klinikum-Krefeld.de
Schmetz
J
J
Bock
S
S
Wirsing von Koenig
C H
CH
D001431:Bacteriological Techniques / Q000379:methods*; D001886:Bordetella pertussis / Q000235:genetics* / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014917:Whooping Cough / Q000175:diagnosis*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial