Simultaneous amplification of Bordetella repeated insertion sequences and toxin promoter region gene by polymerase chain reaction

A polymerase chain reaction was devised to simultaneously detect repeated insertion sequences and the pertussis toxin promoter gene for the diagnostic identification of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. The sensitivity of this method was sufficient to detect one B. pertussis organism using the following cycles and temperatures: 95 degrees C for 15 min, followed by 32 amplification cycles (1 min at 95 degrees C, 1 min at 66 degrees C, I min at 72 degrees C), and finally 5 min at 72 degrees C. Using the primers as a combined set did not affect sensitivity, but required an increased temperature for optimal annealing compared with a single-sequence assay. As nasopharyngeal aspirate and swab materials sometimes contain hemoglobin, we also tested the inhibitory effect of hemoglobin on this assay, which was inhibited completely when using DNA extracts from samples containing hemoglobin at a final concentration >0.015 g/L: this inhibition was reversed by addition of bovine serum albumin to the buffer. Our assay shows promising sensitivity and specificity for clinical use.