Hugo Van Heuverswyn

DNA probes for Bordetella species and a colorimetric reverse hybridization assay for the detection of Bordetella pertussis

Three oligonucleotide probe sequences were inferred from the 16S ribosomal ribonucleic acid (rRNA) and the 16S-23S rRNA spacer sequences of Bordetella pertussis ATCC 10380. These probes were used in hybridization tests with deoxyribonucleic acid from Bordetella species and other relevant bacterial taxa. A probe from the spacer region hybridized exclusively to the B. pertussis strains tested and not to strains from other species. Using a combination of three probes, B. pertussis, B. parapertussis/B. bronchiseptica and B. avium could be specifically identified and differentiated from other taxa. Differentiation between B. parapertussis and B. bronchiseptica was not possible with the probes used. Using the spacer probe, a colorimetric hybridization assay specific for B. pertussis was developed based on enzymatic amplification of the 16S-23S rRNA spacer and reverse hybridization in microtitre wells. As compared with results using agarose gel electrophoresis, and Southern and dot-spot hybridization with a 32P-labelled probe, this assay proved to be faster and easier to perform and was found to be at least as sensitive and specific.