Background and aims: Bordetella pertussis is a gram negative bacterium of the genus Bordetella and is the causative agent of the pertussis disease that affects millions of people worldwide and kills about 200,000 children every year according to the World Health Organization.
It is commonly detected by a singleplex real-time PCR based on the insertion sequence IS481. The use of this sequence is controversial as it also amplifies Bordetella holmesii. In order to avoid false positive results, we developed a new multiplex real time PCR assay. Our method is based on four specific sites for Bordetella species detection and includes an internal control to assure operational effectiveness and efficiency of both bacterial extraction and PCR reaction. The four chosen loci enable the specific detection of the most prevalent clinical strains of Bordetella: B.pertussis, B.parapertussis, B.holmesii and B.bronchiseptica.
Methods: A panel of 83 Bordetella and non-Bordetella samples was tested for amplification with our multiplex method using the IS481, IS1001, BHIS1001 and the ptxS1 loci. The reaction was done on a Rotor-Gene 6000 instrument on four different channels and includes an internal control on a fifth channel.
Results: Specific detection of Bordetella species and identification of pathogenic strains from clinical samples was achieved within less than 4 hours including DNA extraction. Two quality control panels of Bordetella species were also perfectly detected using the multiplex reaction.
Conclusion: This rapid and sensitive detection of Bordetella species should facilitate the diagnosis, help the management of disease and control the outbreaks of this pathogen.
