There are several new methods for the detection of B. pertussis under investigation. Some of these involve the use of DNA probe technology and PCR. We propose a dual approach to the development of a DNA-based diagnostic test(s) specific for B. pertussis. One approach involves the further characterization of sequences associated with the porin gene to identify a short sequence which is unique to B. pertussis. The porin gene of B. pertussis has been cloned and sequenced by LP/CBER. Sequence differences were found when regions adjacent to the 5' end of the porin genes of B. pertussis and B. parapertussis were analyzed. Two pairs of primers were designed that amplified a 159 bp fragment from B. pertussis DNA and a 121 bp fragment from B. parapertussis DNA respectively. PCR was performed on 195 aspirates. It detected 88% of the samples which were both culture and CHO positive with a specificity of 97%. """"""""Shared-primer"""""""" PCR was designed for detecting B. pertussis and B. parapertussis simultaneously. A non-radioactive Digoxigenin detection system which could tremendously increase the sensitivity of the PCR is currently under investigation. Hybridization of Bordetella specific DNA probes to colony lifts from culture plates of suspected pertussis patients is also being developed. The objective of these studies is to develop rapid, sensitive and species-specific clinical assays for the detection of B. pertussis. Hybridomas producing monoclonal antibodies that specifically detect B. pertussis in clinical samples have been developed for the detection, by immunofluorescence, of infection in children suspected of having whooping cough. These monoclonal antibodies are being evaluated in the clinical environment.
