Analytical verification of a multiplex PCR for identification of Bordetella bronchiseptica and Pasteurella multocida...

Bordetella bronchiseptica and Pasteurella multocida are etiologic agents of progressive atrophic rhinitis (PAR) and bronchopneumonia in swine. Only dermonecrotic toxin-producing strains of P. multocida play a role in atrophic rhinitis while both toxigenic and nontoxigenic strains have been associated with pneumonia. Monitoring and investigation of outbreaks involving these bacteria require sensitive and accurate identification and reliable determination of the toxigenic status of P. multocida isolates. In the present study, we report the development, optimization, and performance characteristics of a multiplex PCR assay for simultaneous amplification of up to three different targets, one common to all P. multocida strains, one found only in toxigenic P. multocida strains, and one common to B. bronchiseptica strains. Based on analysis of 94 P. multocida isolates (31 toxigenic) and 126 B. bronchiseptica isolates assay sensitivity is 100% for all amplicons. Evaluation of 22 isolates of other bacterial genera and species commonly found in the swine respiratory tract demonstrated a specificity of 100% for all gene targets. The limit of detection for simultaneous amplification of all targets is 1-10pg of DNA per target, corresponding to a few hundred genomes or less. Amplicon mobility in agarose gels and sequence analysis indicate the amplicons are highly stable. The data presented establish this multiplex PCR as a reliable method for identification of B. bronchiseptica and both toxigenic and nontoxigenic P. multocida that may greatly simplify investigations of swine PAR and bronchopneumonia.