Objective  A duplex PCR assay for rapid detecting Pasteurella multocida serogroup F strain in rabbits was developed.

  Methods  Primer concentration and annealing temperature for the duplex PCR were optimized based upon two sets of specific primers targeting the conserved sequences of kmt1 of P. multocida and fcbD of P. multocida serogroup F strain. Specificity, sensitivity, and accuracy of the methodology were tested for its applicability.

  Results  The optimized conditions for the assay included 0.8 μmol·L⁻¹ on the primer concentration and 60 ℃ for the annealing temperature. The assay detection was specific for P. multocida serogroup A, D, and F strains and free of cross-reactions with Bordetella bronchiseptica, Klebsiella pneumonia, Escherichia coli, Staphylococcus aureus, and negative control. A highly sensitive detection limit up to 1×10³ copies·μL⁻¹ on genomic DNA of P. multocida serogroup F isolate was achieved. In the repeated intra- and inter-assays on 90 lung specimens from dead diseased rabbits, the assay variation coefficients were identical. By using 87 lung samples collected from known infection cases, the newly developed duplex PCR assay and reported multiplex PCR assay yield agreements of 97.70% and 94.25% with the known results, respectively. Moreover, the current duplex PCR determination also yielded an agreement of 93.10% on the measurements with the reported from a multiplex PCR assay.

  Conclusion  The newly established duplex PCR assay based on the specific primers targeting the conserved sequences of kmt1 of P. multocida as well as fcbD of P. multocida serogroup F strain was tested to be highly specific, sensitive, repeatable, and accurate in detecting the pathogenic P. multocida serogroup F strain in rabbits.