Rapid, sensitive and specific detection of Brucella canis by real-time PCR assay using HybProbe

We designed and evaluated a specific HybProbe set for identification of Brucella canis, which is based on a specific single nucleotide polymorphism (SNP) of the omp25 gene. Real-time PCR assay using this probe specifically identified B. canis reference and field strains, however, the other Brucella species and general bacteria strains were not amplified. This real-time PCR assay can re-confirm specific amplification in melting curve analysis. The average melting temperature values for generation of B. canis strains was 67℃, however, that of the other Brucella species was 62℃, due to a single base-pair mismatch at the binding site of the probe. The detection limit appeared to be 30 fg of DNA and 100 CFU by serial dilution of whole blood inoculated with B. canis. In addition, use of the buffy coat resulted in approximately 50 times higher sensitivity than whole blood directly. This technique is widely applicable for use in diagnostic laboratories and is readily discriminative with regard to small amounts of biomass.