Development of a PCR assay for typing and subtyping of Brucella species

In the course of this study, examinations were carried out to develop a PCR-based test which allows discrimination of Brucella species and biovars not targeted by the currently established gel-based PCR assays. Appropriate primers were designed based on specific deletions and insertions in the different Brucella genomes as determined by RAPD-PCR and whole-genome comparisons. After testing the specificity of the primers with a set of 22 Brucella reference strains of all species and biovars, they were used to supplement the existing PCR assays resulting in a 19-primer multiplex PCR. In addition to the commonly used PCR assays, the developed assay specifically identified B. neotomae, B. pinnipedialis, B. ceti, and B. microti. Furthermore, it differentiated B. abortus biovars 1, 2, 4 from biovars 3, 5, 6, 9, as well as between B. suis biovar 1, biovars 3, 4, and biovars 2 and 5. When tested in the multiplex assay, all Brucella type and reference strains and the majority of 118 field strains examined could be accurately identified by their respective banding patterns according to their previous typing. B. canis strains were subdivided into 2 groups, one exhibiting a unique pattern and the other one a banding pattern shared with B. suis biovars 3 and 4. Species of the closely related genus Ochrobactrum and several other clinically relevant bacteria showed no amplification product. Hence, the developed PCR assay is useful for rapid identification of Brucella at the species and at the biovar level.