Background and purpose: Brucellosis is a zoonotic disease. Given that serologic test has some specific characteristics such as difficult growth, problems associated with culture, and cross-reactions with otherbacteria, in this study multiplex real-time polymerase chain reaction (PCR) method was used to detect the most common species of Brucella spp.

Materials and methods: This experimental study was performed in Hamedan University of Medical Sciences, Hamedan, Iran, 2015, in two bioinformatical and analytical stages. A universal primer was designed to detect Brucella genus and two primers were prepared for B. melitensis and B. abortus, which are the most common species. The target genes for universal Brucella, B. melitensis, and B. abortus primers were ITS, BMEII-0466, and BruAB2-0168, respectively. In the analytical stage, specificity and sensitivity of the primers were evaluated.

Results: In this study, the universal primer of Brucella was detected in 83 bp at melt temperature of 82^∘C. Moreover, B. melitensis and B. abortus primers were specifically identified in 121 bp and 285 bp at melt tempretures of 84^∘C and 87^∘C, respectively. The sensitivity for the universal Brucella primer and B. abortus was 50 fg, while it was 100 fg for the B. melitensis primer.

Conclusion: The identified primers revealed high levels of sensitivity and specificity. Therefore, they can be considered along with culture in clinical trials.