Introduction: The PCR-based methods overcome the limitations of conventional methods for detection of brucellosis (culture and serology). Multiplex-PCR is an ideal method for the identification of human brucellosis from different clinical. The most common Primer pairs for identification of genus Brucella include B4-B5 and F4-R2. The several studies has been performed on the identification of Brucella at species level and partly at biovar level using multiplex-PCR, but no studies with this technique has performed at genus level. The purpose of this study was to develop and optimize a suitable method for detection of the genus Brucella by multiplex-PCR. Material and Methods: DNA was isolated from bacteria pure culture by using the boiling method. Two primer pairs B4-B5 and F4-R2 were used to identify the genus Brucella. Primers were assessed using bioinformatics softwares BLAST, Allele ID 7, Base stacking TM and Primer premier 5. Sequences B4 and B5 reported in reference paper were changed so that annealing temperature of primer pair B4-B5 fits with F4-R2 annealing. Finally multiplex-PCR was performed on the DNA. For negative control, sterile distilled water was used instead of the DNA. Result: In theory view, the negative amplification factors for primer B4-B5 were reduced and also B4-B5 was more consistent with F4-R2 primers. In practical view, this method was able to detect Brucella and bands of both primers were visible using gel electrophoresis. Modified B4- B5 primer with these changes was amplified a 222 bp fragment in the same gene. Conclusion: Using this method can overcome the problems related to molecular detection of brucellosis; also detection is done with more sensitivity. Routine diagnosis of brucellosis by PCR assay yet has not been standardization. Therefore, this method can be used to diagnose brucellosis in clinical laboratories routinely and can be alternative substitution for risky culture method and nonspecific serological tests.

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