Aims:The detection of microbial nucleic acid for the diagnosis of infection is dependent on the successful separation of nucleic acid from clinical material. DNA isolation by boiling method for detection of brucella is simple, reproducing, fast and effective. The aim of this study was to optimize PCR with three different primer pairs for detection of brucellosis in serum, using boiling for DNA isolation. Materials and Methods: In this study the serums was used that were taken from human and animal in the acute phase of brucellosis. Three primer pairs B4-B5, F4-R2 and JPF-JPR were used which are able to detect all species of Brucella. DNAs were separated by boiling. To eliminate or reduce the effects of inhibitors of the PCR, The resulting DNA was diluted with ratios of 1/100, 1/200, 1/300 and 1/500 and with each three pairs of primers, PCR was performed on the all dilutions. With the each PCR a positive control and a negative control was set up. Results: In using of each three primer pairs, dilution of 1 (DNA yielded of boiling) was not detectable. The best dilutions was 1/100 and 1/200 for B4-B5 primer, 1/100, 1/200, 1/300 and 1/500 for F4-R2 primer and 1/200 for JPF-JPR primer. In total, dilution of 1/200 of DNA, for each three primer pairs was appropriate. Conclusions: In PCR tests for detection of Brucella, DNA isolation by boiling method can be used that with using this method for DNA isolation, costs associated to PCR tests would be reduced to a considerable amount.

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