Background and Aim: In most studies the molecular detection of Brucella genus used primers B4-B5 that amplify part of the 31-kDa Brucella abortus antigenic protein gene and can detect all species of Brucella. Due to the low levels of bacteria and presence of inhibitors such as antibodies and proteins in serum, detection of Brucella in serum by PCR samples, is often difficult. Therefore division of a low-cost approach with accurate results in molecular diagnostics for Brucella is of great importance. The aim of this study was to increase the sensitivity and specificity of primers B4-B5 and optimize the boiling methods in DNA extraction from serum for the diagnosis of brucellosis. Methods: In this study, 38 human and 30 animals’ sera were examined. All the samples were seropositive, and all were taken in the acute stage of the brucellosis disease. The specimens were tested by Wright, 2ME and Coombs Wright Test (in human samples) and antibody titer of each was determined. Their DNA was extract by the boiling method. By using BLAST software, variations on primers were performed to optimize them. Dilution of the extracted DNA was performed to reduce the effects of PCR inhibitors. The best dilution for PCR was determined and PCR was performed on all samples. For positive control, DNA extracted of the Brucella bacterium and for negative control, distilled water instead of DNA was used in the experiments. Results: Results: This test was able to detect Brucella in serum in Wright’s antibody titers 1/40 and higher, 2ME titers 1/40 or higher and Coombs Wright titers 1/20 and higher (human samples). From 68 positive sera, 54 samples were positive by PCR and the sensitivity of this test was 79.4%. Best dilutions of DNA extracted from the serum 1/200 were determined. Conclusion: Modified primers B4-B5 have a sensitivity and specificity higher than the same primers in other reports. The modified primer can be used in future study to detect Brucella genus in clinical samples and as well as serum samples.

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