Evaluation of Different Primers for Detection of Brucella in Human and Animal Serum Samples by Using PCR Method

Background: The Polymerase Chain Reaction (PC) method-can overcome-the limitations of-Conventional methodology. The aim of this study is to evaluate three primer pairs broadly used B4/B5, F4/R2 and JPF/JPR, for detection of Brucella by PCR, in human and animal serum samples and to determine analytic sensitivity of primers. Methods: Total of 68 serum samples were collected during the acute phase of brucellosis. 10-fold serial-dilutions were prepared from bacterial suspension and serum suspension using Brucella abortus S19. DNA was isolated using boiling. The best dilution from DNA wait determined for PCR with three-primer pairs. PCR was performed using primer pairs on all bacterial dilutions, serum dilutions, 1/200 dilutions and serum samples Comparison of sensitivity between three primer pairs was performed with statistical analysis. Results: The best DNA dilution Was 1/200: From 68 serum samples, 54 cases (79.41%), 44 cases (64.70%) and 35 eases (51.47%), were positive by PCR with B4/B5, F4/R2 and JPF/JPR respectively. B4/B5, F4/R2 and JPF/JPR were able to identify 9 x 10(2), 9 and 9 x 10(5) bacteria in 1-ml of bacterial suspension and 9 x 10(4), 9 x 10(5) and 9 x 10(7) bacteria in 1 ml of dilution 1/200 of Serum dilutions respectively The differences between-primers by statistical analysis were significant for human, animal and total samples. Conclusion: No band was observed in dilutions one of DNA isolated from serum. Therefore, to decrease the effects-of inhibitors, DNA was diluted, When DNA isolation is boiling, F4/R2 and B4/B5 have the greatest sensitivity for purified bacteria and serum in the detection of Brucella respectively. DNA isolation by boiling can decrease the PCR costs.