Evaluation of different primers for detection of Brucella in human and animal serum samples by using PCR method.

Abstract

The Polymerase Chain Reaction (PCR) 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. 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 was 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. The best DNA dilution was 1/200. From 68 serum samples, 54 cases (79.41%), 44 cases (64.70%) and 35 cases (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 × 10(2), 9 and 9 × 10(5) bacteria in 1 ml of bacterial suspension and 9 × 10(4), 9 × 10(5) and 9 × 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. 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.