PCR method is rapid, sensitive, highly specific and low cost for detection of pathogen and can overcome the problems of conventional detection methods. Serum is the preferred specimen for diagnosis of brucellosis due to severe decrease in PCR inhibitors. The purpose of this study was to evaluate three primer pairs B4-B5, F4-R2 and JPF-JPR broadly used, for detection of Brucella genus by PCR in human and animal serum samples and to determine the analytic sensitivity of primers. 38 human and 30 sheep serum samples were collected in acute phase of brucellosis. A cell suspension of Brucella abortus S19 was prepared equivalent with 3 McFarland. 10-fold serial dilutions from cell suspension and serum dilutions [dilution 1+ negative serum] were prepared. Serum dilutions were incubated for 24h. DNA was isolated using boiling method. DNA isolated from serum was diluted to ratios 1/100, 1/200, 1/300 and 1/500 and the best dilution for PCR with three primers was determined. DNA serum dilutions and DNA serum samples were diluted 1/200 and PCR was performed using three primers on all bacterial dilutions, serum dilutions, 1/200 dilutions and serum samples. Comparison of sensitivity between three primers was performed with statistical analysis. A260/A280 of nine DNA by Nanodrop was <1. The best dilution of serum DNA was 1/200 for each three primer. From 68 serum samples, 54 cases (79/41%), 44 cases (64/70%) and 35 cases (51/47%), from 38 human samples, 33 cases (86/84%), 27 cases (71/05%) and 22 cases (57/89 %) and from 30 animal samples, 21 cases (70%), 17 cases (56/67 %) and 13 cases (43/33%) were positive by PCR with B4-B5, F4-R2 and JPF-JPR respectively. B4-B5, F4-R2 and JPF-JPR primers were able to identify 9×102, 9 and 9×105 bacteria in 1ml of bacterial suspension; 9×108, 9×108 and 9×108 bacteria in 1 ml of serum suspension and 9×104, 9×105 and 9×107 bacteria in 1ml of dilution 1/200 of Serum dilutions respectively. The results of analysis showed significant differences between primer pairs for human serum samples, animal serum samples and total samples together. The results of Nanodrop and smear formation on the gel showed the presence of inhibitors. No band was observed in dilutions 1 of serum DNA, but in dilutions 1/100 to 1/500 a band was observed, so the best work to eliminate or reduce the effects of these inhibitors was DNA dilution. B4-B5 could detect the highest Brucella cases in serum samples and all the dilution 1/200 of serum dilutions. Also F4-R2 could detect all the serial dilutions from pure bacteria. So when DNA isolation is boiling method, F4-R2 in purified bacteria and B4-B5 in serum, have the greatest sensitivity for detection of Brucella. Therefore DNA isolation by boiling can reduce the costs associated to PCR.

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