Evaluation of designed IS711 primers and universal primers of B4 and B5 for detection of Brucella spp. in clinical samples
Abstract Background: Brucellosis as a global concern is a zoonotic infectious disease which affects a large number of individuals in developing countries. Microbiological, serological and molecular approaches are useful for detection and identification of Brucella spp. A confirmed diagnosis requires isolation of Brucella from clinical specimens that is the most sensitive method in the acute and sub-acute phases of the diseases. On the other hand, molecular diagnostic techniques are more sensitive and more specific than serological techniques, especially in chronic localized cases because of antigenic cross-reactions or antibody titers lower than 160. Until now different Brucella specific sequences like BCSP 31, IS711 and 16SrRNA have been amplified for detection of Brucella spp. In this study, the sensitivity and specificity of The B4-B5 primers and IS711 designed primers were evaluated for detection of of Brucella Spp. in the clinical samples. Results : Amplification of extracted DNA from serum of 49 suspected patients were tested with two sets of specific primers. The BCSP31 amplicon was 223 bp and all the 49 (100%) serum specimens were positive by B4-B5 primers, including 4 cases with negative 2ME test result. The designed IS711 primers amplified the IS711 product with 448 bp length and 46 of 49 (93.87%) cases were positive. The sensitivity of the applied primers (B4-B5 and IS711) was evaluated by using the serial dilutions of extracted purified DNA molecules of B. melitensis and B. abortus . The B4-B5 primers can detect the least number of both B. melitensis and B. abortus , 0.1 CFU/reaction. However, the designed IS711 set is able to detect 10 CFU/reaction. The B4-B5 primer and IS711 designed primer recognized 100% (49/49) and 94% (46/49) of the cases, respectively. Conclusion: This study indicated that the sensitivity of B4-B5 primer is 100%, while the sensitivity of the designed primer of IS711 is 94%. The laboratory experiment revealed that designed IS711 set is 1×10 2 times more sensitive than sensitivity of the other experiments for detection of IS711 target sequence in the specimens.