Real-time PCR assay based glyceraldehydes 3-phosphate gene for identification of Brucella sp

Brucellosis is one of the most important zoonoses which affects both animals and humans and leads to serious economic and public health problems. The aim of this study was to design, optimize and evaluate real-time PCR assay for Brucella sp. detection by targeting gap gene and to compare to those of conventional PCR assays. A low variation in C_T values was observed for the gap gene target when the same quantity of DNA for 5 Brucella reference strains was used as template in the assays (C_T: 21 -23 with 500 pg of Brucella DNA). No amplification products were observed in real-time PCR whatever the target with any of the 50 non-Brucella organisms tested. In the analytical sensitivity of real-time PCR assay based gap gene of B. abortus biovar 1 RB51, DNA concentration of 5 fg was successfully amplified and the sensitivity of the gap-based TaqMan real-time PCR assay was identical and 10-100 times higher than the sensitivity of the three conventional PCR. In the clinical trial, 9 (16.3%) and 11 (21.2%) among 52 blood samples from cows confirmed with B. abortus infection by Rose Bengal Spot agglutination test were positive in culture of B. abortus and gap real-time PCR, respectively. In conclusion, the use of the gap-based TaqMan real-time PCR assay appears promising due to it high sensitivity for the simple, faster and specific detection of the Brucella sp.