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Brucellosis has been one of the most important reproductive diseases among different livestock species as well as animal handlers and is considered a reemerging infectious disease in many areas of the world. A total of 353 clinical samples of cattle (207) and buffalo (146) were collected for molecular detection of Brucella. Out of 353 clinical samples, 6 samples (vaginal discharge-2, placenta-2, foetal stomach content-1, fetal lung-1) positive for Brucella organism by genus specific PCR based on bscp31 (B4/B5 primer, 223bp). All six genus specific positive sample also positive for B. abortus +IS711 (498bp) species specific PCR. All the samples which were detected positive in conventional PCR also detected positive for Brucella in SYBR green and TaqMan probe based real time PCR.