1Department of Veterinary Microbiology, CCS Haryana Agricultural University, Hisar-125 004.
2College Central Laboratory, College of Veterinary Sciences, CCS Haryana Agricultural University, Hisar, Haryana.
*E-mail: vetyccl@hau.ernet.in
Polymerase chain reaction (PCR) for the diagnosis of brucellosis in sheep was standardized using culture of Brucella melitensis biovar 1. Primers used were directed against the 31kDa Brucella cell surface protein (BCSP-31) gene which yields a product of 223bp. Standardization was done to optimize the Taq DNA polymerase, MgCl2, annealing temperature and template requirement for amplification. It was found that 1.25U of Taq, 1.5mM of MgCl2, 56°C annealing temperature and 2μl of template DNA were ample for amplification of B. melitensis DNA. Detection limit of the assay was found to be as low as 167.6fg of DNA from B. melitensis cells. Being rapid and specific this PCR assay has the potential to be used as a routine diagnostic assay for diagnosing brucellosis in sheep.
Diagnosis, Brucellosis, Sheep, PCR, BCSP-31
