Real time PCR assay for differentiation of Brucella abortus and Brucella melitensis

Brucellosis is one of the zoonotic diseases of major concern and can cause huge economic losses to livestock industry. Serological tests and bacterial isolation are considered as the gold standard assay for diagnosis of Brucella spp. but they are time-consuming, hazardous and lack specificity. To control and eradicate a disease, a confirmatory diagnostic method which is sensitive, quick and specific is the foremost requirement. Therefore in this study, we evaluated the performances of two newly designed TaqMan real-time PCR assays targeting the BruAB_0168 gene and BMEII0466 gene for Brucella abortus and Brucella melitensis (respectively). Both the assays were found to be highly specific in differentiation of respective species. Both the assays can detect as low as 0.02 fg of DNA and there was no detectable difference found in sensitivity of these two tests. R-2 value and efficiency of these tests ranged from 0.992 - 0.998 and 100-106%, respectively showing that these assays are highly efficient. Compared to conventional PCR assays these qPCR assays were 100 times higher sensitive. In conclusion, the present study showed that the developed real-time PCR assays are more sensitive, specific, have high reproducibility and repeatability and are faster than serological and conventional PCR methods for differentiation of Brucella abortus and Brucella melitensis.