Indian Journal of Animal Research

A SYBR Green real time PCR assay amplifying F57 gene of Mycobacterium avium subsp. paratuberculosis (MAP) was developed to evaluate the protective efficacy of the inactivated vaccine to prevent the colonization of MAP in the tissues of BALB/c mice challenge model. Bacterial burden in the liver, spleen and intestine of vaccinated and sham immunized control mice, after intra peritoneal challenge with MAP were quantified by real time PCR assay. A 195 bp fragment of MAP F57 sequence was cloned into TA cloning vector and the resultant recombinant plasmid was used to generate a series of quantification standards with copy number ranging from 10⁹ to 1 copy. This absolute quantification technique is rapid and able to detect as few as 10 MAP organisms per gram of tissue and the assay has the efficiency of 0.982.