Species-specific identification of Mycobacterium bovis by PCR

The Random Amplified Polymorphic DNA (RAPD) technique was used in the identification of a species-specific fragment of Mycobacterium bovis. A fragment of approximately 500 bp was amplified from the genome of 15 different M. bovis strains, including M. bovis BCG Pasteur, but was shown to be absent in 26 different mycobacteria and 20 different clinical isolates of Mycobacterium tuberculosis. When the fragment was used as a probe in a Southern blot analysis, several radioactive bands common to M. tuberculosis and M. bovis were observed. However, this fragment hybridized specifically to a 2900 bp EcoRI fragment in the M. bovis genome, but failed to hybridize in either M. tuberculosis or M. avium chromosomal DNA. Based on a partial nucleotide sequence of the 500 bp fragment, two oligonucleotide primers were designed and a PCR assay was developed. Using purified mycobacterial DNA samples, only M. bovis and M. bovis BCG rendered a unique amplification band. This PCR assay is able to detect down to 10 fg purified M. bovis DNA, which corresponds roughly to two bacilli. The assay is also useful for identifying the bacilli directly from uncultured biological samples, such as milk.