Detection of Mycoplasma bovis with an improved pcr assay

A Mycoplasma bovis species-specific PCR assay has been developed with improvement of a previously described method (Ghadersohi et al., 1997). This test and its semi-nested version (Hayman and Hirst, 2003) did not function at all in our hands. A new reverse primer (Mbr2) was designed using previously published sequence data. For testing specificity, DNA was extracted from the most frequently occurring mycoplasma species and bacteria of bovine origin. The new PCR detected only Mycoplasma bovis. Moreover, no cross-reaction was observed with the genetically closest relative species, M. agalactiae. The target organism could be detected in a dose as low as 150 CFU ml(-1) in broth cultures using ethidium-bromide-stained agarose gels.