Development of a specific DNA probe and PCR for the detection of Mycoplasma bovis

Mycoplasma bovis is responsible for several production diseases in cattle, including mastitis, arthritis, pneumonia, abortion and infertility. Current methodologies for detecting and identifying M. bovis are time consuming and difficult. Tests which rely on antigen or antibody detection have poor sensitivity and specificity. In this paper associated protocols for the development of a hybridization probe and PCR are described. A genomic library (SauIIIA digested) was prepared from M. bovis DNA (Colindale Reference Strain: NC10131:02) and cloned into pUC19. Colony hybridization, using a probe preparation made from purified M. bovis DNA, was used to identify colonies of interest. M. bovis DNA fragments were retrieved from recombinant plasmids by digestion with EcoRI and HindIII. This DNA was used to prepare randomly primed probes for dot blot hybridization analysis with immobilized DNA from M. bovis (two strains), M. dispar, M. agalactiae, M. bovigenitalium (two strains), M. ovipneumoniae, a Group 7 strain, M. arginini and bacteria belonging to different genera. Four probes were found to hybridize only with M. bovis and M. ovipneumoniae DNA, whereas one probe reacted with genomic DNA from only one of the two M. bovis strains. The level of sensitivity of the dot blot hybridization assay was 200 CFU (colony forming units)/mL. To enhance the sensitivity further, an M. bovis-specific PCR assay was developed. The primers were designed using sequences obtained from the probe DNA which discriminated M. bovis from all other Mycoplasma DNA tested. The minimum amount of target DNA that could be detected by the PCR assay was that isolated from 10-20 CFU/mL. The PCR assay was therefore 10 times more sensitive than dot blot hybridization.