Detection of mycoplasma bovis using in vitro deoxyribonucleic acid amplification

Abstract

Detection of Mycoplasma bovis by traditional culture methods is rather time-consuming and is often hampered by bacterial contamination. The development of a rapid and specific alternative is, therefore, an important prerequisite in improving the diagnosis of bovine diseases caused by this agent. The authors have successfully used nucleic acid probes containing genomic restriction fragments of M. bovis cloned into the plasmid vector pUC19 for species-specific detection by dot blot hybridisation of small quantities of M. bovis deoxyribonucleic acid (DNA). The problem of direct M. bovis detection from contaminated milk could not be solved using this procedure. Therefore, further research was conducted using in vitro DNA amplification by polymerase chain reaction (PCR). Species-specific nucleic acid probes were sequenced and suitable PCR primers selected. Using the PCR procedure, ten colony-forming units (CFU) were detected from broth cultures and, after DNA isolation, the equivalent of 1 CFU was detected. Direct detection of M. bovis from biological samples proved extremely difficult due to protein interference. It was shown, however, that direct PCR detection from milk is possible after effective protein removal by combined extraction and protease digestion.