Use of Polymerase Chain Reaction for Detection of Mycobacteria.

The polymerase chain reaction (PCR) has been widely described as a possible test format for mycobacterial diseases. This thesis investigates the suitability of PCR for the detection of mycobacterial disease in several formats. Firstly how it may be simplified for use in developing countries, through the use of non-radioactive detection based on fluorescein or biotin labelling during PCR. Secondly the possible use of PCR in tuberculosis diagnostic laboratories as investigated in the collaborative study designed by Noordhoek et al. 1994, where Southern blotting and probing with digoxygenin labelled probe were investigated. Thirdly its possible use as an in-situ method for the detection of mycobacteria in tissue sections, this work demonstrated direct incorporation of digoxygenin into PCR products. The final part of this thesis describes the investigation of the use of PCR based on the gene coding for the M. bovis protein antigen MPB70 for the detection of M. bovis infection in cattle. The investigation of cross-reactivity of the PCR test with other mycobacteria indicated the presence of an hitherto undiscovered Mycobacterium kansasii MPB70 gene analog. This analog was partly sequenced and characterised. Single stranded conformation polymorphism (SSCP) analysis and sequence determination of the MPB70 analog in a range of M. kansasii strains showed significant variation in the DNA sequence and predicted amino acid sequence among different M. kansasii isolates