IDENTIFICATION OF MYCOBACTERIA TO THE SPECIES LEVEL BY AUTOMATED RESTRICTION ENZYME FRAGMENT LENGTH POLYMORPHISM ANALYSIS

An automated method for the restriction fragment length polymorphism ( RFLP) analysis for the differentiation of mycobacteria to the species level is described, After polymerase chain reaction (PCR) amplificatio n of a sequence of the gene encoding the 65-kDa surface antigen common to all mycobacteria the product was investigated by RFLP analysis. Fo r accurate determination of fragment sizes the asymmetrically fluoresc ein-labelled PCR product was partially digested with restriction site enzymes BstEII and HaeIII. The fragments obtained were analysed electr ophoretically using an automated laser fluorescence DNA sequencer. Det ermination of fragment sizes revealed a deviation of +/-1 base pair (b p; 0.6%) when compared to expected sizes. The validity of this approac h was confirmed by analysing mycobacterial DNA obtained from pure cult ures of Mycobacterium (M.) tuberculosis and alcohol-fixed smears as we ll as paraffin embedded sputa of patients with culture-proven tubercul osis. Additionally a diagnostic algorithm was established by investiga tion of cultured M. bovis, M. bovis bacille Calmette-Guerin, M. avium, M. intracellulare and M. fortuitum. The method allows the identificat ion of restriction enzyme sites which are only 40 bp apart. Partial re striction enzyme digestion of asymmetrically fluorescence-labelled PCR products will presumably lead to the discovery of new restriction enz yme sites.