IDENTIFICATION OF MYCOBACTERIUM SPECIES BY USING AMPLIFIED RIBOSOMAL DNA RESTRICTION ANALYSIS

A rapid procedure for the identification of cultured Mycobacterium iso lates, based on the combination of enzymatic amplification and restric tion analysis, is described. The 16S rRNA genes (rDNA) of 99 strains b elonging to 18 different species of the genus Mycobacterium were enzym atically amplified. Amplified rDNA restriction analysis with the enzym es CfoI, MboI, and RsaI was carried out. The combination of the amplif ied rDNA restriction analysis patterns obtained after restriction with CfoI and MboI enabled differentiation between Mycobacterium asiaticum (number of strains = 4), M. avium (n = 22), M. chelonae (n = 5), M. f lavescens (n = 1), M. fortuitum (n = 6), M. gordonae (n = 6), M. intra cellulare (n = 13), M. marinum (n = 7), M. nonchromogenicum (n = 1), M . simiae (n = 5), M. terrae (n = 5), the M. tuberculosis complex (n = 11), and 2 of 4 strains of M. xenopi. Further restriction with RsaI wa s necessary to differentiate between the species M. kansasii (n = 5), M. scrofulaceum (n = 4), and the 2 other M. xenopi strains. The M. avi um-M. intracellulare complex was characterized by a specific MboI patt ern, and M. avium and M. intracellulare strains could further be diffe rentiated by restriction with CfoI. The whole procedure, including sam ple preparation prior to the polymerase chain reaction, can be carried out within 8 h, starting from a pure culture.