NUCLEIC-ACID SEQUENCE-BASED AMPLIFICATION (NASBA) FOR THE IDENTIFICATION OF MYCOBACTERIA

Nucleic acid sequence-based amplification (NASBA), an isothermal ampli fication technique for nucleic acids (NA), was investigated for the sp ecies-specific identification of mycobacteria. A set of primers was se lected from a highly conserved region of the 16S rRNA sequence of myco bacteria sandwiching a variable sequence to perform amplification of m ycobacterial RNA. Species-specific probes for the M. tuberculosis comp lex, M. avium-paratuberculosis, M. intracellulare and M. leprae were h ybridized in-solution with the amplified nucleic acids of 10 pathogeni c mycobacteria and 11 closely related bacteria, as well as with human- derived NA in an enzyme-linked gel assay (ELGA). Each probe was shown to hybridize specifically to the amplified single-stranded RNA of the corresponding species. Thirty-two clinical isolates of M. tuberculosis strains from different parts of the world were correctly identified b y NASBA using the M. tuberculosis-complex-specific probe. In combinati on with the ELGA, NASBA could identify mycobacteria rapidly, i.e. in l ess than 6 h. The relative simplicity and rapidity of this technique m akes it an attractive tool for species-specific identification of myco bacteria.