Identification of Mycobacteria by Comparative Sequence Analysis and PCR - Restriction Fragment Length Polymorphism Analysis

Diagnosis of mycobacterial infection is dependent upon the isolation and identification of causative agents. The procedures involved are time consuming and technically demanding. To improve the laborious identification process mycobacterial systematics supported by gene analysis is feasible, being particularly useful for slowly growing or uncultivable mycobacteria. To complement genetic analysis for the differentiation and identification of mycobacterial species, an alternative marker gene, rpoB encoding the B subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 52 reference strains of mycobacteria including Mycobacterium tuberculosis H37Rv (ATCC 27294) and clinical isolates by the PCR. The nucleotide sequences were directly determined (306 bp) and aligned using the multiple alignment algorithm in the MegAlign package (DNASTAR) and MEGA program. A phylogenetic tree was constructed with a neighborhood joining method. Comparative sequence analysis of rpoB DNA provided the basis for species differentiation. By being grouped into species-specific clusters with low sequence divergence among strains belonging to same species, all the clinical isolates could be easily identified. Furthermore RFLP analysis enabled rapid identification of clinical isolates.

Identification of Mycobacterium avium complex ( MAC ) clinical Strains to a Species Level by Sequencing and PCR - SSCP Analysis of rpoB DNA

A recent study showed that comparative sequence analysis of rpoB DNAs could reveal natural relationships in genus Mycobacterium [J Clin Microbiol. 37 (6). 1999]. rpoB DNAs showed interspecies variation and intraspecies conservation, Based on these data, we developed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) protocols which enable species differentiation in genus Mycabacterium. When this assay was applied to 24 clinical isolates identified as M. avium complex (MAC) by biochemical test, these were successfully differentiated into M. avium and M. intracellulare. These results were concordant with those obtained by 16s rDNA analysis. It is the first report that PCR-SSCP analysis of rpoB DNA could be used for species differentiation of MAC strains.