J Korean Soc Microbiol.  1999 Dec;34(6):561-571.

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

• Kook YH

Abstract

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.

Keyword

rpoB DNA; sequence analysis; PCR-RFLP

MeSH Terms

Base Sequence
Classification
Complement System Proteins
Diagnosis
DNA
DNA-Directed RNA Polymerases
Mycobacterium tuberculosis
Polymerase Chain Reaction*
Polymorphism, Restriction Fragment Length*
Residence Characteristics
Sequence Analysis*
Complement System Proteins
DNA
DNA-Directed RNA Polymerases