J Korean Soc Microbiol.  1997 Aug;32(4):389-399.

Amino Acid Substitution Caused by Mutated rpoB Gene of Rifampin - Resistant Mycobacterium tuberculosis

• Cho DT
• Lee YC
• Bang SH
• Kim JM

Abstract

Mobility shifts in non-denatured gel electrophoresis of PCR-amplified Rif' region in each of fifteen different mutants of M. tuberculosis were discerned by single strand conformation polymorphism (SSCP) analysis. The findings of mobility differences between rifampin-resistant and susceptible strains showed an excellent agreement with data obtained by traditional susceptibility test. SSCP-PCR seemed to replace the cultivation method of susceptibility test that was known to be time-consuming, labor wasting, and skeptical in quality control. After screening of rpoB gene mutation by SSCP-PCR, detection of specific sequence changes in the region of rpoB gene was attempted through the procedures of PCR-amplification, cloning of PCR-products using pGEM-T vector and DNA thermocycling sequencing. Fifteen different types of mutations were identified among fifty strains of rifampin-resistant strains while five rifampin-susceptible control strains showed no sequence changes of rpoB gene as well as reference strain H37rv. Most mutation appeared to be a point mutation due to substitution or deletion except seven mutants showing somewhat complex mutation. Each of mut#ated loci inclined to clustering within a region of eighteen amino acids involving eight codons. The most common mutation of Ser425 shared among twenty-nine mutants and followed by eleven mutants of His420. Several mutants alleles identified in this study appeared to be dissimilar to those of previous reports.

MeSH Terms

Alleles
Amino Acid Substitution*
Amino Acids
Clone Cells
Cloning, Organism
Codon
DNA
Electrophoresis
Mass Screening
Mycobacterium tuberculosis*
Mycobacterium*
Point Mutation
Quality Control
Rifampin*
Tuberculosis
Amino Acids
Codon
DNA
Rifampin