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

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.