Abstract

Polymerase chain reaction (PCR) mediated direct DNA sequencing was evaluated for rapid detection of Rifampicin resistance (RMP^r) of Mycobacterium tuberculosis. After amplification of the rpoB gene, the product was sequenced using ABI 310 Genetic Analyzer and the rifampicin resistance in M. tuberculosis were assessed using the sequence pattern. Among the 100 isolates, 83 isolates were rifampicin sensitive, exhibited a wild-type pattern on PCR mediated direct sequencing, and remaining 17 isolates were resistant. All the RMP^r isolates were multidrug resistant, that is, resistant to at least Rifampicin and isoniazid. The mutation rate at the most effected codon 526 in the 81 bp rifampin resistance-determining region (RRDR) of rpoB gene was determined to be 35.29% in isolates from South India. In addition, the mutation rate at the most affected codon 451 was in the out side of the 81bp RRDR region ofrpoB gene. A synonym mutation was detected (Thr→Thr) at codon 526 in one isolate, which was inside the active site (codons 507-533) of RMP binding in the rpoB gene region. The sequencing analysis for genotypic evaluation of Rifampicin resistance in highly sensitive assay provides a practical alternative to in vitro testing in M. tuberculosis.

Key words: Rifampin resistance, Mycobacterium tuberculosis, rpoB gene, sequencing, mutation.

Abbreviation

Abbreviations: RMP, Rifampin; RMP^r, resistance to rifampin; rpoB, ribonucleic acid polymerase; PCR, polymerase chain reaction; TB, tuberculosis; RRDR, rifampin resistance-determining region; SDS, sodium dodecyl sulfate; CTAB, cetyl trimethylammonium bromide; BLAST, basic local alignment search tool; NCBI,National Center for Biotechnology Information; RMP^s, rifampicin sensitive; MIC,minimum inhibitory concentration; LJ, Lowenstein-Jensen.