CHARACTERIZATION OF RPO B GENE FOR DETECTION OF RIFAMPICIN DRUG RESISTANCE BY SSCP AND SEQUENCE ANALYSIS

Abstract

Purpose: Because of the emergence of multidrug-resistant tuberculosis in recent times, the rapid detection of resistance to the first-line anti-tuberculosis drug rifampicin was felt worldwide. Accordingly, this study was conducted to evaluate the diagnostic potential of polymerase chain reaction–single strand conformation polymorphism (PCR–SSCP) for checking its utility as a rapid screening test for determination of rifampicin drug resistance. Materials and Methods: A total of 34 isolates of Mycobacterium tuberculosis (M. tuberculosis) (22 rifampicin resistant, 11 rifampicin sensitive and one control H37Rv) strains were analysed by PCR–SSCP and DNA sequencing within the 157-bp region of the rpo B gene (Ala500–Val550). Results: Rifampicin resistance was detected successfully by PCR–SSCP in 20/22(90.90%) of rifampicin-resistant strains showing a total of nine different mutations in seven codon positions: codon 513 (CAA→CCA), 516 (GAC→GTC), 507 (GGC→GAC), 526 (CAC→GAC, TAC), 531 (TCG→TTG, TGG), 522 (TCG→TGG) and 533 (GTG→CCG). Two rifampicin-resistant strains showed an identical PCR–SSCP pattern with the wild type H37Rv; 77.27% rifampicin-resistant strains showed a single point mutation and 9.09% had no mutation. Three rifampicin-resistant strains showed characteristic double mutations at codon positions 526 and 531. Sensitivity and specificity were calculated as 90.90% and 100%. Conclusions: Rifampicin-resistant genotypes were mainly found in codon positions 516, 526 and 531. PCR–SSCP seems to be an efficacious method of predicting rifampicin resistance and substantially reduces the time required for susceptibility testing from 4 to 6 weeks to a few weeks.