Characterization of RPO B gene for detection of rifampicin drug resistance by SSCP and sequence analysis.
OBJECTIVE
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.


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 (Ala 500-Val 550).


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.
Negi
S S
SS
National Institute of Communicable Diseases, Ministry of Health and FW, Government of India, 22-Shamnath Marg, Delhi, India.
Singh
U
U
Gupta
S
S
Khare
S
S
Rai
A
A
Lal
S
S
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D017354:Point Mutation; D018807:Polymorphism, Single-Stranded Conformational; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis
D000995:Antitubercular Agents; D001426:Bacterial Proteins; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin