Molecular analysis of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates by multiple allele specific polymerase chain reaction in Puducherry, South India

ABSTRACT

rpoB gene mutations in Mycobacterium tuberculosis [MTB] make the bacteria resistant to rifampicin. Thus, these mutations are surrogate markers for multi-drug resistance [MDR]. The objective of this study was to evaluate an allele-specific multiplex-polymerase chain reaction [MAS-PCR] assay to detect mutations at codons 516, 526 and 531 of the rpoB gene. In total, 127 M. tuberculosis clinical isolates were subjected to standard drug susceptibility tests. A MAS-PCR assay was then performed to detect mutations in the rpoB gene. Three different allele-specific PCR assays were performed [single-step MAS-PCR] and the amplified products were sequenced. Of the 127 isolates, 69 [54.3%] were multidrug resistant M. tuberculosis [MDR-TB], 21 [16.5%] were rifampicin mono-resistant and 37 [29.1%] were drug susceptible. The frequency of mutations at codons 531, 526 and 516 was 54.4%, 18.9% and 5.6%, respectively. A triple mutation was found in 4 [4.4%] isolates. Mutations in regions other than the 81-bp region were observed at codons 413 [11.1%], 511 [12.2%] and 521 [15.6%] of the rpoB gene. The simplicity and specificity of the MAS-PCR assay allows for easy implementation in clinical laboratories to detect rifampicin drug resistance in MDR-TB strains