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

Summary Background 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. Methods 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. Results 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. Conclusions 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.