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

Abstract

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.

• Authors: Ravibalan Thirumurugan, Maruthai Kathirvel, Kommoju Vallayyachari, Kesavan Surendar, Antony V Samrot, Muthuraj Muthaiah
• Journal: Journal of infection and public health (J Infect Public Health) 2015 Nov-Dec Vol. 8 Issue 6 Pg. 619-25 ISSN: 1876-035X [Electronic] England
• PMID: 26117709 (Publication Type: Journal Article)
• Copyright: Copyright © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Chemical References

• Antitubercular Agents
• DNA-Directed RNA Polymerases
• RNA polymerase beta subunit
• Rifampin

Topics

• Alleles
• Antitubercular Agents (pharmacology)
• DNA-Directed RNA Polymerases (genetics)
• Drug Resistance, Bacterial
• Genetic Variation
• Humans
• India
• Microbial Sensitivity Tests (methods)
• Molecular Diagnostic Techniques (methods)
• Multiplex Polymerase Chain Reaction (methods)
• Mutation
• Mycobacterium tuberculosis (enzymology, genetics, isolation & purification)
• Rifampin (pharmacology)
• Sequence Analysis, DNA
• Tuberculosis, Multidrug-Resistant (diagnosis, microbiology)