title: 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.affiliation: Department of Microbiology, Intermediate Reference Laboratory, State TB Training and Demonstration Centre, Government Hospital for Chest Diseases, Gorimedu, Puducherry 605006, India; Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Sholinganallur, Chennai 600119, Tamil Nadu, India.
authors.forename: Ravibalan
authors.initials: R
authors.lastname: Thirumurugan
authors.affiliation: Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Sholinganallur, Chennai 600119, Tamil Nadu, India.
authors.forename: Maruthai
authors.initials: M
authors.lastname: Kathirvel
authors.affiliation: Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Sholinganallur, Chennai 600119, Tamil Nadu, India.
authors.forename: Kommoju
authors.initials: K
authors.lastname: Vallayyachari
authors.affiliation: Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Sholinganallur, Chennai 600119, Tamil Nadu, India.
authors.forename: Kesavan
authors.initials: K
authors.lastname: Surendar
authors.affiliation: Department of Microbiology, Intermediate Reference Laboratory, State TB Training and Demonstration Centre, Government Hospital for Chest Diseases, Gorimedu, Puducherry 605006, India.
authors.forename: Antony V
authors.initials: AV
authors.lastname: Samrot
authors.affiliation: Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Sholinganallur, Chennai 600119, Tamil Nadu, India. Electronic address: stdcirlpdy@gmail.com.
authors.forename: Muthuraj
authors.initials: M
authors.lastname: Muthaiah
mesh_terms: D000483:Alleles; D000995:Antitubercular Agents / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D014644:Genetic Variation; D006801:Humans; D007194:India; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D012293:Rifampin / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin
keywords: Drug resistance; Multiple allele-specific PCR; Mycobacterium tuberculosis; Rifampicin; rpoB gene