title: Development and evaluation of a novel multiple-primer PCR amplification refractory mutation system for the rapid detection of mutations conferring rifampicin resistance in codon 425 of the rpoB gene of Mycobacterium leprae.
abstract: Rifampicin-resistant Mycobacterium leprae is regularly reported and drug resistance is a major threat for the elimination of leprosy. There is an urgent need for a simple method that can detect rifampicin resistance in clinical isolates. This study developed a multiple-primer PCR amplification refractory mutation system, a simple, reliable and economical method for clinical specimens that allowed the rapid detection of mutations in the nucleotides of the codon for Ser425 of the M. leprae rpoB gene, mutation of which to Leu, Met or Phe is associated with rifampicin resistance. The approach involved a multiple-primer PCR in which both mutant-specific and normal sets of primers were included in the reaction. The mutant-specific primer was complementary to the corresponding sequence of the wild-type gene except for one additional deliberate mismatch at the fourth nucleotide from the 3'-OH terminus. A single mismatch has little influence on the yield of PCR products, but if there are two mismatches as a result of mutation at the position being tested, the mutant-specific primer will not function in PCR under appropriate conditions, leading to no yield of PCR product from the mutant allele. The assay was evaluated successfully using a panel of plasmids and M. leprae reference strains carrying the wild-type or known rpoB mutations. The assay was subsequently applied to M. leprae DNA extracts from skin biopsies taken from patients. In all biopsy samples, the wild-type allele was detected for Ser425. The PCR results correlated with rifampicin susceptibility, as also measured by the traditional in vivo mouse footpad technique.
authors.affiliation: Mycobacterial Research Laboratory, Anandaban Hospital, Kathmandu, Nepal.
authors.forename: Bishwa Raj
authors.initials: BR
authors.lastname: Sapkota
authors.affiliation: Mycobacterial Research Laboratory, Anandaban Hospital, Kathmandu, Nepal.
authors.forename: Chaman
authors.initials: C
authors.lastname: Ranjit
authors.affiliation: Mycobacterial Research Laboratory, Anandaban Hospital, Kathmandu, Nepal.
authors.forename: Kapil Dev
authors.initials: KD
authors.lastname: Neupane
authors.affiliation: Mycobacterial Research Laboratory, Anandaban Hospital, Kathmandu, Nepal.
authors.forename: Murdo
authors.initials: M
authors.lastname: Macdonald
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D006801:Humans; D007918:Leprosy / Q000382:microbiology; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009154:Mutation; D009166:Mycobacterium leprae / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013223:Statistics as Topic
chemical_list: D000900:Anti-Bacterial Agents; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin