J Med Microbiol International Journal of Systematic and Evolutionary Microbiology
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J Med Microbiol 57 (2008), 179-184; DOI: 10.1099/jmm.0.47534-0
© 2008 Society for General Microbiology
ISSN 1473-5644

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

Bishwa Raj Sapkota, Chaman Ranjit, Kapil Dev Neupane and Murdo Macdonald

Mycobacterial Research Laboratory, Anandaban Hospital, Kathmandu, Nepal

Correspondence
Bishwa Raj Sapkota
immuno{at}tlmnepal.org

Received 25 July 2007
Accepted 31 October 2007

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.

Abbreviations: AFB, acid-fast bacilli; CFP, control forward primer; CRP, common reverse primer; MARS, multiple-primer PCR amplification refractory mutation system; MB, multibacillary; MDT, multi-drug therapy; MFP, mouse footpad.




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M. Matsuoka, K. S. Aye, K. Kyaw, E. V. Tan, M. V. Balagon, P. Saunderson, R. Gelber, M. Makino, C. Nakajima, and Y. Suzuki
A novel method for simple detection of mutations conferring drug resistance in Mycobacterium leprae, based on a DNA microarray, and its applicability in developing countries
J. Med. Microbiol., October 1, 2008; 57(10): 1213 - 1219.
[Abstract] [Full Text] [PDF]


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