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

Journal of Medical Microbiology
Bishwa Raj SapkotaMurdo Macdonald

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 ...Continue Reading

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Citations

Jan 16, 2009·Journal of Medical Case Reports·Bishwa R SapkotaRam K Maharjan
Aug 4, 2012·PLoS Neglected Tropical Diseases·Emmanuelle CambauVincent Jarlier
Mar 29, 2011·Lung Cancer : Journal of the International Association for the Study of Lung Cancer·Jinyin ZhaoWeijun Chen
Mar 10, 2015·Indian Journal of Dermatology, Venereology and Leprology·Abu Hena Hasanoor RejaBasudev Bhattacharya
Apr 12, 2014·PLoS Neglected Tropical Diseases·Alejandra Nóbrega MartinezSinésio Talhari
Sep 25, 2016·Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases·E AndreE Cambau
Jun 9, 2017·Journal of Feline Medicine and Surgery·Carolyn R O'BrienJanet A Fyfe

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