Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types
- 1 March 2012
- journal article
- research article
- Published by American Society for Microbiology in Journal of Clinical Microbiology
- Vol. 50 (3), 742-753
- https://doi.org/10.1128/jcm.05183-11
Abstract
Drug resistance surveillance and strain typing ofMycobacterium lepraeare necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR–high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) offolP1,rpoB, andgyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included threefolP1, tworpoB, and onegyrAmutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all thefolP1mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations.Keywords
This publication has 44 references indexed in Scilit:
- Transmission of Dapsone-Resistant Leprosy Detected by Molecular Epidemiological ApproachesAntimicrobial Agents and Chemotherapy, 2011
- Molecular Drug Susceptibility Testing and Genotyping of Mycobacterium leprae Strains from South AmericaAntimicrobial Agents and Chemotherapy, 2011
- High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical IsolatesJournal of Clinical Microbiology, 2010
- Rapid Detection of Multidrug-Resistant Mycobacterium tube rculosis by Use of Real-Time PCR and High-Resolution Melt AnalysisJournal of Clinical Microbiology, 2010
- Long-Term Relapse Risk of Multibacillary Leprosy after Completion of 2 Years of Multiple Drug Therapy (WHO-MDT) in Cebu, PhilippinesThe American Journal of Tropical Medicine and Hygiene, 2009
- Population-Based Molecular Epidemiology of Leprosy in Cebu, PhilippinesJournal of Clinical Microbiology, 2009
- Rapid Variable-Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical SpecimensJournal of Clinical Microbiology, 2009
- Evaluation of high-resolution melting analysis for screening the LDL receptor geneClinical Biochemistry, 2009
- Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locusBMC Genomic Data, 2009
- Diversity of Potential Short Tandem Repeats in Mycobacterium leprae and Application for Molecular TypingJournal of Clinical Microbiology, 2005