1. Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types
- Author
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Pratibha Thapa, Masanori Matsuoka, Saraswoti Khadge, Patrick J. Brennan, Masanori Kai, Wei Li, Varalakshmi D. Vissa, and Deanna A. Hagge
- Subjects
Microbiology (medical) ,Mutant ,Mutation, Missense ,Single-nucleotide polymorphism ,Drug resistance ,Microbial Sensitivity Tests ,Biology ,medicine.disease_cause ,Real-Time Polymerase Chain Reaction ,Polymorphism, Single Nucleotide ,Sensitivity and Specificity ,Mice ,Drug Resistance, Bacterial ,medicine ,Animals ,Humans ,Transition Temperature ,Typing ,Mycobacterium leprae ,Genetics ,Mutation ,Mycobacteriology and Aerobic Actinomycetes ,rpoB ,biology.organism_classification ,Anti-Bacterial Agents ,High Resolution Melt Analysis ,Molecular Diagnostic Techniques - Abstract
Drug resistance surveillance and strain typing of Mycobacterium leprae are 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) of folP1 , rpoB , and gyrA , targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1 , two rpoB , and one gyrA mutation 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 the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (
- Published
- 2012