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J Clin Microbiol. 2011 Oct;49(10):3450-7. doi: 10.1128/JCM.01068-11. Epub 2011 Aug 10.

Rapid detection of isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis clinical isolates using high-resolution melting analysis.

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1
Centre for Infectious Diseases and Microbiology-Public Health, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales, Australia.

Abstract

A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis by targeting resistance-associated mutations in the katG, mabA-inhA promoter, rpoB, and gyrA genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical M. tuberculosis isolates was selected for development and evaluation of HRMA. PCR amplicons from the katG, mabA-inhA promoter, rpoB, and gyrA genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified katG and/or mabA-inhA promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates, rpoB mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and gyrA mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in M. tuberculosis which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.

PMID:
21832014
PMCID:
PMC3187326
DOI:
10.1128/JCM.01068-11
[Indexed for MEDLINE]
Free PMC Article
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