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. 2010 Nov;48(11):3893-8.
doi: 10.1128/JCM.00396-10. Epub 2010 Sep 15.

High-resolution melting curve analysis for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates

Go Eun Choi  1 , Sun Min Lee, Jongyoun Yi, Sang Hyun Hwang, Hyung Hoi Kim, Eun Yup Lee, Eun Hae Cho, Jee Hee Kim, Hwa-Jung Kim, Chulhun L Chang
Affiliations

High-resolution melting curve analysis for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates

Go Eun Choi et al. J Clin Microbiol. 2010 Nov.

Abstract

We evaluated high-resolution melting (HRM) curve analysis as a tool for detecting rifampin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis in an accurate, affordable, and rapid manner. Two hundred seventeen M. tuberculosis clinical isolates of known resistance phenotype were used. Twenty-nine known rpoB mutant DNAs, including rare mutations, were also included. Four pairs of primers were designed: rpoB-F/R (for codons 516 to 539 of rpoB), rpoB-516F/R (for codons 508 to 536 of rpoB), katG-F/R (for the codon 315 region of katG), and inhA-F/R (for the nucleotide substitution of C to T at position -15 of inhA). An HRM curve was generated for each isolate after real-time PCR differentiated the mutant from the wild-type strains. DNA sequencing of the target regions was performed to confirm the results of the HRM curve analysis. All but one of the 73 RIF-resistant (RIF-R) strains and all 124 RIF-susceptible (RIF-S) isolates were correctly identified by HRM curve analysis of rpoB. Twenty-seven of 29 known rpoB mutants were detected. In HRM curve analysis of katG and inhA, 90 INH-R strains that harbored katG or inhA mutations, or both, and all INH-S strains were correctly identified. Ten phenotypically INH-R strains not harboring katG or inhA mutations were not detected. The HRM curve analysis will be a useful method for detection of RIF and INH resistance in M. tuberculosis in a rapid, accurate, simple, and cost-effective manner.

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Figures

FIG. 1.
FIG. 1.
HRM curve (a) and difference plot (b) of the 109-bp fragment of rpoB. Each line indicates the melting curve for an individual sample. In the difference plot, the curve profile of M. tuberculosis H37Rv was compared with the curve profiles of all other sample. The baseline represents M. tuberculosis H37Rv and all RIF-S isolates, as well as five RIF-R isolates. Some curves for specific mutations are indicated. The melting curves that are clearly different from those of the wild and mutant types were defined as variant-type curves. These curves are not easily differentiated from the other curves but are clearly distinguishable by a difference plot.
FIG. 2.
FIG. 2.
HRM curve (a) and difference plot (b) of the 121-bp fragment of rpoB. Each line indicates the melting curve for an individual sample. In the difference plot, the curve profile of M. tuberculosis H37Rv was compared with the curve profiles of all other samples. Some curves indicate specific mutations.
FIG. 3.
FIG. 3.
HRM curve (a) and difference plot (b) of katG. The HRM curve indicates three different profiles. Each line indicates the melting curve for an individual sample. In the difference plot, the curve profile of M. tuberculosis H37Rv was compared with the curve profiles of all other samples. The baseline represents M. tuberculosis H37Rv and all INH-S isolates as well as isolates without mutations of katG. The mutant isolates showed melting curve profiles different from the M. tuberculosis H37Rv melting curve profile.
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