The emergence and transmission of
multidrug-resistant tuberculosis (MDR-TB) and
extensively drug-resistant tuberculosis (
XDR-TB) have raised concern about diagnostic delay associated with culture-based
drug susceptibility testing methods. The association between
rifampin resistance and MDR-TB or
XDR-TB makes it an important
genetic marker for genotypic
drug susceptibility testing. In this article, we describe the analysis of the physical properties of the
rifampin resistance-determining region (RRDR) in the rpoB gene by high-resolution thermal melt analysis as a method for detecting
rifampin resistance in Mycobacterium tuberculosis complex. The RRDR from the M.
tuberculosis complex was amplified by PCR from
DNA templates extracted from sputum cultures of M.
tuberculosis or the laboratory strain (H37Rv) in the presence of a fluorescent
DNA binding
dye. Subsequent mixing of the amplification products from the respective sputum cultures and the laboratory strain and thermocycling allowed the formation of
DNA duplexes. The thermal denaturation properties of these
DNA duplexes were determined by measuring the derivative of the intensity of fluorescence at different temperatures. Analysis of
DNA extracted from 153 sputum cultures showed a sensitivity of 98% and a specificity of 100% for the detection of
rifampin resistance compared to the "gold standard" culture-based phenotyping method. No statistical difference was detected in the performance of the method when applied to crude
DNA from 134 boiled cultures. This method, named "FAST-Rif" ("fluorometric assay for susceptibility testing of
rifampin"), allowed the rapid, reliable, and easy detection of genotypic
rifampin resistance as a marker for MDR-TB and
XDR-TB.