Fluorometric assay for testing rifampin susceptibility of Mycobacterium tuberculosis complex

Hoek K.G.P.
Gey Van Pittius N.C.
Moolman-Smook H.
Carelse-Tofa K.
Jordaan A.
Van Der Spuy G.D.
Streicher E.
Victor T.C.
Van Helden P.D.
Warren R.M.
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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 iesting of rifampin"), allowed the rapid, reliable, and easy detection of genotypic rifampin resistance as a marker for MDR-TB and XDR-TB. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
rifampicin, RNA polymerase beta subunit, bacterial protein, primer DNA, rpoB protein, Mycobacterium tuberculosis, tuberculostatic agent, unclassified drug, antibiotic sensitivity, article, complex formation, controlled study, DNA binding, DNA extraction, DNA structure, fluorometry, gene amplification, multidrug resistance, Mycobacterium tuberculosis, nonhuman, polymerase chain reaction, priority journal, sensitivity and specificity, sputum culture, antibiotic resistance, DNA sequence, drug effect, evaluation, genetics, human, isolation and purification, methodology, microbiological examination, microbiology, single nucleotide polymorphism, sputum, transition temperature, tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis complex, Antibiotics, Antitubercular, Bacterial Proteins, DNA Primers, Drug Resistance, Bacterial, Fluorometry, Humans, Microbial Sensitivity Tests, Mycobacterium tuberculosis, Polymerase Chain Reaction, Polymorphism, Single Nucleotide, Rifampin, Sensitivity and Specificity, Sequence Analysis, DNA, Sputum, Transition Temperature, Tuberculosis, Multidrug-Resistant
Journal of Clinical Microbiology