title: Fluorometric assay for testing rifampin susceptibility of Mycobacterium tuberculosis complex.
abstract: 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.
authors.affiliation: DST/NRF Centre of Excellence for Biomedical Tuberculosis Research/MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Faculty of Health Sciences, Stellenbosch University, Tygerberg, Cape Town, South Africa
authors.forename: K G P
authors.initials: KG
authors.lastname: Hoek
authors.forename: N C
authors.initials: NC
authors.lastname: Gey van Pittius
authors.forename: H
authors.initials: H
authors.lastname: Moolman-Smook
authors.forename: K
authors.initials: K
authors.lastname: Carelse-Tofa
authors.forename: A
authors.initials: A
authors.lastname: Jordaan
authors.forename: G D
authors.initials: GD
authors.lastname: van der Spuy
authors.forename: E
authors.initials: E
authors.lastname: Streicher
authors.forename: T C
authors.initials: TC
authors.lastname: Victor
authors.forename: P D
authors.initials: PD
authors.lastname: van Helden
authors.forename: R M
authors.initials: RM
authors.lastname: Warren
mesh_terms: D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013183:Sputum / Q000382:microbiology; D044366:Transition Temperature; D018088:Tuberculosis, Multidrug-Resistant / Q000382:microbiology
chemical_list: D000904:Antibiotics, Antitubercular; D001426:Bacterial Proteins; D017931:DNA Primers; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin