Fluorometric assay for testing rifampin susceptibility of Mycobacterium tuberculosis complex

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dc.contributor.authorHoek K.G.P.
dc.contributor.authorGey Van Pittius N.C.
dc.contributor.authorMoolman-Smook H.
dc.contributor.authorCarelse-Tofa K.
dc.contributor.authorJordaan A.
dc.contributor.authorVan Der Spuy G.D.
dc.contributor.authorStreicher E.
dc.contributor.authorVictor T.C.
dc.contributor.authorVan Helden P.D.
dc.contributor.authorWarren R.M.
dc.date.accessioned2011-05-15T15:56:55Z
dc.date.available2011-05-15T15:56:55Z
dc.date.issued2008
dc.description.abstractThe 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.
dc.description.versionArticle
dc.identifier.citationJournal of Clinical Microbiology
dc.identifier.citation46
dc.identifier.citation4
dc.identifier.issn951137
dc.identifier.other10.1128/JCM.02343-07
dc.identifier.urihttp://hdl.handle.net/10019.1/10109
dc.subjectrifampicin
dc.subjectRNA polymerase beta subunit
dc.subjectbacterial protein
dc.subjectprimer DNA
dc.subjectrpoB protein, Mycobacterium tuberculosis
dc.subjecttuberculostatic agent
dc.subjectunclassified drug
dc.subjectantibiotic sensitivity
dc.subjectarticle
dc.subjectcomplex formation
dc.subjectcontrolled study
dc.subjectDNA binding
dc.subjectDNA extraction
dc.subjectDNA structure
dc.subjectfluorometry
dc.subjectgene amplification
dc.subjectmultidrug resistance
dc.subjectMycobacterium tuberculosis
dc.subjectnonhuman
dc.subjectpolymerase chain reaction
dc.subjectpriority journal
dc.subjectsensitivity and specificity
dc.subjectsputum culture
dc.subjectantibiotic resistance
dc.subjectDNA sequence
dc.subjectdrug effect
dc.subjectevaluation
dc.subjectgenetics
dc.subjecthuman
dc.subjectisolation and purification
dc.subjectmethodology
dc.subjectmicrobiological examination
dc.subjectmicrobiology
dc.subjectsingle nucleotide polymorphism
dc.subjectsputum
dc.subjecttransition temperature
dc.subjecttuberculosis
dc.subjectMycobacterium tuberculosis
dc.subjectMycobacterium tuberculosis complex
dc.subjectAntibiotics, Antitubercular
dc.subjectBacterial Proteins
dc.subjectDNA Primers
dc.subjectDrug Resistance, Bacterial
dc.subjectFluorometry
dc.subjectHumans
dc.subjectMicrobial Sensitivity Tests
dc.subjectMycobacterium tuberculosis
dc.subjectPolymerase Chain Reaction
dc.subjectPolymorphism, Single Nucleotide
dc.subjectRifampin
dc.subjectSensitivity and Specificity
dc.subjectSequence Analysis, DNA
dc.subjectSputum
dc.subjectTransition Temperature
dc.subjectTuberculosis, Multidrug-Resistant
dc.titleFluorometric assay for testing rifampin susceptibility of Mycobacterium tuberculosis complex
dc.typeArticle
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