Fluorometric Assay for Testing Rifampin Susceptibility of Mycobacterium tuberculosis Complex

Abstract

The emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and extensively drugresistant 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.

Molecular Characterization of Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Strains in Jiangxi, China

“…Second, mutations within the RRDR of rpoB have been reported to occur in 95% or higher of the RIF-resistant isolates (9,36,54). Third, RIF resistance is an excellent surrogate marker for MDR M. tuberculosis (26,37). In the present study, six novel rpoB mutations were identified, and they all fell outside of the RRDR region of the rpoB gene.…”

Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system

“…Melting of the PCR product is also influenced by various factors, such as salt in the buffer solution, DNA quality, DNA concentration and DNA extraction methods, along with other factors that can influence the behaviour of the melting curves. A previous study suggested another twostep HRMA technique that alters the shape of the melting transition to increase the sensitivity and specificity by forming heteroduplexes (Hoek et al, 2008). This two-step technique may in fact cause cross-contamination and may be cumbersome.…”

Gene Scanning of an Internalin B Gene Fragment Using High-Resolution Melting Curve Analysis as a Tool for Rapid Typing of Listeria monocytogenes

“…Nevertheless, a differentiation by a single PCR step may be achieved through the addition of a reference DNA or of unlabeled probes (spiking) to the reaction, which will form heteroduplex amplicons that might exhibit differentiable HRM curve profiles. 23,31,32 In conclusion, mutation scanning by HRM curve analysis of a 500-bp inlB fragment identified the clinically most relevant L. monocytogenes serotypes and enabled rapid, reliable, simple, and cost-effective typing of L. monocytogenes. Application of this technique differentiated 15 HRM subtypes and enabled the analysis of 384 samples in less than 2 hours using an LightCycler 480 instrument.…”