Fast detection of frequent multidrug resistance conferring mutations in Mycobacterium tuberculosis isolates using a duplex high-resolution melting curve assay
Abstract number: P2036
Pietzka A., Stöger A., Allerberger F., Ruppitsch W.
Objective: Rapid diagnosis of multidrug-resistant (MDR) tuberculosis has substantial impact on progression and spreading of the disease. A widely used surrogate marker for MDR-TB testing is resistance against rifampicin, which is caused by diverse mutations in the RNA polymerase b-subunit gene (rpoB). The aim of this study was a general improvement in multidrug-resistance testing of Mycobacterium tuberculosis isolates by optimisation and simplification of PCR based methods. As a model we used a duplex high-resolution melting (HRM) curve analysis approach to scan for the most frequent mutations in cluster I and outside cluster I of the rpoB.
Methods: Thirty-four MDR-TB and nineteen fully susceptible Mycobacterium tuberculosis strains were used to develop a duplex HRM PCR assay to screen simultaneously for the V176F mutation and cluster I codon 456 mutations of the rpoB on a LightCycler480 instrument using LightCycler 480 software 1.5 (Roche Diagnostics, Penzberg, Germany). For analysis melting curves were normalised, temperature-shifted, and a difference plot was generated (Figure 1).
Results: Parallel gene scanning of two distinct regions within the rpoB allowed the correct identification of 34 MDR-TB isolates and all 19 non-MDR-TB isolates in a single closed-tube assay format. HRM curve analysis generated four distinct and highly reproducible curve profiles each specific for the respective SNP. Thus, non MDR-TB isolates were easily distinguishable from MDR-TB isolates independent of the kind of mutation. In addition the method allowed a differentiation between MDR-TB isolates due to the specific SNP conferring rifampicin resistance (Figure 1).
Conclusions: The developed duplex high resolution melting assay represents a diagnostic improvement in terms of speed, simplicity, accuracy and cost-effectiveness. Moreover, since the entire amplification product is scanned for sequence variations any mutation can be detected by HRM due to melting curve profiles that will be unrelated to the non-MDR-TB melting curve profile used as a standard.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
|Back to top|