Rapid identification of multidrug-resistant Mycobacterium tuberculosis isolates by rpoB gene scanning using high-resolution melting curve PCR analysis

J Antimicrob Chemother. 2009 Jun;63(6):1121-7. doi: 10.1093/jac/dkp124. Epub 2009 Apr 15.


Background: Multidrug-resistant (MDR) Mycobacterium tuberculosis poses a serious threat to the control of tuberculosis (TB) and constitutes an increasing public health problem. The availability of rapid in vitro susceptibility tests is a prerequisite for optimal patient treatment. Rifampicin resistance caused by diverse mutations in the rpoB gene is an established and widely used surrogate marker for MDR-TB. We used a high-resolution melting (HRM) curve analysis approach to scan for mutations in the rpoB gene.

Methods: A total of 49 MDR-TB and 19 fully susceptible non-MDR-TB isolates, as determined by conventional drug susceptibility testing using the BACTEC-MGIT960 system, were used to evaluate the suitability of HRM curve analysis as a rapid and accurate screening system for rifampicin resistance.

Results: HRM analysis of the rpoB cluster I site allowed the correct allocation of 44 of the 49 MDR-TB isolates and all non-MDR-TB isolates. Three of the five MDR-TB isolates (60%) falsely identified as non-MDR-TB harboured the V176F mutation that could be specifically detected by an additional HRM assay. The combined HRM analysis of all strains and isolates exhibited 95.9% sensitivity and 100% specificity.

Conclusions: With a positive predictive value of 100% and a negative predictive value of at least 99.9%, this combined HRM curve analysis is an ideal screening method for the TB laboratory, with minimal requirements of cost and time. The method is a closed-tube assay that can be performed in an interchangeable 96- or 384-well microplate format enabling a rapid, reliable, simple and cost-effective handling of even large sample numbers.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics*
  • DNA, Bacterial / genetics*
  • DNA-Directed RNA Polymerases
  • Humans
  • Microbial Sensitivity Tests / methods
  • Mutation, Missense
  • Mycobacterium tuberculosis / genetics*
  • Mycobacterium tuberculosis / isolation & purification
  • Polymerase Chain Reaction / methods*
  • Predictive Value of Tests
  • Sensitivity and Specificity
  • Transition Temperature*
  • Tuberculosis, Multidrug-Resistant / diagnosis*


  • Bacterial Proteins
  • DNA, Bacterial
  • rpoB protein, Mycobacterium tuberculosis
  • DNA-Directed RNA Polymerases