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Journal of Clinical Microbiology, April 2008, p. 1369-1373, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02343-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Fluorometric Assay for Testing Rifampin Susceptibility of Mycobacterium tuberculosis Complex{triangledown} ,{dagger}

K. G. P. Hoek, N. C. Gey van Pittius, H. Moolman-Smook, K. Carelse-Tofa, A. Jordaan, G. D. van der Spuy, E. Streicher, T. C. Victor, P. D. van Helden, and R. M. Warren*

DST/NRF Centre of Excellence for Biomedical Tuberculosis Research/MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, Stellenbosch University, Tygerberg, Cape Town, South Africa

Received 6 December 2007/ Returned for modification 2 January 2008/ Accepted 18 February 2008

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.

* Corresponding author. Mailing address: 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, P.O. Box 19063, Tygerberg, Cape Town, South Africa 7505. Phone: 021 938 9073. Fax: 021 938 9476. E-mail: rw1{at}sun.ac.za

{triangledown} Published ahead of print on 27 February 2008.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

Journal of Clinical Microbiology, April 2008, p. 1369-1373, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02343-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Pietzka, A. T., Indra, A., Stoger, A., Zeinzinger, J., Konrad, M., Hasenberger, P., Allerberger, F., Ruppitsch, W. (2009). Rapid identification of multidrug-resistant Mycobacterium tuberculosis isolates by rpoB gene scanning using high-resolution melting curve PCR analysis. J Antimicrob Chemother 63: 1121-1127 [Abstract] [Full Text]  


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