This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mohamed, A. M.
Right arrow Articles by Hinrichs, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mohamed, A. M.
Right arrow Articles by Hinrichs, S. H.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, March 2004, p. 1016-1023, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1016-1023.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Temperature-Mediated Heteroduplex Analysis for Detection of pncA Mutations Associated with Pyrazinamide Resistance and Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by Denaturing High- Performance Liquid Chromatography

Amr M. Mohamed,1 Dhundy R. Bastola,1 Glenn P. Morlock,2 Robert C. Cooksey,2 and Steven H. Hinrichs1*

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6495,1 Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303332

Received 8 April 2003/ Returned for modification 19 August 2003/ Accepted 5 October 2003

The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates and simultaneously differentiate between M. tuberculosis and Mycobacterium bovis. Features that contributed to an optimal assay included the use of two different reference probes for the pncA gene targets from wild-type M. tuberculosis and wild-type M. bovis, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type M. tuberculosis strains (13 were PZA-resistant strains) and 21 M. bovis strains (8 were BCG strains). In all isolates tested, wild-type M. tuberculosis generated a single-peak pattern when mixed with the M. tuberculosis probe and a double-peak pattern with the M. bovis probe. In contrast, all M. bovis isolates generated a double-peak pattern when mixed with the M. tuberculosis probe and a single-peak pattern with the M. bovis probe. PZA-resistant mutant M. tuberculosis isolates generated characteristic patterns that were easily distinguishable from both wild-type M. tuberculosis and M. bovis isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between M. tuberculosis and M. bovis. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.

* Corresponding author. Mailing address: Department of Pathology and Microbiology, University of Nebraska Medical Center, 986495 Nebraska Medical Center, Omaha, NE 68198-6495. Phone: (402) 559-4116. Fax: (402) 559-4077. E-mail: shinrich{at}unmc.edu.

Journal of Clinical Microbiology, March 2004, p. 1016-1023, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1016-1023.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Shi, R., Zhang, J., Li, C., Kazumi, Y., Sugawara, I. (2006). Emergence of Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates from China as Determined by gyrA Mutation Analysis Using Denaturing High-Pressure Liquid Chromatography and DNA Sequencing. J. Clin. Microbiol. 44: 4566-4568 [Abstract] [Full Text]  
  • Posteraro, P., Branca, G., Sanguinetti, M., Ranno, S., Cammarota, G., Rahimi, S., De Carlo, M., Posteraro, B., Fadda, G. (2006). Rapid detection of clarithromycin resistance in Helicobacter pylori using a PCR-based denaturing HPLC assay. J Antimicrob Chemother 57: 71-78 [Abstract] [Full Text]  
  • Mishra, A., Singhal, A., Chauhan, D. S., Katoch, V. M., Srivastava, K., Thakral, S. S., Bharadwaj, S. S., Sreenivas, V., Prasad, H. K. (2005). Direct Detection and Identification of Mycobacterium tuberculosis and Mycobacterium bovis in Bovine Samples by a Novel Nested PCR Assay: Correlation with Conventional Techniques. J. Clin. Microbiol. 43: 5670-5678 [Abstract] [Full Text]  
  • McCammon, M. T., Gillette, J. S., Thomas, D. P., Ramaswamy, S. V., Rosas, I. I., Graviss, E. A., Vijg, J., Quitugua, T. N. (2005). Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region. Antimicrob. Agents Chemother. 49: 2210-2217 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»