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Journal of Clinical Microbiology, February 2002, p. 735, Vol. 40, No. 2
0095-1137/01/$04.00+0     DOI: 10.1128/JCM.40.2.735.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Detection of rpoB Mutations in Mycobacterium tuberculosis with LightCycler Technology


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In a recent article, Edwards et al. evaluated three biprobes to detect mutations in rifampin-resistant isolates of Mycobacterium tuberculosis (1). The authors claimed that their approach allowed the detection of all mutations in the 46 isolates studied. We read their paper with interest, because we have used LightCycler PCR to detect resistance to rifampin and isoniazid in M. tuberculosis (3). However, there are aspects of their work that warrant an explanation. The first and major issue concerns biprobe C, which spans codon 526. The authors observed three melting peaks with this probe, one for the wild-type strains (61°C), one for the strains with mutations at codon 526 (49°C), and one for the strains with mutations at codon 531 (66°C). The third melting peak was confirmed by testing 27 strains. Given that biprobe C spans codons 523 to 529, it can hardly be used to detect a mutation at codon 531 (see GenBank accession number L27989 for rpoB). The third peak may, however, correspond to an unreported mutation within codons spanned by the probe (i.e., 523 to 529). The authors should also explain how it is possible that the melting temperature for this unreported mutation is 5°C higher than that for the wild-type. The only valid explanation is that the sequence of the probe they used did not perfectly match the wild-type sequence. In that case, the mutant would indeed have been a better match and have therefore had a higher melting temperature. However, according to the published sequence (5), the perfect match occurs in fact between the probe and the wild-type sequence. Yet another point that requires clarification is the reported melting peaks of biprobes B and C. Edwards et al. reported a melting temperature for the mutants at a certain codon, independent of the type of mutation. In our experience, distinguishing a shift in the melting peak when there is a different single substitution at the same codon is difficult, though some authors have been able to detect different mutations at the same position due to differences in melting temperature (2, 4). In strains with a double mutation, and the author reported several of them, a shift in the melting temperature ought to be observed.


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  1. Edwards, K. J., L. A. Metherell, M. Yates, and N. A. Saunders. 2001. Detection of rpoB mutations in Mycobacterium tuberculosis by biprobe analysis. J. Clin. Microbiol. 39:3350-3352.[Abstract/Free Full Text]
  2. Lindler, L. E., W. Fan, and N. Jahan. 2001. Detection of ciprofloxacin-resistant Yersinia pestis by fluorogenic PCR using the LightCycler. J. Clin. Microbiol. 39:3649-3665.[Abstract/Free Full Text]
  3. Torres, M. J., A. Criado, J. C. Palomares, and J. Aznar. 2000. Use of real-time PCR and fluorometry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis. J. Clin. Microbiol. 38:3194-3199.[Abstract/Free Full Text]
  4. Walker, R. A., N. Saunders, A. J. Lawson, E. A. Lindsay, M. Dassama, L. R. Ward, M. J. Woodward, R. H. Davies, E. Liebana, and J. Threlfall. 2001. Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype Typhimurium DT104 isolates. J. Clin. Microbiol. 39:1443-1448.[Abstract/Free Full Text]
  5. Williams, D. L., C. Waguespack K., K. Eisenach, J. T. Crawford, F. Portaels, M. Salfinger, C. M. Nolan, C. Abe, V. Sticht-Groh, and T. P. Gillis. 1994. Characterization of rifampin resistance in pathogenic mycobacteria. Antimicrob. Agents Chemother. 38:2380-2386.[Abstract/Free Full Text]
Maria J. Torres*
Antonio Criado
Jose C. Palomares
Javier Aznar
Departamento de Microbiología
Unidad de Microbiología Molecular
Facultad de Medecina
Universidad de Sevilla
Apartado 914
41080 Sevilla, Spain

* Phone: 34-54557448 Fax: 34-54377413 E-mail: mjtorres{at}0040us.es


Authors' Reply


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In our paper we reported that Mycobacterium tuberculosis rpoB gene codon 531 mutations increase the apparent melting temperature of biprobe C, which spans codons 523 to 529. Torres et al. point out that biprobe C does not cover codon 531 and query our observations.

Prior to performing the work, we had not anticipated that biprobe C would reveal codon 531 mutations; however, the results were robust and were obtained for all 27 such strains tested. This sequence locus has the potential to form a relatively stable hairpin structure when in single-stranded form. We suggest that this structure can destabilize the biprobe C-amplicon duplex. In strains with codon 531 mutations, the single-stranded structures are less stable themselves and so are less able to compete with the duplex structure. This hypothesis neatly explains the higher melting temperature of biprobe C on the codon 531 mutant sequence compared with the wild type. It may also explain the larger than expected (12°C) effect of a single codon 526 mutation on the biprobe C melting temperature. It is unclear why the codon 526 mutants and double mutants could not be distinguished by biprobe C; however, in our experience, the precise effect of mismatches on melting temperatures is always sequence dependent.

Nick Saunders
Kirstin Edwards
Molecular Biology Unit
Central Public Health Laboratory
London, United Kingdom

Journal of Clinical Microbiology, February 2002, p. 735, Vol. 40, No. 2
0095-1137/01/$04.00+0     DOI: 10.1128/JCM.40.2.735.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.




This Article
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