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 Lefmann, M.
Right arrow Articles by Göbel, U. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lefmann, M.
Right arrow Articles by Göbel, U. B.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, January 2004, p. 339-346, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.339-346.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Novel Mass Spectrometry-Based Tool for Genotypic Identification of Mycobacteria

Michael Lefmann,1 Christiane Honisch,2 Sebastian Böcker,2 Niels Storm,3 Friedrich von Wintzingerode,1 Cord Schlötelburg,1,{dagger} Annette Moter,1 Dirk van den Boom,2* and Ulf B. Göbel1

Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, 10117 Berlin,1 SEQUENOM GmbH, 22761 Hamburg, Germany,3 SEQUENOM Inc., San Diego, California 921212

Received 11 April 2003/ Returned for modification 1 August 2003/ Accepted 26 September 2003

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR amplified and in vitro-transcribed 16S rRNA gene (rDNA) was used for the identification of mycobacteria. Full-length 16S rDNA reference sequences of 12 type strains of Mycobacterium spp. frequently isolated from clinical specimens were determined by PCR, cloning, and sequencing. For MALDI-TOF MS-based comparative sequence analysis, mycobacterial 16S rDNA signature sequences (~500 bp) of the 12 type strains and 24 clinical isolates were PCR amplified using RNA promoter-tagged forward primers. T7 RNA polymerase-mediated transcription of forward strands in the presence of 5-methyl ribo-CTP maximized mass differences of fragments generated by base-specific cleavage. In vitro transcripts were subsequently treated with RNase T1, resulting in G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 type strains, allowing unambiguous identification. All 24 clinical isolates were identified unequivocally by comparing their detected mass signal pattern to the reference sequence-derived in silico pattern of the type strains and to the in silico mass patterns of published 16S rDNA sequences. A 16S rDNA microheterogeneity of the Mycobacterium xenopi type strain (DSM 43995) was detected by MALDI-TOF MS and later confirmed by Sanger dideoxy sequencing. In conclusion, analysis of 16S rDNA amplicons by MS after base-specific cleavage of RNA transcripts allowed fast and reliable identification of the Mycobacterium tuberculosis complex and ubiquitous mycobacteria (mycobacteria other than tuberculosis). The technology delivers an open platform for high-throughput microbial identification on the basis of any specific genotypic marker region.

* Corresponding author. Mailing address: SEQUENOM Inc., 3595 John Hopkins Court, San Diego, CA 92121. Phone: (858) 202-9066. Fax: (858) 202-9084. E-mail: dvandenboom{at}sequenom.com.

{dagger} Present address: VDI/VDE-Technologiezentrum Informationstechnik GmbH, 14513 Teltow, Germany.

Journal of Clinical Microbiology, January 2004, p. 339-346, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.339-346.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Matthiesen, R., Kirpekar, F. (2009). Identification of RNA molecules by specific enzyme digestion and mass spectrometry: software for and implementation of RNA mass mapping. Nucleic Acids Res 37: e48-e48 [Abstract] [Full Text]  
  • Jackson, G. W., McNichols, R. J., Fox, G. E., Willson, R. C. (2008). Toward Universal Flavivirus Identification by Mass Cataloging. J. Mol. Diagn. 10: 135-141 [Abstract] [Full Text]  
  • Honisch, C., Chen, Y., Mortimer, C., Arnold, C., Schmidt, O., van den Boom, D., Cantor, C. R., Shah, H. N., Gharbia, S. E. (2007). Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing. Proc. Natl. Acad. Sci. USA 104: 10649-10654 [Abstract] [Full Text]  
  • Sturenburg, E., Storm, N., Sobottka, I., Horstkotte, M. A., Scherpe, S., Aepfelbacher, M., Muller, S. (2006). Detection and Genotyping of SHV {beta}-Lactamase Variants by Mass Spectrometry after Base-Specific Cleavage of In Vitro-Generated RNA Transcripts.. J. Clin. Microbiol. 44: 909-915 [Abstract] [Full Text]  
  • Honisch, C., Raghunathan, A., Cantor, C. R., Palsson, B. O., van den Boom, D. (2004). High-throughput mutation detection underlying adaptive evolution of Escherichia coli-K12. Genome Res 14: 2495-2502 [Abstract] [Full Text]  
  • Spottke, B., Gross, J., Galla, H.-J., Hillenkamp, F. (2004). Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after solid phase purification. Nucleic Acids Res 32: e97-e97 [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»