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Journal of Clinical Microbiology, September 2005, p. 4558-4566, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4558-4566.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Evaluation of tRNA Gene PCR for Identification of Mollicutes

Tim Stakenborg,1 Jo Vicca,2 Rita Verhelst,3 Patrick Butaye,1 Dominiek Maes,2 Anne Naessens,4 Geert Claeys,3 Catharine De Ganck,3 Freddy Haesebrouck,2 and Mario Vaneechoutte3*

Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium,1 Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium,2 Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium,3 Department of Microbiology, University of Brussels (VUB) Hospital, Laarbeeklaan 101, 1090 Brussels, Belgium4

Received 15 December 2004/ Returned for modification 14 February 2005/ Accepted 2 June 2005

We evaluated the applicability of tRNA gene PCR in combination with fluorescent capillary electrophoresis with an ABI310 genetic analyzer (Applied Biosystems, Calif.) for the identification of different mollicute species. A total of 103 strains and DNA extracts of 30 different species belonging to the genera Acholeplasma, Mycoplasma, and Ureaplasma were studied. Reproducible peak profiles were generated for all samples, except for one M. genitalium isolate, the three M. gallisepticum isolates, and 8 of the 24 Ureaplasma cultures, where no amplification could be obtained. Clustering revealed numerous discrepancies compared to the identifications that had been previously obtained by means of biochemical and serological tests. Final identification was obtained by 16S rRNA gene amplification followed by sequence analysis and/or restriction digestion. This confirmed the identification obtained by tRNA gene PCR in all cases. Seven samples yielded an unexpected tRNA gene PCR profile. Sequence analysis of the 16S rRNA genes showed that six of these samples were mixed and that one had a unique sequence that did not match any of the published sequences, pointing to the existence of a not-yet-described species. In conclusion, we found tRNA gene PCR to be a rapid and discriminatory method to correctly identify a large collection of different species of the class of Mollicutes and to recognize not-yet-described groups.

* Corresponding author. Mailing address: Department of Clinical Chemistry, Microbiology, and Immunology, University Hospital, De Pintelaan, 185 9000 Ghent, Belgium. Phone: 32 9 240 3692. Fax: 32 9 240 3659. E-mail: Mario.Vaneechoutte{at}UGent.be.

Journal of Clinical Microbiology, September 2005, p. 4558-4566, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4558-4566.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Baele, M., Stakenborg, T., Lopes dos Santos Santiago, G., Haesebrouck, F., Vaneechoutte, M. (2009). tDNA-PCR Followed by Automated Fluorescent Capillary Electrophoresis for Identification of Bacterial Species. CSH Protocols 2009: pdb.prot5196-pdb.prot5196 [Abstract] [Full Text]  


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