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Applied and Environmental Microbiology, November 2003, p. 6380-6385, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6380-6385.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

R. Temmerman,1* L. Masco,1 T. Vanhoutte,1 G. Huys,1 and J. Swings1,2

Laboratory of Microbiology, Ghent University,1 Belgian Coordinated Collections of Microorganisms/Laboratory of Microbiology Ghent Bacterial Collection, B-9000 Ghent, Belgium2

Received 27 May 2003/ Accepted 19 August 2003

The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a complete community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

* Corresponding author. Mailing address: Laboratory of Microbiology, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Phone: 32-9-264-52-49. Fax: 32-9-264-50-92. E-mail: Robin.Temmerman{at}rug.ac.be.

Applied and Environmental Microbiology, November 2003, p. 6380-6385, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6380-6385.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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