title: Development and validation of a nested-PCR-denaturing gradient gel electrophoresis method for taxonomic characterization of bifidobacterial communities.
abstract: 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.
authors: Laboratory of Microbiology, Ghent University, Belgium. Robin.Temmerman@rug.ac.be
authors: R
authors: R
authors: Temmerman
authors: L
authors: L
authors: Masco
authors: T
authors: T
authors: Vanhoutte
authors: G
authors: G
authors: Huys
authors: J
authors: J
authors: Swings
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques; D001644:Bifidobacterium / Q000145:classification* / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis; D017753:Ecosystem; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005243:Feces / Q000382:microbiology; D020459:Genes, rRNA; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S; D012417:Rumen / Q000382:microbiology; D017422:Sequence Analysis, DNA; D012722:Sewage / Q000382:microbiology; D013045:Species Specificity
chemical_list: D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012722:Sewage