High-throughput method for comparative analysis of denaturing gradient gel electrophoresis profiles from human fecal samples reveals significant increases in two bifidobacterial species after inulin-type prebiotic intake.
Denaturing gradient gel electrophoresis (DGGE) is one of the most commonly used molecular tools to study complex microbial communities. Despite its widespread use, meaningful interpretative analysis remains a major drawback of this method. We evaluated the combination of computer-assisted band-matching with nonparametric statistics for comparative analysis of DGGE banding patterns. Fecal samples from 17 healthy volunteers who consumed 20 g of the prebiotic compound oligofructose-enriched inulin (OF-IN) for 4 weeks were analyzed before and after treatment. DGGE fingerprinting profiles were analyzed using bionumerics software version 4.6., which resulted in a data matrix that was used for statistical analysis. When comparing DGGE profiles before and after OF-IN intake with a Wilcoxon nonparametric test for paired data, two band-classes increased significantly after OF-IN intake (P<0.003 and <0.02). These two band-classes could be assigned to the species Bifidobacterium longum and Bifidobacterium adolescentis by band-sequencing analysis, and their significant increase was quantitatively confirmed with real-time PCR using species-specific primers (respectively P<0.012 and <0.010). Therefore, the nonparametric analysis of a data matrix obtained by computer-assisted band-matching of complex profiles facilitated the interpretative analysis of these profiles and provided an objective and high-throughput method for the detection of significant taxonomic differences in larger numbers of complex profiles.
Gastroenterology and Leuven Food Science and Nutrition Centre (LFoRCe), University Hospital Gasthuisberg, Leuven, Belgium.
Marie
M
Joossens
Geert
G
Huys
Kristel
K
Van Steen
Margo
M
Cnockaert
Séverine
S
Vermeire
Paul
P
Rutgeerts
Kristin
K
Verbeke
Peter
P
Vandamme
Vicky
V
De Preter
D000328:Adult; D001644:Bifidobacterium / Q000302:isolation & purification*; D016172:DNA Fingerprinting; D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007422:Intestines / Q000382:microbiology*; D007444:Inulin / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction; D056692:Prebiotics; D018709:Statistics, Nonparametric
D056692:Prebiotics; D007444:Inulin