title: Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota.
abstract: Some compounds originating from the human gut microbial metabolism of exogenous and endogenous substrates may have properties that profoundly affect the host's physiological processes. The influence of these metabolites on differences in disease risk among individuals could be mediated by metabolism specific to the gut microbial community composition. In this study, we evaluated the effectiveness of terminal restriction fragment polymorphism (TRFLP) as a biomarker of the fecal microbial community (as a surrogate of gut microbiota) for application in human population-based studies. We tested the effects of experimental conditions on DNA quality, DNA quantity, and TRFLP patterns derived from gut bacterial communities. Genomic DNA was extracted from fecal slurries and the bacterial 16S rDNA genes were amplified and analyzed by TRFLP. We found that the composition of the TRFLP fingerprints varied by different extraction procedure. The best quality and quantity of community DNA extracted from fecal material was obtained by using the QIAamp DNA stool minikit (Qiagen, Valencia, CA) with 95 degrees C incubation and moderate bead beating treatment during the cell-lysis step. Homogenization of fecal samples reduced variation among replicates. Once the TRFLP procedure was optimized, we assessed the methodological and inter-individual variation in gut microbial community fingerprints. The methodological variation ranged from 4.5-8.1% and inter-individual variation was 50.3% for common peaks. In conclusion, standardized TRFLP is a robust, reproducible, and high-throughput method that will provide a useful biomarker for characterizing gut microbiota in human fecal samples.
authors.affiliation: Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Li
authors.forename: Meredith A J
authors.initials: MA
authors.lastname: Hullar
authors.forename: Johanna W
authors.initials: JW
authors.lastname: Lampe
mesh_terms: D000328:Adult; D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D016172:DNA Fingerprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000382:microbiology; D005260:Female; D041981:Gastrointestinal Tract / Q000382:microbiology*; D014644:Genetic Variation; D006801:Humans; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S