title: Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota.
abstract: The porcine gastrointestinal tract (GIT) microbiota has been studied to increase production efficiency, improve product quality, and help attempt to reduce disease. During the developmental period from birth through weaning, the intestinal microbiota undergoes a rapid ecological succession. There is interest in developing a monitoring technique that allows for analysis of bacterial population levels and shifts within the pig intestine. The objective of this study was to determine if denaturant gradient gel electrophoresis (DGGE) could be effectively applied to measure changes in bacterial populations of the pig GIT, as influenced by age, diet or compartment. Bacterial genetic diversity was determined using DGGE analysis of the V3 region of 16S rDNA PCR products (approximately 200 bp) obtained from primers specific for the domain Bacteria. Protocol development included optimization of: DNA extraction procedures, PCR amplification, removal of PCR artifacts, and optimization of gel preparation and image capture. DGGE analysis revealed diverse bacterial populations between pigs of different ages and among individual gut compartments. Comparison of fecal DNA from different aged pigs revealed several unique PCR product bands indicating the presence of unique bacterial populations. Comparison of different gut compartments demonstrated that bacterial populations were most similar (C, value > 50%) within a single compartment and between adjacent ones. Thus, DGGE can be used to examine bacterial diversity and population shifts in the pig GIT.
authors.affiliation: Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana 61801, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Simpson
authors.forename: V J
authors.initials: VJ
authors.lastname: McCracken
authors.forename: B A
authors.initials: BA
authors.lastname: White
authors.forename: H R
authors.initials: HR
authors.lastname: Gaskins
authors.forename: R I
authors.initials: RI
authors.lastname: Mackie
mesh_terms: D000375:Aging; D000818:Animals; D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D004032:Diet; D004064:Digestive System / Q000382:microbiology*; D017753:Ecosystem; D004586:Electrophoresis / Q000379:methods*; D005243:Feces / Q000382:microbiology; D014644:Genetic Variation; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013552:Swine / Q000254:growth & development / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D004277:DNA, Single-Stranded; D012336:RNA, Ribosomal, 16S