Application of denaturant gradient gel electrophoresis for the analysis ofthe porcine gastrointestinal microbiota

The porcine gastrointestinal tract (GIT) microbiota has been studied to inc rease production efficiency, improve product quality, and help attempt to r educe 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 object ive of this study was to determine if denaturant gradient gel electrophores is (DGGE) could be effectively applied to measure changes in bacterial popu lations of the pig GIT, as influenced by age, diet or compartment. Bacteria l genetic diversity was determined using DGGE analysis of the V3 region of 16S rDNA PCR products (similar to 200 bp) obtained from primers specific fo r the domain Bacteria. Protocol development included optimization of: DNA e xtraction procedures, PCR amplification, removal of PCR artifacts, and opti mization of gel preparation and image capture. DGGE analysis revealed diver se bacterial populations between pigs of different ages and among individua l gut compartments. Comparison of fecal DNA from different aged pigs reveal ed several unique PCR product bands indicating the presence of unique bacte rial populations. Comparison of different gut compartments demonstrated tha t bacterial populations were most similar (C-s value > 50%) within a single compartment and between adjacent ones. Thus, DGGE can be used to examine b acterial diversity and population shifts in the pig GIT. (C) 1999 Elsevier Science B.V. All rights reserved.