Design and in vitro evaluation of new rpoB-DGGE primers for ruminants

• Published in: FEMS microbiology ecology Vol. 76; no. 1; pp. 156 - 169
• Main Authors: Perumbakkam, Sudeep, Craig, A. Morrie
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.04.2011; Oxford University Press
• Subjects: Animals; Bacteria - classification; Bacteria - genetics; Bacterial Proteins - genetics; Cattle - microbiology; Community structure; Denaturing Gradient Gel Electrophoresis; DGGE; DNA Primers - genetics; DNA, Bacterial - genetics; Ecology; Genes; Genes, Bacterial; Microbiology; phylogenetics; Phylogeny; Proteobacteria; RNA, Ribosomal, 16S - genetics; rpoB; rumen; Rumen - microbiology; Ruminantia; Ruminants - microbiology; Sequence Analysis, DNA; Sheep - microbiology; phylogenetics; DGGE; rumen
• ISSN: 0168-6496; 1574-6941
• DOI: 10.1111/j.1574-6941.2011.01042.x

Two new primer sets based on the rpoB gene were designed and evaluated with bovine and ovine rumen samples. The newly developed rpoB-DGGE primer set was used along with the 16S rRNA gene-V3, and another (old) rpoB-DGGE-based primer set from a previous study to in vitro compare the bovine and ovine rumen ecosystems. The results indicate a significant (P<0.001) difference in the microbial population between the two ruminants irrespective of the primers used in the analysis. Qualitative comparison of the data provides evidence for the presence of similar phyla profiles between the 16S rRNA gene and the newly developed rpoB primers. A comparison between the two rpoB-based primer sets (old and new) showed that the old rpoB-based primers failed to amplify phylum Bacteroidetes (a common phylum in the rumen) in both bovine and ovine rumen samples. The old and new rpoB-DGGE-based primers amplified a large number of clones belonging to phylum Proteobacteria, providing a useful insight into the microbial structure of the rumen. ChaoI, ACE, Simpson, and Shannon-Weaver index analysis estimated the bovine rumen to be more diverse than the ovine rumen for all three primer sets. These results provide a new insight into the community structure among ruminants using the newly developed primers in this study.