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United States Department of Agriculture

Agricultural Research Service

Title: Group Specific Amplified Ribosomal-Dna Restriction Analysis to Evaluate Microbial Community Changes among Swine Feces and Storage Pit Samples

item Ziemer, Cherie
item Cotta, Michael
item Whitehead, Terence

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: May 25, 2000
Publication Date: N/A

Technical Abstract: Anaerobic digestion of swine waste results in the production of various odorous compounds including ammonia, organic acids, and sulfides; however, little is known about microorganisms involved in these processes. Group specific amplified ribosomal-DNA restriction analysis (GS-ARDRA) was used to determine changes in microbial community structure among swine fecal and dstorage pit samples. PCR primer and probe sequences were evaluated for their potential for targeted DNA amplification by optimizing annealing temperatures and determining specificity using a set of target and non-target organisms. A number of primer sets were identified, and two targeting Bacteroides-Prevotella (BP) and Streptococcus-Lactococcus (Str) groups were initially evaluated, as well as a universal (Un) primer pair. Seven tetrameric restriction enzymes (RE) were screened for their ability to differentiate among organisms within target groups in order to optimize a set of three RE for each target group. Two RE were suitable for both target groups HaeIII and RsaI, while the third RE was Sau3AI for BP and Un groups and AluI for Str group. PCR amplification reactions, using total DNA isolated from swine fecal and storage pit samples, were performed. The amplified target DNA was subjected to digestion with the appropriate RE set and analyzed by agarose gel electrophoresis. Differentiation between swine fecal and storage pit samples using GS-ARDRA was possible when the BP group was targeted but not when the Str or Un groups were used. These results are consistent with 16S rDNA sequencing data from bacterial isolates and clones obtained from swine feces and waste storage pits. Screening of targeted primer pairs continues in order to optimize GS-ARDRA analysis of environmental swine waste samples.

Last Modified: 4/21/2015