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United States Department of Agriculture

Agricultural Research Service

Title: Application of Group Specific Amplified Rdna Restriction Analysis to Characterize Swine Fecal and Manure Storage Pit Samples

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

Submitted to: Anaerobe
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 23, 2004
Publication Date: July 20, 2004
Citation: Ziemer, C.J., Cotta, M.A., Whitehead, T.R. 2004. Application of group specific amplified rDNA restriction analysis to characterize swine fecal and manure storage pit samples. Anaerobe. 10:217-227.

Interpretive Summary: Incomplete anaerobic digestion of swine manure by microorganisms results in 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 was evaluated as a method to determine changes in microbial community structure among swine fecal and manure pit samples. PCR primer sets were identified targeting the following groups: Bacteroides- Prevotella, Clostridial Clusters I and II, Clostridial Clusters IX and XI, Clostridial Clusters XIVa and XIVb, Lactobacillus, Desulfovibrionaceae and Streptococcus-Lactococcus, as well as a primer set targeting total 16S rDNA. Restriction enzymes, which cut DNA at different sites and results in different size pieces of DNA that can be visualized by agarose gel electrophorsis, were used. Total DNA from swine feces, manure storage pits sand human fecal samples was extracted and amplified using group targeted primers. The resulting banding patterns after restriction enzyme treatment were evaluated for differences among our samples. Fecal and manure storage pit samples obtained on the same day were more similar to each other than to any other samples. The GS-ARDRA technique provides a rapid method to differentiate among swine fecal and manure storage pit samples. Laboratories evaluating manure-handling systems will be able to use this technique to screen multiple samples for microbial community changes.

Technical Abstract: Incomplete anaerobic digestion of swine manure by microorganisms results in 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 evaluated as a method to determine changes in microbial community structure among swine fecal and manure pit samples. A number of PCR primer sets were identified targeting the following groups: Bacteroides-Prevotella, Clostridial Clusters I and II, Clostridial Clusters IX and XI, Clostridial Clusters XIVa and XIVb, Lactobacillus, Desulfo- vibrionaceae, and Streptococcus-Lactococcus, as well as a primer set targeting total 16S rDNA. Seven tetrameric restriction enzymes (RE) were screened for their ability to differential among species within each target group, in order to optimize a set of at least three RE for each target group. Total DNA from swine feces, manure storage pits and human fecal samples was extracted and amplified using group targeted primers. After digestion with RE, banding patterns were visualized using agarose gel electrophoresis. Fecal and manure storage pit samples obtained on the same day were more similar to each other than to any other samples. The GS- ARDRA technique will be used to differentiate among swine fecal and waste storage pit samples.

Last Modified: 7/30/2014
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