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United States Department of Agriculture

Agricultural Research Service

Title: Development of Clostridial Specific Denaturing Gradient Gel Electrophoresis Analysis for Assessment of Swine Intestinal, Fecal and Manure Samples

Authors
item Ziemer, Cherie
item Atherly, Todd

Submitted to: American Society for Microbiology Branch Meeting
Publication Type: Abstract Only
Publication Acceptance Date: September 24, 2005
Publication Date: September 24, 2005
Citation: Ziemer, C.J., Atherly, T.A. 2005. Development of clostridial specific denaturing gradient gel electrophoresis analysis for assessment of swine intestinal, fecal and manure samples. American Society for Microbiology Branch Meeting. p.63.

Technical Abstract: Denaturing gradient gel electrophoresis (DGGE) has been accepted as a method to assess bacterial community profiles in environmental samples. Recently, it has been adapted to examine a specific bacterial group. In swine intestinal, fecal and manure samples, members of clostridial groups predominate. In order to investigate the dynamics of these groups in more detail, we developed DGGE methods for two of the predominant clostridial groups; Clostridia clusters I and II which include C. perfringens, C. acetobutylicum, C. histolyticum and C. pfenningii and Clostridia clusters XI and IX which include C. difficile, C. sticklandii, Eubacterium tenue, Megasphera elsdenii and Selenomonas spp. DNA extracts were initially amplified using a 'universal' primer set (S-*-Univ-0007-a-S-20 and S-*-Univ-1401-a-A-18). After cleaning the amplified product, group specific primers were use to amplify the target bacterial groups; for Clostridia clusters I and II primers were S-*-Chis-0150-a-S-23 and S-*-Univ-1401-a-A-18 and for Clostridia clusters IX and XI primers were S-*-Cpar-0180-a-S-23 and S-*-Univ-1401-a-A-18. The final amplification step used the DGGE primers 357f-gc and 519r. Samples of swine cecal, fecal, and manure samples were amplified using these primers. Gels were 10% polyacrylamide (37.5:1 acrylamide-bisacrylamide) with 45-60% urea-formamide denaturing gradients (100% denaturant is defined as 7 M urea and 40% formamide). Denaturing gradient gel electrophoresis was performed on final PCR products with samples electrophoresed at 85 V for 16 hours in 0.5 x TAE buffer at a constant temperature of 60°C. Gels were stained with SYBR Gold and photographed with a FluorS Imager. Bands were then excised from the gel and sequenced in order to confirm target group specificity. This method allows for higher resolution comparisons of the dynamics of targeted bacterial groups in swine intestinal, fecal, and manure samples.

Last Modified: 4/20/2014
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