Comparison of rapid methods for the extraction of bacterial DNA from colonic and caecal lumen contents of the pig.
OBJECTIVE
The increasing uses of DNA methodologies to study the micro flora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from the intestinal sample. Thus, the objective of this study was to determine which DNA extraction methods are most effective for luminal samples from pigs. Several routinely used nucleic acid extraction procedures were compared based upon quantity and purity of extracted DNA.


RESULTS
DNA was extracted from pig colonic and caecal lumen samples using 19 methods for bacterial DNA extraction. The quantity of total DNA recovered by each extraction method was determined and compared. Two methods using extraction with polyvinylpolypyrrolidone (PVPP) or phenol and two methods involving bead mill homogenization were found to provide the greatest quantity of extracted DNA for both colonic and caecal lumen. Extracted DNA from these four methods was further analysed for purity based upon the presence of PCR inhibitors, which was ascertained by determining the efficiency of amplification of a segment of the 16S rDNA. PCR amplification could be readily achieved with DNA extracted by each of these four methods, but efficiency of amplification tended to be higher with DNA from two of the methods (one extracted with PVPP and one with bead mill homogenization).


CONCLUSIONS
Four extraction methods proved to be significantly superior in quantity of DNA extracted from luminal samples. Of these four, no strong inhibitors of PCR amplification were detected in any of the extracted DNA. However, the efficiency of amplification tended to be lower in DNA samples from two of the methods, suggesting the presence of low levels of PCR inhibitors.


CONCLUSIONS
Results of this study provide a basis for choosing which DNA extraction procedures are most effective for use with samples of pig lumen.
Anderson
K L
KL
USDA/ARS, National Swine Research and Information Center, Ames, IA 50011, USA. anderson@nsric.ars.usda.gov
Lebepe-Mazur
S
S
D000818:Animals; D002432:Cecum; D003106:Colon; D004269:DNA, Bacterial / Q000032:analysis*; D005766:Gastrointestinal Contents / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013552:Swine
D004269:DNA, Bacterial