Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2003
Publication Date: 9/1/2003
Citation: ANDERSON, K.L., LEBEPE MAZUR, S.M. COMPARISON OF RAPID METHODS FOR THE EXTRACTION OF BACTERIAL DNA FROM COLONIC AND CECAL LUMEN CONTENTS OF THE PIG. JOURNAL OF APPLIED MICROBIOLOGY. 2003. v. 94. p.988-993. Interpretive Summary: Molecular biological techniques are rapidly becoming very popular in the study of the pigs gastrointestinal micro-flora. Many of these techniques require extracting DNA from intestinal samples. A number of different laboratory protocols have been developed for extracting microbial DNA from various types of environmental samples. However, it is not certain which of these techniques are most effective for extracting bacterial DNA from pig intestinal contents. Thus, we compared the effectiveness of a number of popular DNA extraction techniques. Of these, four provided the largest quantity of extracted DNA from intestinal samples. DNA from these four extraction procedures was further evaluated for the presence of substances that would inhibit polymerase chain reaction (PCR). By amplifying a segment of the 16S ribosomal gene, the efficiency of PCR amplification was determined for each DNA extract. We were able to obtain PCR amplification of DNA extract from these four extraction procedures, which indicated the absence of strong inhibitors of PCR in any of the extracted DNA. However, the efficiency of amplification tended to be lower in DNA from two of the four extraction methods. This indicated that low levels of PCR inhibitors were present in DNA from two of the extraction methods. The results of this study provide a basis for choosing which DNA extraction protocol to use when studying bacteria present in the contents of the pig gastrointestinal tract.
Technical Abstract: The increasing use of DNA methodologies to study the microflora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from intestinal samples. Thus, the objective of this study was to determine which extraction methods are most effective for colonic and cecal lumen samples from pigs. Several routinely used nucleic acid extraction procedures were compared based upon quantity and purity of extracted DNA. Four methods were found to provide the greatest quantity of extracted DNA for both colonic and cecal samples. These methods were further analyzed for 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. Thus, no strong inhibitors of PCR amplification were detected in any of the extracted DNA. However, the efficiency of amplification was lower in DNA samples from two of the methods, suggesting the presence of low levels of PCR inhibitors. Results of this study provide a basis for choosing which DNA extraction procedures are most effective for pig lumen samples.