Author
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ANDERSON, KEVIN |
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Submitted to: Rowett Research Institute Institute National De La Resherche Agronomique Joint Symposium
Publication Type: Abstract Only Publication Acceptance Date: 6/15/2002 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: The swine gastrointestinal tract contains a large number of bacterial species that are not detected by standard cultivation procedures. Thus, non-culture methods must be employed. One such method, the polymerase chain reaction (PCR), enables the amplification and specific identification of small quantities of DNA from samples of digesta. This permits PCR to not only provide a means of detecting the presence of uncultured species of bacteria, but also of estimating their population size and distribution. However, the results obtained from PCR amplification are directly affected by the efficiency of extracting genomic DNA from the sample. This extraction efficiency can be affected by various factors, including incomplete cell lysis, DNA sorption to particulate material, and degradation or damage of the DNA. A comparative analysis of various extraction methods was achieved by obtaining luminal samples from both the cecum and colon of three pigs. Total DNA was extracted from these samples using 19 different DNA extraction methods. These methods consisted of contrasting physical, chemical, and enzymatic protocols. Of these 19 methods, four recovered a significantly greater (P < 0.05) quantity of total DNA from both cecum and colon samples than the other methods. In addition, using a universal primer for PCR amplification of eubacterial 16S rDNA, no PCR inhibitory agents were detected in DNA obtained by any of the four extraction methods. The results of this study provide a guideline for determining the most appropriate DNA extraction methods for luminal contents from the swine. |
