Development of Simple Methods of DNA Extraction from Environmental Samples for Monitoring Microbial Community Based on PCR.
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概要
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Studies were made so as to establish simple and rapid DNA extraction methods for PCR-based monitoring of microbial community in the water/soil environment. Several kinds of cell-lysis enzyme, chemical agents, and mechanical treatments (proteinase K, SDS (sodium dodecyl sulfate), CTAB (cetyltrimethyl ammonium bromide), PVPP (polyvinylpolypyrrolidone), freeze-and-thaw, and ultrasonication) were comparatively investigated solely or in combinations for their DNA extracting capability against each 3 water and soil samples inoculated with the PCR-targeting bacterium, <I>Pseudomonas putida</I> BH. For water samples, cell lysis with proteinase K allowed to detect the target bacterium at a sensitivity at 10<SUP>1</SUP>cells/m<I>l</I> against backgrounds of indigenous bacteria at 10<SUP>4</SUP>-10<SUP>5</SUP> cfu/m<I>l</I> with the DNA recovery of ca.25-55%, when coupled with the phenol-chloroform extraction and ethanol precipitation. However, the other alternatives investigated showed considerable inhibitory effects on the PCR amplification and were, therefore, less sensitive. For soil samples, ultrasonication in addition to the uses of proteinase K and SDS in the presence of a high concentration of chelating agent wasthe most effective, although purification of the DNA extracts with a spun column were required in addition to the phenol-chloroform extraction and ethanol precipitation. This method enabled the PCR-mediated detection of the target bacterium at 10<SUP>1</SUP>-10<SUP>2</SUP> cells/g of the soil samples where 10<SUP>7</SUP>-10<SUP>9</SUP> cfu/g of indigenous bacteria existed and the DNA yield was 80-95%. The methods established here seem to be able to extract a most or considerable portion of the DNA from a variety of environmental samples with a sufficiently high purity for PCR amplification. These methods also seem routinely applicable, because the procedures are very simple and do not contain time-consuming and labor-full operations.
- 日本水処理生物学会の論文
日本水処理生物学会 | 論文
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