Development of Simple Methods of DNA Extraction from Environmental Samples for Monitoring Microbial Community Based on PCR
Development of Simple Methods of DNA Extraction from Environmental Samples for Monitoring Microbial Community Based on PCR
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基于 PCR 的环境样本 DNA 提取监测微生物群落简单方法的开发
DOI：
10.2521/jswtb.36.193
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
M. Fujita
中科院分区：
文献类型：
--
作者：
K. Sei;Kenichi Asano;Naohiro Tateishi;K. Mori;M. Ike;T. Kohno;M. Fujita
本研究旨在建立简便、快速的DNA提取方法，用于基于pcr的水/土壤环境微生物群落监测。比较研究了几种细胞裂解酶、化学试剂和机械处理（蛋白酶K、十二烷基硫酸钠、十六烷基三甲基溴化铵、聚氯乙烯聚吡咯烷酮、冻融和超声波）单独或联合对接种了pcr靶向细菌恶臭假单胞菌BH的3种水和土壤样品的DNA提取能力。对于水样，用蛋白酶K进行细胞裂解，检测目标细菌的灵敏度为101个细胞/ml，而本地细菌的灵敏度为104-105个cfu/ml， DNA回收率为ca。苯酚氯仿萃取和乙醇沉淀，25-55%。然而，研究的其他替代品对PCR扩增表现出相当大的抑制作用，因此不太敏感。对于土壤样品，超声波除使用蛋白酶K和SDS在高浓度螯合剂存在下是最有效的，尽管除了苯酚-氯仿提取和乙醇沉淀外，还需要用旋转柱纯化DNA提取物。该方法可在本土细菌含量为107 ~ 109 cfu/g的土壤样品中，以101 ~ 102个细胞/g的速度对目标细菌进行pcr检测，DNA产率为80 ~ 95%。这里建立的方法似乎能够从各种环境样品中提取大部分或相当一部分DNA，具有足够高的PCR扩增纯度。这些方法似乎也经常适用，因为程序非常简单，不包含耗时和费力的操作。
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, Pseudomonas putida BH. For water samples, cell lysis with proteinase K allowed to detect the target bacterium at a sensitivity at 101cells/ml against backgrounds of indigenous bacteria at 104-105 cfu/ml with the DNA recovery of ca .25-55%, when coupled with the phenolchloroform 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 was the 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 101-102 cells/g of the soil samples where 107-109 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.
影响因子：
9.4
作者：
BOOM, R;SOL, CJA;VANDERNOORDAA, J
通讯作者：
VANDERNOORDAA, J
影响因子：
4.4
作者：
MUYZER, G;DEWAAL, EC;UITTERLINDEN, AG
通讯作者：
UITTERLINDEN, AG