Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565–0871, Japan.
Biol Pharm Bull. 2013;36(5):872-6. doi: 10.1248/bpb.b13-00012.
In Southeast Asian countries, industrialization and urbanization is occurring rapidly, and water pollution in rivers and canals poses serious problems in some areas, especially in cities. Excess inflow of domestic, agricultural, and industrial wastewater to freshwater environments disturbs the aquatic microbial ecosystem, which can further pollute water by inhibiting biodegradation of pollutants. Therefore, monitoring of microbes in freshwater environment is important to identify changes in indigenous microbial populations and to estimate the influence of wastewater inflows on them. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis is suitable for monitoring changes in microbial communities caused by human activities, but this method can be difficult in eutrophic freshwater samples that contain PCR inhibitors. In this study, we optimized DNA extraction procedures and PCR conditions for DGGE analysis of bacterial populations in freshwater samples (canal, river, and tap water) collected in Bangkok, Thailand. A simple freeze-thaw procedure was effective for extracting DNA from bacterial cells in the samples, and LA Taq with added bovine serum albumin provided the best PCR amplification. The PCR-DGGE approach revealed that the most common bacteria in freshwater samples belonged to Gammaproteobacteria, while a Gram-positive bacterium was present at Bangkok Noi Canal. Temporally and spatially continuous analyses of bacterial populations in Bangkok canals and rivers by PCR-DGGE approach should be useful to recognize disturbances of microbial ecosystems caused by excess inflows of wastewater.
在东南亚国家，工业化和城市化进程迅速发展，河流和运河的水污染在一些地区，尤其是在城市，造成了严重的问题。过多的生活污水、农业和工业废水流入淡水环境，扰乱了水生微生物生态系统，从而通过抑制污染物的生物降解进一步污染水。因此，监测淡水环境中的微生物对于识别土著微生物种群的变化以及估计废水流入对它们的影响非常重要。聚合酶链反应（PCR）-变性梯度凝胶电泳（DGGE）分析适用于监测人类活动引起的微生物群落变化，但在含有 PCR 抑制剂的富营养化淡水样本中，这种方法可能很困难。在本研究中，我们优化了用于泰国曼谷采集的淡水样本（运河、河流和自来水）中细菌种群的 DGGE 分析的 DNA 提取程序和 PCR 条件。简单的冻融程序可有效提取样本中细菌细胞的 DNA，添加牛血清白蛋白的 LA Taq 提供了最佳的 PCR 扩增效果。PCR-DGGE 方法表明，淡水样本中最常见的细菌属于γ变形菌门，而在曼谷诺伊运河中存在一种革兰氏阳性菌。通过 PCR-DGGE 方法对曼谷运河和河流中细菌种群进行时空连续分析，有助于识别由于过量废水流入而对微生物生态系统造成的干扰。
