16S ribosomal DNA-based analysis of bacterial diversity in purified water used in pharmaceutical manufacturing processes by PCR and denaturing gradient gel electrophoresis
16S ribosomal DNA-based analysis of bacterial diversity in purified water used in pharmaceutical manufacturing processes by PCR and denaturing gradient gel electrophoresis
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DOI：
10.1128/aem.68.2.699-704.2002
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发表时间：
2002-02-01
影响因子：
4.4
通讯作者：
Nasu, M
中科院分区：
文献类型：
--
作者：
Kawai, M;Matsutera, E;Nasu, M
采用变性梯度凝胶电泳（DGGE）对自来水离子交换制备的部分纯净水中的细菌群落进行了分析。用通用引物扩增16S核糖体DNA片段，包括V6、-7和-8区，并进行DGGE分析。用PCR-DGGE条带图谱测定的纯净水中细菌多样性明显低于其他水生环境。用流式细胞术对大豆酪蛋白消化（SCD）和R2A培养基上分离的酯酶活性菌群进行分析。纯化水中的优势菌具有酯酶活性，但在SCD或R2A培养基上未检测到。DGGE凝胶上主条带的DNA序列分析显示，在这些培养基上可培养的细菌为缓生根瘤菌、黄单胞菌和窄养单胞菌，而优势菌与之前鉴定的细菌关系不密切。这些数据表明了独立培养方法对制药用水质量控制的重要性。
The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.