Analysis of Microbial Flora in Activated Sludge Scum Using Immunochemical and Molecular-Biological Methods

使用免疫化学和分子生物学方法分析活性污泥浮渣中的微生物菌群

• 批准号: 14550546
• 负责人: IWAHORI Keisuke
• 金额: $ 2.24万
• 依托单位: University of Shizuoka
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14550546/
• 关键词: mycolic acid-containing bacteria; scum; immunomagnetic separation; hybridization; PCR-DGGE method; fluorescence in situ hybridization; Wuhrmann process; Eikelboom TYPE1851; Gordonia sp.; Mycobacterium sp.; ミコール酸含有菌; オリゴヌクレオチドプローブ; プライマー; 16S

This research aimed at the applicability of immunochemical and molecular-biological methods to analyse the microbial flora in the scum of activated sludge. The obtained results can be summarized as follows;Operational modes of the immunomagnetic separation (IMS) using an anti-mycolic acid polyclonal antibody from Gordonia amarae SCI were investigated, and it was shown that 1 hr-treatment of PBS solution with Tween 20 of 0.2% was effective for the microbial dispersion and that proper dillution ratio of the polyclonal antibody was about 50 times. Though variation of the recovery for each mycolata was dependent on the specificity of the antibody, mycolata was found to be effectively recovered by this IMS procedure optimized with SCI cells. From the similar investigation on the hybridization, 45 min-ultrasonication with 20W and 20KHz for the cell fragmentation and spin colum method for the extraction of RNA fraction were applicable to the detection and determination of the mycolata. In the DGGE analysis with PCR-amplified fraction from the scum sample, denaturing gradient gel concentration of 30〜60% was shown to be suitable. The analytical investigations in the scum samples of Wuhrmann process, using the above-mentioned various metods, were carried out. As the results, ten or over bands were detected, and Gordonia alkalivorans, G. nitida, G. amicalis and Mycobacterium mucogenicum in mycolic acid-containing bacteria were identified from BLAST homologous detection to the base sequences of several bands with the higher fluorescence intensity. Existence of Eikelboom TYPE1851 in filamentous bacteria was simultaneously shown by the fluorescence in situ hybridization.Judging from these results, the immunochemical and molecular-biological methods were concluded to be applicable and useful to analyse the microbial flora in the scum of activated sludge.

本研究旨在探讨免疫化学和分子生物学方法分析活性污泥浮渣中微生物植物群的适用性。所获得的结果可以总结如下：使用来自Gordonia amarae SCI的抗分枝菌酸多克隆抗体的免疫磁性分离（IMS）的操作模式进行了研究，并且表明，用0.2%的Tween 20的PBS溶液处理1小时对于微生物分散是有效的，并且多克隆抗体的适当稀释比为约50倍。尽管每种真菌的回收率的变化取决于抗体的特异性，但发现通过用SCI细胞优化的IMS程序可以有效地回收真菌。从相似的杂交研究结果来看，20 W、20 KHz的45 min超声破碎法和旋转柱法提取RNA片段适用于真菌的检测和鉴定。在用来自浮渣样品的PCR扩增组分的DGGE分析中，显示30 - 60%的变性梯度凝胶浓度是合适的。用上述方法对乌尔曼法浮渣样品进行了分析研究。结果表明，在10条以上的条带中，戈登氏菌（Gordonia alkalivorans）、G. nitida、黑腹隐翅虫G.经BLAST同源性检测发现，含分枝菌酸的细菌中有amicalis和Mycobacterium mucogenicum的碱基序列具有较高的荧光强度。荧光原位杂交结果同时表明丝状菌中存在Eikelboom TYPE 1851，说明免疫化学和分子生物学方法是分析活性污泥浮渣中微生物植物群的有效方法。

项目成果

Immunomagnetic separation of scum-forming bacteria using polyeclonal antibody that recognizes mycolic acids

使用识别霉菌酸的多克隆抗体对浮渣形成细菌进行免疫磁性分离

• 发表时间: 2002
• 期刊: Journal of Microbiological Methods 51
• 作者: S.Morisada

S.Morisada, N.Miyata, K.Iwahori: "Immunomagnetic separation of scum-foming bacteria using polyclonal antibody that recognizes mycolic acids"J. Microbiol. Methods. 51. 141-148 (2002)

S.Morisada、N.Miyata、K.Iwahori：“使用识别霉菌酸的多克隆抗体对浮渣形成细菌进行免疫磁性分离”J.