[1]朱琳,尹立红,浦跃朴,等.荧光原位杂交法检测环境硝化细菌实验条件优化及应用[J].东南大学学报(自然科学版),2005,35(2):266-270.[doi:10.3969/j.issn.1001-0505.2005.02.023]
　Zhu Lin,Yin Lihong,Pu Yuepu,et al.Optimization and application of fluorescence in situ hybridization assay for detecting nitrifying bacteria in environmental samples[J].Journal of Southeast University (Natural Science Edition),2005,35(2):266-270.[doi:10.3969/j.issn.1001-0505.2005.02.023]
点击复制

荧光原位杂交法检测环境硝化细菌实验条件优化及应用()
分享到：

《东南大学学报(自然科学版)》[ISSN:1001-0505/CN:32-1178/N]

卷:
35
期数:
2005年第2期
页码:
266-270
栏目:
环境科学与工程
出版日期:
2005-03-20

文章信息/Info

Title:
Optimization and application of fluorescence in situ hybridization assay for detecting nitrifying bacteria in environmental samples
作者:
朱琳1;  尹立红1;  浦跃朴1;  吕锡武2;  李先宁2
1 东南大学公共卫生学院, 南京 210009; 2 东南大学土木工程学院, 南京 210096
Author(s):
Zhu Lin1;  Yin Lihong1;  Pu Yuepu1;  Lü Xiwu2;  Li Xianning2
1 School of Public Health, Southeast University, Nanjing 210009, China
2 College of Civil Engineering, Southeast University, Nanjing 210096, China
关键词:
荧光原位杂交法;  硝化细菌;  亚硝化细菌;  硝酸菌
Keywords:
fluorescence in situ hybridization;  nitrifying bacteria;  nitrosobacteria;  nitrobacteria
分类号:
X172
DOI:
10.3969/j.issn.1001-0505.2005.02.023
摘要:
通过正交试验筛检荧光原位杂交(FISH)技术在检测环境中硝化细菌时的最佳及适合的实验条件.结果显示,样品预处理较优的条件为:热固定2 h,乙醇脱水3 min; FISH技术检测环境样本中亚硝化细菌的最佳实验条件为:杂交温度46 ℃,杂交时间3 h,洗脱液中NaCl浓度20 mmol/L; 检测硝酸菌最佳实验条件为:杂交温度46 ℃,杂交时间5 h,洗脱液中NaCl浓度60 mmol/L.应用该方案对太湖、玄武湖、南京锁金村污水处理厂活性污泥中硝化细菌分布进行分析,结果显示,环境样本中亚硝化细菌和硝酸菌含量分别是:太湖1.68×103, 0.54×103cells/mL; 玄武湖1.34×103, 0.38×103cells/mL; 锁金村污水处理厂活性污泥8.30×106, 0.52×106cells/g.FISH技术检测环境样品中硝化细菌与传统检测方法相比具有快速、简便、准确的优点,在研究环境微生物方面有较好的应用前景.
Abstract:
Experimental conditions of fluorescence in situ hybridization(FISH)for detecting nitrifying bacteria in environmental sample is optimized through orthogonal test. Results show that the optimal conditions for sample preparation are heat-fixing time of 2 h and ethanol-dehydration time of 3 min. The optimized conditions for detecting nitrosobacteria include a hybridized temperature of 46 ℃, a hybridized time of 3 h, and concentration of 20 mmol/L NaCl in hybridized solution. While for detecting nitrobacteria the conditions are a hybridized temperature of 46 ℃, a hybridized time of 5 h, and concentration of 60 mmol/L NaCl in hybridized solution. An effective procedure was established and used to detect nitrifying bacteria in Taihu Lake, Xuanwu Lake and activated sludge of the Suojincun sewage treatment plant in Nanjing. The detected contents of nitrobacteria and nitrosobacteria are respectively as follows: 1.68×103, 0.54×103cells/mL in Taihu; 1.34×103, 0.38×103cells/mL in Xuanwu Lake; 8.30×106, 0.52×106cells/g in activated sludge. Compared with traditional culture method, FISH assay is faster and more accurate. Therefore it has good application prospect in detection of environmental microorganisms.

参考文献/References:

[1] 谢冰,徐亚同.环境微生物的分子生物学研究方法[J].世界科技研究与发展,2003,25(2):48-53.
　　Xie Bing,Xu Yatong.The molecular biological methods on environmental microorganisms studies[J]. World Science-Technology R & D,2003, 25(2):48-53.(in Chinese)
[2] 谢冰,徐华,徐亚同.荧光原位杂交法在活性污泥硝化细菌检测中的应用[J].上海环境科学,2003,22(5):363-366.
　 Xie Bin,Xu Hua,Xu Yatong.Application of fluorescence in situ hybridization to detecting nitrifying bacteria in activated sludge[J].Shanghai Environmental Sciences, 2003,22(5):363-366.(in Chinese)
[3] 卢圣栋.现代分子生物学实验技术[M].北京:中国协和医科大学出版社,1999.220-229.
[4] Domingues M R,Araujo J C,Varesche M B,et al.Evaluation of thermophilic anaerobic microbial consortia using fluorescence in situ hybridization(FISH)[J].Water Sci Technol, 2002,45(10):2733.
[5] Lorenzen J,Larsen L H,Kjær T,et al.Biosensor determination of the microscale distribution of nitrate,nitrate assimilation,nitrification,and denitrification in a diatom-inhabited freshwater sediment[J].Appl Environ Microbial, 1998,64(9):3264-3269.
[6] Amann R,Krumholz L,Stahl D A,et al.Fluorescent oligonucleotide probing of whole cells for determinative,phylogenetic,and environmental studies in microbiology[J].J Bacteriol,1990,172(15):762-770.
[7] Okabe S,Satoh H,Watanabe Y,et al.In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes[J]. Appl Environ Microbial,1999,65(7):3182-3191.
[8] 郑天凌,王斐,徐美珠,等.台湾海峡海域细菌产量、生物量及其在微食物环中的作用[J].海洋与湖沼,2002,33(4):415-423.
　 Zheng Tianlin,Wang Fei,Xu Meizhu,et al.Bacterial production,biomass and role in microbial loop in Taiwan strait[J]. Oceanologia Et Limnologia Sinica,2002,33(4):415-423.(in Chinese)
[9] Beatty B,Mai S,Squire S,等.荧光原位杂交技术[M].王瑛等译.天津:天津科技翻译出版社,2002.44-45.
[10] 吴根福,宣晓冬,汪富三.杭州西湖水体中微生物生理群生态分布的初步研究[J].生态学报,1999,19(3):435-440.
　　Wu Genfu,Xuan Xiaodong,Wang Fusan.Population distribution of microorganisms in the water body of west lake,Hangzhou[J]. Acta Ecologica Sinica,1999,19(3):435-440.(in Chinese)
[11] 王国祥,濮培民,黄宜凯,等.太湖反硝化、硝化、亚硝化及氨化细菌分布及其作用[J].应用与环境生物学报,1998,5(2):190-194.
　　Wang Guoxiang,Pu Peimin,Huang Yikai,et al.Distribution and role of denitrifying,nitrifying,nitration and ammonifying bacteria in the Taihu Lake[J].Chin J Appl Environ Biol, 1998, 5(2):190-194.(in Chinese)
[12] 王国惠.东湖细菌生理类群的分布与主要环境因素的相关性[J].华北水利水电学院学报,1996,17(4):48-54.
　　Wang Guohui.The correlation between the distribution of physiological groups of bacteria and main environmental factors in Donghu Lake[J].Journal of North China in Statute of Water Conservancy and Hydroelectric Power, 1996,17(4):48-54.(in Chinese)

相似文献/References:

[1]杨小丽,周娜,陈明,等.FISH技术解析不同氨氮浓度MBR中的微生物群落结构[J].东南大学学报(自然科学版),2013,43(2):380.[doi:10.3969/j.issn.1001-0505.2013.02.029]
　Yang Xiaoli,Zhou Na,Chen Ming,et al.Analysis of microbial community structure in MBR with different ammonia concentrations using fluorescence in situ hybridization[J].Journal of Southeast University (Natural Science Edition),2013,43(2):380.[doi:10.3969/j.issn.1001-0505.2013.02.029]

备注/Memo

备注/Memo:
基金项目: 国家高技术研究发展计划(863计划)资助项目(2002AA601011).
作者简介: 朱琳(1975—),女,硕士生; 尹立红(联系人),女,博士,教授,博士生导师, lhyin@seu.edu.cn.
更新日期/Last Update: 2005-03-20