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超声波破碎法提取活性污泥DNA及其DGGE分析
2013-03-22 15:02:18 益择发布万晶晶,张汝嘉,邢德峰,谢天卉,赵丽华,任南琪
(哈尔滨工业大学市政环境工程学院, 哈尔滨150090, E2mail:wjj_1984_1999@126. com)
摘 要: 为快速提取活性污泥中的微生物DNA,进一步应用于变性梯度凝胶电泳(DGGE)技术分析其群落
结构,采用细胞超声波破碎法直接提取反应器活性污泥DNA,以16S rRNA V3区域通用引物进行PCR扩增,
随后利用DGGE技术对扩增产物的多样性进行评估. 结果表明,超声波破碎法可以快速地提取活性污泥
DNA,用超声波破碎法提取到的活性污泥克服了PCR扩增困难的问题. 在一定的超声波功率下,超声时间对
DNA产率、PCR扩增效率和DGGE分析均有很大影响,实验的最佳超声时间为27 s. DGGE分析表明,用该法
提取到的DNA种类较为丰富,多样性较好,种群强度最高能达到017 OD,能够进一步应用于群落结构分析.
关键词: 超声波破碎法;活性污泥;DNA提取; PCR;变性梯度凝胶电泳(DGGE)
中图分类号: Q93811 文献标识码: A 文章编号: 0367 - 6234 (2008) 04 - 0559 - 04
DNA extraction of activa ted sludge by ultra son ic
fragmenta tion for DGGE ana lyses
WAN J ing2jing, ZHANG Ru2jia, XING De2feng, XIE Tian2hui, ZHAO L i2hua, REN Nan2qi
( School ofMunicipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090,
China, E2mail:wjj_1984_1999@126. com)
Abstract: In order to quickly extract the microbialDNA from the activated sludge and app ly it subsequently in
community analyses by using denature gradient gel electrophoresis (DGGE) technology, DNA of the activated
sludge from reactors was directly extracted by ultrasonic fragmentation, and 16S rRNA genes were amp lified
with a pair of universal p rimers of the 16S rRNA V3 region by polymerase chain reaction ( PCR). The diversi2
ty of the amp lification p roductswas evaluated byDGGE. The results indicate that ultrasonic fragmentation can
extract DNA from activated sludge quickly and the sludge extracted by ultrasonic fragmentation overcomes the
difficulty of PCR amp lification. Ultrasonic time has obvious influence on DNA yield, PCR amp lification effi2
ciency and DGGE p rofiles under constant power and the best time is 27 seconds. DGGE analysis indicates that
the DNA from the activated sludge has high population diversitywith the highest intensity of 017 OD, and has
further app lication in the community analyses.
Key words: ultrasonic fragmentation; activated sludge; DNA extraction; PCR; denaturing gradient gel elec2
trophoresis (DGGE)
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