超声波破碎法提取活性污泥DNA及其DGGE分析

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0.7 OD,能够进一步应用于群落结构分析.

DNA extraction of activated sludge by ultrasonic fragmentation for DGGE analyses

In order to quickly extract the microbial DNA from the activated sludge and apply 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 amplified with a pair of universal primers of the 16S rRNA V3 region by polymerase chain reaction(PCR).The diversity of the amplification products was evaluated by DGGE.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 amplification.Ultrasonic time has obvious influence on DNA yield,PCR amplification efficiency and DGGE profiles under constant power and the best time is 27 seconds.DGGE analysis indicates that the DNA from the activated sludge has high population diversity with the highest intensity of 0.7 OD,and has further application in the community analyses.