厌氧活性污泥微生物群落的SSCP分析条件优化  

Optimization of Single-Strand Conformation Polymorphism Technology for Analyzing Microbial Community of Anaerobic Activated Sludge

作　　者：鲍立新[1] 李建政[1] 赵焱[1] 郑国臣[1] 

机构地区：[1]哈尔滨工业大学市政环境工程学院,哈尔滨150090

出　　处：《科技导报》2008年第2期28-32,共5页Science & Technology Review

基　　金：国家高技术研究发展计划(863计划)项目(2006AA05Z109)

摘　　要：针对厌氧活性污泥微生物群落结构复杂,群落中相似基因序列较多等特点,对影响单链构像多态性(SSCP)图谱分辨率的电泳温度、甘油、交联度和影响聚合酶链式反应(PCR扩增)的BSA等因素进行了实验研究。结果表明,选择细菌16SrDNAV3区(约200bp)PCR扩增片段,在凝胶浓度为12%、丙烯酰胺与双丙烯酰胺质量比为49:1、无甘油等条件下,恒压260V,4℃电泳17h,可获得较为理想的SSCP图谱;BSA的使用,有助于降低样品中残留腐殖酸和棕黄酸等杂质对PCR扩增的抑制作用,提高SSCP图谱的分辨率和可读性,为厌氧活性污泥细菌群落结构解析提供了有效手段。The microbial community structure of anaerobic activated sludge is very complex with many similar microbe gene sequences. The Single-Strand Conformation Polymorphism （SSCP） technology is proposed in this respect. In order to obtain an SSCP profile with a perfect resolution detail, the factors, including electrophoresis temperature, glycerin and gel strength that may influence the resolution of the SSCP profile, and Bovine Serum Albumin （BSA） that may influence the amplification of the Polymerase-Chain-Reaction （PCR）, are investigated in this paper. The results show that SSCP profiles of anaerobic activated sludge microbial community, ,amplified from the V3 region of 16S rDNA genes （about 200 bp）, are perfect in resolution sensitivity under the following conditions： the gel concentration of 12%, the ratio of acrylamide （AM） and N, N＇-Methylenebisacrylamide （DMAM） of 49： 1, without glycerin, electrophoresis at 4℃ and 260 V for 17 hours. The use of BSA in pretest treatment of anaerobic activated sludge could weaken the inhibiting effect of humic acid and fulvic acid on PCR amplification and improve the resolution sensitivity of SSCP profiles. The optimized technique can be used for microbial community analysis of anaerobic activated sludge.

关 键 词：分子生物学 聚合酶链式反应 单链构像多态性 厌氧活性污泥 群落结构 牛血清白蛋白 

分 类 号：X703[环境科学与工程—环境工程]