引物选择对污泥微生物多样性分析的影响

研究不同引物对PCR-DGGE和PCR-RFLP技术分析污泥群落结构的影响,选取8对通用引物扩增16S rDNA不同可变区序列并对细菌多样性进行DGGE分析,也选取11对通用引物扩增16S rDNA片段并对细菌多样性进行RFLP分析.通过分析PCR产物琼脂糖凝胶电泳图谱、DGGE图谱和酶切图谱,评价不同引物的扩增效果及其对污泥群落多样性的表征能力.结果表明,采用不同引物进行污泥群落结构DGGE分析或RFLP分析时,污泥群落多样性差异显著.PCR-DGGE分析中,引物B341F/B534R(V3区)分析效果较好,条带较丰富,能够充分反映污泥群落多样性.PCR-RFLP分析中,引物27f/8f和1500R扩增效果较好,酶切位点较多,酶切图谱条带丰富,差异性显著,能够充分表征污泥群落多样性.因此,活性污泥细菌多样性分析时,PCR-DGGE分析较优的引物为B341F和B534R,PCR-RFLP分析较优的引物为27f/8f和1500R.

Effect of Different Primers on Microbial Community of Activated Sludge

The aim of this study was to investigate the effect of different primers in PCR-DGGE and PCR-RFLP on the analysis of microbial community in activated sludge. 8 pairs of primers were chosen to amplify the variable region of 16S rDNA for PCR-DGGE analysis, while 11 pairs of primers were used to amplify the total length of 16S rDNA for PCR-RFLP analysis. The effect of different primers on the analysis of microbial community in activated sludge was determined by electrophoresis analysis of the PCR products. The microbial community of the activated sludge was different when different primers were used. For PCR-DGGE analysis, the primers B341F/B534R had good amplification results and the bands were excessive; while for PCR-RFLP, the primers 27f/8f and 1500R had good amplification results and the bands digested by the two enzymes had the highest diversity. The primers B341F/B534R and 27f/8f/1500R were relatively good for PCR-DGGE and PCR-RFLP, respectively, in the analysis of microbial community in activated sludge.