PCR-DGGE技术在活性污泥细菌多样性中的条件研究

Optimization of PCR-DGGE conditions in the investigation of activated sludge bacterial diversity

探讨了适用于分析研究活性污泥中细菌多样性的PCR-DGGE实验条件。采用NestedPCR（巢式PCR）技术扩增污泥中细菌的16S rDNA V3区域,利用该扩增产物对污泥中细菌DGGE条件进行优化。结果表明,所采用的Nested-PCR技术准确性较高,可从微量的DNA中扩增出分子大小约190 bp的目标片段。经DGGE条件优化,确定其最佳DGGE条件为丙烯酰胺浓度8.0%,PCR产物作为DGGE上样样品,凝胶变性梯度范围30%～60%,电泳电压140 V,电泳时间8 h,电泳温度60℃。该研究为活性污泥中细菌的PCR-DGGE技术的后序研究提供有效途径。

PCR-DGGE experimental conditions of bacteria in activated sludge were investigated in this work. Sludge bacteria V3 domain sequences of 16S rDNA region were amplified in Nested-PCR. The amplified products of the sludge bacteria were used for DGGE analysis. The experimental conditions for DDGE analysis were optimized. The experimental results showed that the Nested-PCR technique was highly accurate; about 190 bp target segments were amplified from the trace DNA. The optimized condi- tions for DGGE analysis were as following： polyacrylamide concentration 8%, the denaturing gradient from 30% to 60%. The electrophoresis was running at a fixed voltage of 140 V for 8 h at 60 ℃. This work is helpful for further study on RCR-DGGE analysis of activated sludge.