应用于PCR-DGGE分析的活性污泥微生物总DNA提取方法比较

Comparison of extraction methods of activated sludge microbial DNA for PCR-DGGE analysis

探讨适用于PCR-DGGE分析研究的活性污泥细菌和真菌的DNA提取方法。采用5种方法提取活性污泥微生物基因组DNA,以DNA纯度、含量、片段大小及DGGE条带多样性作为考察指标评价提取方法的优劣,以确定最佳实验方案。紫外吸收法和琼脂糖凝胶电泳结果显示,试剂盒法提取的DNA含量最低,其余4种方法获得的DNA含量无显著差异,就DNA纯度而言,试剂盒法最优;除高温裂解法对真菌细胞壁裂解效果较差外,其他4种方法均能不同程度地裂解细菌和真菌细胞;DGGE结果表明,高温裂解法获得的细菌条带最多,基于SDS的细胞裂解法得到的真菌条带最多。综合分析,高温裂解法更适合于活性污泥中细菌的PCR-DGGE分析,基于SDS的细胞裂解法则更适合于污泥中真菌的PCR-DGGE分析。

The aim of this study was to develop a method of extracting total microbial DNA from activated sludge for PCR-DGGE analysis. Five methods were used to extract total microbial DNA from activated sludge. The DNA purity, yield, fragment size, and microbial diversity reflected by DGGE technique were used to evaluate the extracted DNA. The results of UV absorbance and agarose gel electrophoresis showed that the yield of DNA extracted by the kit method was lowest among the five methods. There was no significant difference in the yield of DNA for the other four DNA extraction methods. In terms of the DNA purity, the kit method was the best one. Four DNA extraction methods could effectively lysis both bacterial and fungal cells except the high tempera- ture lysis method for fungal cells. According to the DGGE analysis, more diverse bacterial communities were obtained with the DNA extracted by the high temperature lysis method, and more diverse fungal communities were obtained with the DNA extracted by the SDS lysis method. High temperature lysis method could be more suitable for bacterial PCR-DGGE analysis of activated sludge, while SDS lysis method could be more suitable for fungal PCR-DGGE analysis.