工业化废水处理反应器污泥总DNA提取方法

A Method for Total DNA Extraction of Sludge Samples from Full-scale Wastewater Treatment Bioreactors

摘要:

根据工业化废水处理反应器污泥特性, 对常规的溶菌酶-SDS-酚/氯仿环境样品总DNA提取方法进行改进, 增强样品预处理, 强化细胞裂解, 提高杂质去除效率, 获得了一种工业化污泥总DNA提取的通用方法, 并采用该方法对石家庄若干实际运行的工业化厌氧、好氧反应器的污泥样品进行了总DNA提取研究。结果表明, 该方法对所选污泥样品均有效, 具有普适性。提取的污泥总DNA杂质含量少, 纯度高, A260/ A280在1.8左右; 提取效率较高, 总DNA产率都在0.7 mg/g以上, 最大产率可达0.85 mg/g。所提取的污泥总DNA可以直接作为模板进行PCR反应, PCR产物直接进行变性梯度凝胶电泳(DGGE), 能够得到较好的DGGE谱图, 表明该方法提取的污泥总DNA样品可满足后续分析研究的要求。

Abstract:

According to the characteristics of sludge samples from full-scale wastewater treatment bioreactors, the essential total DNA extraction method for most environmental samples, lysozyme-SDS-phenol/ chloroform method, was modified to improve sample pretreatment, intensify cell lysis and enhance the efficiency of impurity removal. Obtain a general total DNA extraction method for industrial sludge samples. Such a method was applied for total DNA extraction of sludge samples from several running full-scale anaerobic or aerobic bioreactors in Shijiazhuang, China. The results indicated that the modified method was suitable for all the sludge samples in this study, showing the satisfying generality. The extracted total DNA of all sludge samples were pure, with about 1.8 of A260/ A280 ratio. The method was also efficient; with average total DNA yield of over 0.7 mg/g and maximum yield of 0.85 mg/g. Moreover, all the extracted total DNA samples could serve as templates directly to amplify 16S rDNA by PCR. The PCR products could be separated well by denaturing gradient gel electrophoresis (DGGE) and the DGGE band patterns were clear enough to be used for further analysis. All these facts indicated that the total DNA extraction method provided in this study could meet the requirements of sludge samples research, from full-scale wastewater treatment bioreactors, using molecular biology technologies.