基于定量PCR的活性污泥DNA提取方法研究  

Comparative study of the three current DNA extraction methods from the activated sludge for genome DNA based on the quantitative PCR

作　　者：黄培[1,2,3] 叶飞[2,3] 吴胜军[2,3] 黄平[2,3] 杨杉[2,3] 谭远友[1] 王雨[2,3] 

机构地区：[1]武汉纺织大学环境工程学院,武汉430200 [2]中国科学院重庆绿色智能技术研究院,重庆400714 [3]中国科学院水库水环境重点实验室,重庆400714

出　　处：《安全与环境学报》2016年第3期221-226,共6页Journal of Safety and Environment

基　　金：国家自然科学基金项目(41303053);中国科学院西部行动计划项目(KZCX2-XB3-14-01);中国科学院"西部之光"人才培养计划"西部博士资助项目";重庆市基础与前沿研究计划项目(cstc2013jcyj A20003)

摘　　要：分别采用基于复合酶＋化学裂解（BTK）和玻璃珠＋SDS（十二烷基硫酸钠）裂解（PS）的试剂盒法及基于溶菌酶裂解的CTAB（十六烷基三甲基溴化铵）法3种方法提取活性污泥基因组DNA,以所得DNA质量浓度、纯度和细菌16S rRNA基因丰度为考察指标对各提取方法进行评价,以确定提取活性污泥DNA的最适方法。结果表明,CTAB法所得DNA质量浓度（496.3~1 715.3μg/m L）显著高于其他两种试剂盒法,BTK试剂盒法所得DNA纯度（A260/A280〉1.76,A260/A230〉1.9）高于其他两种方法。实时荧光定量PCR结果表明,3种方法提取的DNA样品均能在稀释500倍时作为模板对细菌16S rRNA基因丰度进行定量分析;其中CTAB法所能得到的基因丰度最高（1.9×108~1.4×1010copies/g）,显著高于BTK试剂盒法（3.7×106~8.6×106copies/g）和PS试剂盒法（7.1×106~1.3×107copies/g）所得。因此,从定量PCR的结果来看,CTAB法获得的DNA模板可得到更高的细菌16S rRNA基因丰度,与该方法获得的较高DNA产率一致;由于该方法所得DNA纯度不高,进行定量PCR分析时可通过较高倍数稀释得到解决。同时CTAB法所需试剂均为常用生化试剂,方便获得且价格低廉,优先推荐。The paper aimed to present the results of our comparative study of the three DNA extractive methods from the activated sludge based on the quantitative PCR for genome DNA in hoping to choose the most suitable one for the purpose.We have carefully compared the three methods for DNA isolation on the basis of comparing the concentrations and purity of the DNA yielded as well as the results of the quantitative PCR of the bacteria 16 S rRNA.The three methods include BTK DNA isolation kit based on the complex enzymatic-chemical lysis,the PS DNA isolation kit method based on the beads beating-SDS lysis and the CTAB（Hexadecyltrimethyl Ammonium Bromide） assay based on the lysozyme lysis.According to the results of comparison in the concentration and yield of DNA that have been extracted by using the three methods,the yield of CTAB assay accounts for 496.3-1 715.3 μg/mL,whereas the purest DNA can be gained by the BTK kit（A260/A280 1.76,A260/A230 1.9）.What is more,the bacterial 16 S rRNA gene was retrieved successfully by the quantitative PCR with 500-fold diluted DNA from the aforementioned three methods.The DNA from the method of BTK kit（3.7 × 106-8.6 × 106 copies/g） was compared with that from PS kit（7.1 ×106-1.3 × 107 copies/g）.In comparison with the other two methods,the DNA gained from the method of CTAB（1.9 × 108-1.4× 1010 copies/g） turns to be higher than that from the other two methods.Besides,little difference has been found of the quantitative results in diluting multiple with the DNA from PS kit,in which the results of 500-fold dilution have been found 22.01,3.46,3.3,1.81 times higher than those of 100-fold from the other two methods,respectively.The amount of bacterial 16 S rRNA gained through the CTAB method tends to be conspicuously higher than the rest two commercial kits,whose abundance was comparable to the concentration of the yielded DNA,though the purity from CTAB doesn＇t seem so good as those from BTK method.However,the impact of the method tends to be diminished due to the

关 键 词：环境工程学 实时荧光定量PCR 活性污泥 DNA提取 16SrRNA 

分 类 号：Q987[生物学—遗传学]