高含固率木质纤维素厌氧发酵物总DNA的4种提取方法比较  

Comparison of Four Methods for Extracting Microbial Total DNA from Lignocellulosic Digestate with High Solid Content

作　　者：马旭光[1] 刘素君[1] 江滔[1] 常佳丽 唐琼[1] MA Xu-guang;LIU Su-jun;JIANG Tao;CHANG Jia-li;TANG Qiong(College of Chemistry, Leshan Normal University, Leshan 614000, China)

机构地区：[1]乐山师范学院化学学院,四川乐山614000

出　　处：《中国沼气》2020年第2期28-35,共8页China Biogas

基　　金：国家自然基金项目(51508258);四川省科技厅应用基础项目(2017JY0175);乐山市科技局重点项目(15NZD106);乐山师范学院人才引进计划项目(Z1410)。

摘　　要：不同的样本特性和提取方法对获得微生物总DNA的质量有重要影响。文章基于高含固率木质纤维素厌氧发酵物腐殖酸、酚类物质含量高、质地均一性差、微生物浓度低的特点,研究了4种方法提取不同高含固率粪秸厌氧发酵物中微生物总DNA的效果。结果表明,常规的十二烷基磺酸钠法(sodium dodecyl sulfate,SDS)、十二烷基磺酸钠和溴化十六烷基三甲铵结合法(sodium dodecyl sulfate and cetyltrimethyl ammonium bromide,SDS-CTAB)和商业的粪便试剂盒法提取的DNA质量均较差,SDS法和试剂盒法未能获得聚合酶链式反应(polymerase chain reaction,PCR)扩增目的条带,SDS-CTAB法得到的条带较模糊;改进SDS-CTAB法获得的DNA杂质少、纯度高,具有较好的稳定性,A260/A280和A260/A230值分别为1.74~1.86和1.65~1.86,每克样品的DNA浓度在50 ng·μL^-1以上,电泳条带单一齐整、清晰明亮,PCR扩增的目的条带清晰度高,适宜后续分子生物学技术的分析。林格氏液洗脱、聚乙烯吡咯烷酮-40(Polyvinyl Pyrrolidone-40,PVP-40)洗涤液除杂以及裂解液和多种酶联合破壁是改进SDS-CTAB法获得该类专一性样本高质量微生物总DNA的关键步骤。Different sample characteristics and extraction methods have important effects on the quality of extracted microbial DNA.Based on the characteristics of high content of humic acid,phenolic substances,poor homogeneity of texture and low concentration of microorganisms in high solid lignocellulosic digestate,this paper studied the effects of four extraction methods on the extraction of total microbial DNA from straw-manure fermentation digestate with different high solid concentrations.The results showed that the DNA was of poor quality extracted by conventional SDS(sodium dodecyl sulfate)method,SDS-CTAB(sodium dodecyl sulfate and cetyltrimethyl ammonium bromide)method and commercial fecal kit method.The PCR(polymerase chain reaction)amplification target band was not found or ambiguous by methods of SDS,fecal kit and SDS-CTAB.On the other hand,the improved SDS-CTAB method could obtain microbial DNA with less impurities,high purity and better stability.The quantitative detection results showed that A260/A280,A260/A230 were 1.74~1.86,1.65~1.86,respectively,and the DNA concentration per gram of sample was above 50 ng·μL^-1.The electrophoretic band of DNA was uniform and clear,and the PCR amplified target band had high definition,suitable for subsequent molecular analysis.The key steps for improved SDS-CTAB method could obtain the high-quality microbial DNA were:elution by Ringer's solution,impurity removal by Polyvinyl Pyrrolidone 40(pvp-40)washing solution,and breaking the cell wall by lysate solution combined with various enzymes.

关 键 词：高含固率 木质纤维素厌氧发酵物 微生物DNA提取方法 纯度 PCR扩增 

分 类 号：Q781[生物学—分子生物学] X712[环境科学与工程—环境工程]