Modified pretreatment method for total microbial DNA extraction from contaminated river sediment
Modified pretreatment method for total microbial DNA extraction from contaminated river sediment
复制标题
DOI：
10.1007/s11783-014-0679-4
复制
发表时间：
2015-06-01
影响因子：
6.4
通讯作者：
Liu, Xueduan
中科院分区：
文献类型：
--
作者：
Fang, Yun;Xu, Meiying;Liu, Xueduan
从污染环境样品中提取高质量的微生物DNA是微生物生态学研究的重要步骤。基于已有的土壤和沉积物样品预处理方法，通过选择最佳预处理参数（即试剂溶液、反应时间和温度），开发了一种改进的预处理方法，用于从严重污染的河流沉积物样品中提取微生物DNA。预处理过程为：在采用标准DNA提取程序之前，用含有0.1 mol center dot L-1乙二胺四乙酸（EDTA）、0.1 mol center dot L-1 Tris （pH 8.0）、1.5 mol center dot L-1 NaCl、0.1 mol center dot L-1 NaH2PO4和Na2HPO4的溶液在65A℃、180 r center dot min（-1）下洗涤三次，以去除腐臭物质和重金属。我们比较了预处理后的标准程序DNA提取结果，未预处理的结果，以及使用商业PowerSoil (TM) DNA分离试剂盒的结果。结果表明，从DNA产率、DNA片段长度和原核生物多样性的测定来看，预处理显著提高了DNA质量。预处理后的DNA所显示的原核生物多样性也明显高于单独使用PowerSoil (TM) DNA分离试剂盒提取的DNA。对于少量（0.25 g甚至更低）的沉积物样品，该预处理方法效果良好。该方法为环境监测和修复过程中微生物群落分析提供了一种新颖、简单、经济的DNA提取工具。
Extraction of high-quality microbial DNA from contaminated environmental samples is an essential step in microbial ecological study. Based on previously published methods for soil and sediment samples, a modified pretreatment method was developed for extracting microbial DNA from heavily contaminated river sediment samples via selection of optimal pretreatment parameters (i.e., reagent solution, reaction duration, and temperature). The pretreatment procedure involves washing the river sediment sample for three times with a solution containing 0.1 mol center dot L-1 ethylene diamine tetraacetic acid (EDTA), 0.1 mol center dot L-1 Tris (pH 8.0), 1.5 mol center dot L-1 NaCl, 0.1 mol center dot L-1 NaH2PO4, and Na2HPO4 at 65A degrees C with 180 r center dot min(-1) for 15 min to remove humic materials and heavy metals prior to the employment of standard DNA extraction procedures. We compared the results of standard procedure DNA extraction following pretreatment, without pretreatment, and with using a commercial PowerSoil (TM) DNA Isolation Kit. The results indicated that the pretreatment significantly improved the DNA quality based on DNA yield, DNA fragment length, and determination of prokaryotic diversity. Prokaryotic diversity exhibited in the DNA with the pretreatment was also considerably higher than that extracted with the PowerSoil (TM) DNA Isolation Kit only. The pretreatment method worked well even with a small amount of sediment sample (0.25 g or even lower). The method provides a novel, simple, cost-effective tool for DNA extraction for microbial community analysis in environmental monitoring and remediation processes.