Maciel B M, Santos A C F, Dias J C T, Vidal R O, Dias R J C, Gross E, Cascardo J C M, Rezende R P
Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Salobrinho, Ilhéus, BA, Brazil.
Genet Mol Res. 2009 Mar 31;8(1):375-88. doi: 10.4238/vol8-1gmr559.
Landfarm soil is used to bioremediate oil wastes from petrochemical industries. We developed a simplified protocol for microbial DNA extraction of tropical landfarm soil using only direct lysis of macerated material. Two samples of tropical landfarm soil from a Brazilian refinery were analyzed by this protocol (one consisted of crude oil-contaminated soil; the other was continuously enriched for nine months with petroleum). The soil samples were lysed by maceration with liquid nitrogen, eliminating the need for detergents, organic solvents and enzymatic cell lysis. Then, the DNA from the lysed soil sample was extracted using phenol-chloroform-isoamyl alcohol or guanidium isothiocyanate, giving high DNA yields (more than 1 micro g DNA/g soil) from both soil types. This protocol compared favorably with an established method of DNA template preparation that included mechanical, chemical and enzymatic treatment for cell lysis. The efficiency of this extraction protocol was confirmed by polymerase chain reaction amplification of the 16S rRNA gene, denaturing gradient gel electrophoresis and cloning assays. Fifty-one different clones were obtained; their sequences were classified into at least seven different phyla of the Eubacteria group (Proteobacteria - alpha, gamma and delta, Chloroflexi, Actinobacteria, Acidobac teria, Planctomycetes, Bacteroidetes, and Firmicutes). Forty percent of the sequences could not be classified into these phyla, demonstrating the genetic diversity of this microbial community. Only eight isolates had sequences similar to known sequences of 16S rRNA of cultivable organisms or of known environmental isolates and therefore could be identified to the genus level. This method of DNA extraction is a useful tool for analysis of the bacteria responsible for petroleum degradation in contaminated environments.
陆地农场土壤用于对石化工业产生的油类废物进行生物修复。我们开发了一种简化方案，仅通过对研磨后的材料进行直接裂解来提取热带陆地农场土壤中的微生物DNA。采用该方案分析了来自巴西一家炼油厂的两个热带陆地农场土壤样本（一个样本是原油污染土壤；另一个样本用石油连续富集了九个月）。通过用液氮研磨使土壤样本裂解，无需使用洗涤剂、有机溶剂和酶促细胞裂解。然后，使用苯酚 - 氯仿 - 异戊醇或异硫氰酸胍从裂解后的土壤样本中提取DNA，两种土壤类型均获得了高DNA产量（超过1μg DNA/g土壤）。该方案与一种既定的DNA模板制备方法相比具有优势，既定方法包括用于细胞裂解的机械、化学和酶处理。通过16S rRNA基因的聚合酶链反应扩增、变性梯度凝胶电泳和克隆分析证实了该提取方案的效率。获得了51个不同的克隆；它们的序列被归类为真细菌组的至少七个不同门（变形菌门 - α、γ和δ、绿弯菌门、放线菌门、酸杆菌门、浮霉菌门、拟杆菌门和厚壁菌门）。40%的序列无法归类到这些门中，这表明了该微生物群落的遗传多样性。只有八个分离株的序列与可培养生物或已知环境分离株的16S rRNA已知序列相似，因此可以鉴定到属水平。这种DNA提取方法是分析污染环境中负责石油降解的细菌的有用工具。
