Efficient isolation of target DNA is a crucial first step of DNA-based metagenomic analyses of environmental samples. Insufficient quantity and purity of DNA isolated using commercial kits result in missing genetic information, especially for large-diameter substrates in constructed wetlands (CWs). Here, we addressed this problem by devising a cost-effective calcium chloride lysozyme-sodium dodecyl sulfate (SDS) method (CCLS), with key improvements in the steps of humic acid removal and cell lysis. The buffer comprising Tris, EDTA, Na2O2P7 and PVPP (TENP), and skim milk, could reduce adsorption between microorganisms and substrates, and calcium chloride precipitated and removed over 94% of humic acid. This humic acid removal step, when compared to the PowerSoil DNA kit (MO BIO Laboratories Inc.) (MBKIT), significantly enhanced the DNA purity (A260/230) from 0.68 to 1.63 (p < 0.01). When gentle and extended cell lysis in CCLS replaced the short but violent bead-beating in the MBKIT, DNA yield and the amount of lysed bacteria detected by quantitative real-time polymerase chain reaction (qPCR) on average increased by 2 and 4 folds, respectively, compared to that obtained using the MBKIT (p < 0.01). Furthermore, the full-length bacterial 16S rRNA gene and nirK gene from denitrifying microorganisms were successfully amplified from CCLS-generated DNA. Additionally, bacterial diversity indices of richness, Shannon, and evenness examined by denaturing gradient gel electrophoresis (DGGE) increased by 75, 30, and 7%, respectively, by CCLS compared to that using the MBKIT. Hence, the CCLS method enables improved evaluation of microbial density and diversity in CW systems.

中文翻译：

---

改进的DNA纯化，具有质量保证，可评估人工湿地的微生物遗传含量。

有效分离目标DNA是环境样品基于DNA宏基因组学分析的关键第一步。使用商业试剂盒分离的DNA数量和纯度不足会导致缺少遗传信息，尤其是对于人工湿地（CW）中的大直径底物。在这里，我们通过设计一种经济高效的氯化钙溶菌酶-十二烷基硫酸钠（SDS）方法（CCLS）解决了这个问题，并对腐殖酸去除和细胞裂解步骤进行了重大改进。包含Tris，EDTA，Na2O2P7和PVPP（TENP）的缓冲液以及脱脂乳可以减少微生物与底物之间的吸附，氯化钙沉淀并除去94％以上的腐殖酸。与PowerSoil DNA试剂盒（MO BIO Laboratories Inc.）（MBKIT）相比，该去除腐殖酸的步骤，将DNA纯度（A260 / 230）从0.68显着提高到1.63（p <0.01）。当CCLS中的温和扩展细胞裂解代替MBKIT中短而剧烈的珠击时，通过定量实时聚合酶链反应（qPCR）检测到的DNA产量和裂解细菌数量平均分别增加了2倍和4倍。 ，与使用MBKIT获得的结果相比（p <0.01）。此外，从CCLS生成的DNA中成功地扩增了反硝化微生物的全长细菌16S rRNA基因和nirK基因。此外，与使用MBKIT相比，通过变性梯度凝胶电泳（DGGE）检测的细菌丰度，香农和均匀度的细菌多样性指数分别由CCLS提高了75、30和7％。因此，