Leibniz-Institut für Agrartechnik Potsdam-Bornim eV, Abteilung Bioverfahrenstechnik, Potsdam, Germany.
Syst Appl Microbiol. 2010 Mar;33(2):78-84. doi: 10.1016/j.syapm.2009.11.004. Epub 2010 Jan 22.
Quantitative real-time PCR (Q-PCR) is commonly applied for the detection of certain microorganisms in environmental samples. However, some environments, like biomass-degrading biogas fermenters, are enriched with PCR-interfering substances. To study the impact of the DNA extraction protocol on the results of Q-PCR-based analysis of the methane-producing archaeal community in biogas fermenters, nine different protocols with varying cell disruption and DNA purification approaches were tested. Differences in the quantities of the isolated DNA and the purity parameters were found, with the best cell lysis efficiencies being obtained by a combined lysozyme/SDS-based lysis. When DNA was purified by sephacryl columns, the amount of DNA decreased by one log cycle but PCR inhibitors were eliminated sufficiently. In the case of detection of methanogenic Archaea, the chosen DNA isolation protocol strongly influenced the Q-PCR-based determination of 16S rDNA copy numbers. For example, with protocols including mechanical cell disruption, the 16S rDNA of Methanobacteriales were predominantly amplified (81-90% of the total 16S rDNA copy numbers), followed by the 16S rDNA of Methanomicrobiales (9-18%). In contrast, when a lysozyme/SDS-based cell lysis was applied, the 16S rDNA copy numbers determined for these two orders were the opposite (Methanomicrobiales 82-95%, Methanobacteriales 4-18%). In extreme cases, the DNA isolation method led to discrimination of some groups of methanogens (e.g. members of the Methanosaetaceae). In conclusion, for extraction of high amounts of microbial DNA with high purity from samples of biogas plants, a combined lysozyme/SDS-based cell lysis followed by a purification step with sephacryl columns is recommended.
定量实时 PCR（Q-PCR）常用于检测环境样品中的某些微生物。然而，某些环境，如生物质降解沼气发酵器，富含 PCR 干扰物质。为了研究 DNA 提取方案对沼气发酵器中产甲烷古菌群落 Q-PCR 分析结果的影响，测试了 9 种不同的方案，这些方案在细胞破碎和 DNA 纯化方法上有所不同。发现分离出的 DNA 数量和纯度参数存在差异，通过组合溶菌酶/SDS 基础裂解获得了最佳的细胞裂解效率。当通过 sephacryl 柱纯化 DNA 时，DNA 量减少了一个对数周期，但 PCR 抑制剂被充分去除。在检测产甲烷古菌时，所选的 DNA 分离方案强烈影响基于 Q-PCR 的 16S rDNA 拷贝数的测定。例如，在包括机械细胞破碎的方案中，甲烷杆菌的 16S rDNA 主要被扩增（总 16S rDNA 拷贝数的 81-90%），其次是甲烷微菌的 16S rDNA（9-18%）。相比之下，当应用溶菌酶/SDS 基础细胞裂解时，这两个菌门的 16S rDNA 拷贝数测定结果相反（甲烷微菌 82-95%，甲烷杆菌 4-18%）。在极端情况下，DNA 分离方法导致一些产甲烷菌群（例如甲烷八叠球菌科的成员）的区分。总之，建议从沼气厂的样品中提取高纯度和高数量的微生物 DNA 时，采用组合溶菌酶/SDS 基础细胞裂解，然后通过 sephacryl 柱进行纯化步骤。
