Doctorado en Ciencias Mención Biología Celular y Molecular Aplicada, Universidad de La Frontera, Temuco 4811230, Chile.
Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
Int J Mol Sci. 2023 Oct 17;24(20):15243. doi: 10.3390/ijms242015243.
Obtaining sufficient and high-quality genomic DNA from sludge samples is a fundamental issue of feasibility and comparability in genomic studies of microbial diversity. Commercial kits for soil are often used for the extraction of gDNA from sludge samples due to the lack of specific kits. However, the evaluation of the performance of commercial kits for sludge DNA extraction is scarce and optimization of these methods to obtain a high quantity and quality of DNA is necessary, especially for downstream genomic sequencing. Sequential batch reactors (SBRs) loaded with lignocellulosic biomass are used for the synthesis of renewable resources such as levulinic acid (LA), adipic acid (AA), and polyhydroxyalkanoates (PHAs), and the biochemical synthesis of these compounds is conducted through the inoculation of microbes present in the residual activated sludge (AS) obtained from a municipal wastewater treatment plant. To characterize these microbes, the extraction of DNA from residual sewage sludge was conducted with three different commercial kits: Nucleospin Soil from Macherey-Nagel, DNEasy PowerSoil from Qiagen, and E.Z.N.A. Plant DNA Kit from Omega BIO-TEK. Nevertheless, to obtain the highest load and quality of DNA for next-generation sequencing (NGS) analysis, different pretreatments and different combinations of these pretreatments were used. The pretreatments considered were an ultrasonic bath and a temperature of 80 °C, together and separately with different incubation time periods of 30, 60, and 90 min. The results obtained suggest a significant improvement in the efficiency and quality of DNA extraction with the three commercial extraction kits when used together with the ultrasonic bath and 80 °C for 60 min. Here, we were able to prove that physical pretreatments are a viable alternative to chemical lysis for DNA extraction from complex samples such as sludge.
从污泥样品中获得足够且高质量的基因组 DNA 是微生物多样性基因组研究可行性和可比性的基础问题。由于缺乏专用试剂盒，因此经常使用用于土壤的商业试剂盒来提取 gDNA 从污泥样品中。然而，对污泥 DNA 提取用商业试剂盒性能的评估很少，需要对这些方法进行优化以获得高质量和高产量的 DNA，特别是对于下游基因组测序。用于木质纤维素生物质的序批式反应器 (SBR) 用于合成可再生资源，如乙酰丙酸 (LA)、己二酸 (AA) 和聚羟基烷酸酯 (PHA)，并且这些化合物的生物化学合成是通过接种市政污水处理厂剩余活性污泥 (AS) 中存在的微生物进行的。为了表征这些微生物，使用三种不同的商业试剂盒从剩余污水污泥中提取 DNA：来自 Macherey-Nagel 的 Nucleospin Soil、Qiagen 的 DNEasy PowerSoil 和 Omega BIO-TEK 的 E.Z.N.A. Plant DNA 试剂盒。然而，为了获得用于下一代测序 (NGS) 分析的最高负载和质量的 DNA，使用了不同的预处理方法和这些预处理方法的不同组合。考虑的预处理方法是超声浴和 80°C 的温度，单独使用和一起使用不同的孵育时间 30、60 和 90 分钟。结果表明，当与超声浴和 80°C 一起使用 60 分钟时，三种商业提取试剂盒的效率和 DNA 提取质量显著提高。在这里，我们证明了物理预处理是从复杂样品（如污泥）中提取 DNA 的一种可行的化学裂解替代方法。
