不同粪便DNA提取方法比较分析

Comparative analysis of different fecal DNA extraction methods

摘要:

肠道微生物群落结构和多样性与人体疾病密切相关。然而，相关群落结构分析结果可能受到DNA提取质量等实验因素影响。因此，评估不同DNA提取方法对肠道特定种属的提取效果，对于全面、准确获取人体肠道微生物谱，深入探究肠道微生物群落结构具有指导意义。本研究旨在借助实时荧光定量PCR (real-time quantitative polymerase chain reaction,RT qPCR)技术，以DNA提取纯度、浓度，以及对肠道中特定种属微生物基因组DNA的提取丰度为指标，对5种DNA提取方法进行比较分析。结果表明，试剂盒Q的提取效果最佳，特别是对乳杆菌属和双歧杆菌属等革兰氏阳性菌的提取效果较好。N试剂盒的平均DNA提取浓度较Q试剂盒低，但在纯度方面，二者无显著性差异。与其他3种商用试剂盒(M、PSP、TG)相比，N方法对肠道内指定微生物基因组的提取效果仅次于Q试剂盒，位居第二。相比之下，M试剂盒提取所得DNA，质量较高，但浓度偏低，对于肠道内革兰氏阳性菌的提取效果不很理想。TG试剂盒和PSP试剂盒提取所得DNA在浓度、质量以及细菌丰度方面均不及其他验证的试剂盒。综上，Q试剂盒可作为肠道微生态研究相关实验中获取高质量基因组DNA的提取方法。本研究结果为肠道微生态研究相关实验中基因组DNA提取方法的选择提供参考依据。

Abstract:

The community structure and diversity of the gut microbiota are associated with human diseases. However, the analysis of different community structure might be influenced by experimental approaches such as the quality of DNA extraction. Therefore, evaluating the efficiency of different DNA extraction methods for specific intestinal species is a guideline for obtaining a comprehensive human gut microbial profile, which may assist the in-depth investigation into the structure of the gut microbial community. The aim of this study was to perform a comparative analysis of five different DNA extraction methods. With the aid of qPCR, the efficiency of five DNA extraction kits was evaluated in terms of the purity of the extracted DNA, the DNA concentration, and the abundance of genomic DNA extracted from specific intestinal species. The results showed that the kit Q gave the best extraction results, especially for Gram-positive bacteria such as Lactobacillus and Bifidobacterium. The average DNA concentration of the N kit was lower than that of the Q kit, but there was no significant difference between the two in terms of the purity. Compared to the other three commercial kits (M, PSP, TG), the efficiency of the N kit in extracting the genomic DNA of the specified microorganisms were the least different from those of the Q kit. In contrast, the DNA extracted by the M kit was of higher quality but of lower concentration, and was not very efficient for Gram-positive bacteria. The DNA extracted by the TG and PSP kits was inferior to the other validated kits in terms of the concentration, quality and bacterial abundance. These results provide a basis for the selection of genomic DNA extraction methods in microecological research experiments.