Department of Foods and Nutrition, Purdue University, 700 West State St, West Lafayette, IN 47907-2059, USA.
Nutr J. 2010 May 22;9:23. doi: 10.1186/1475-2891-9-23.
The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean(R) Fecal DNA Isolation Kit, M; QIAamp DNA Stool Mini Kit, Q; FastDNA SPIN Kit, FSp; FastDNA SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles.
Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.
Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.
We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.
人们主要采用传统微生物方法研究饮食对肠道微生物群的影响。虽然这些研究推动了人类肠道微生物群的相关知识，但应用新方法促进科学进步势在必行。聚合酶链反应和变性梯度凝胶电泳（PCR-DGGE）的无培养分子指纹图谱方法已被用于研究各种环境样本中的微生物群落。然而，在将这些方案应用于下游分析之前，必须对其进行优化，以提高质量和准确性。在这项研究中，评估了四种商业 DNA 提取试剂盒（Mobio Ultra Clean（R）粪便 DNA 分离试剂盒、M；QIAamp DNA 粪便试剂盒、Q；FastDNA SPIN 试剂盒、FSp；FastDNA SPIN 试剂盒，用于土壤，FSo）的相对功效。此外，还评估了 PCR-DGGE 技术在检测人类肠道细菌指纹图谱差异方面的可行性。
使用四种不同的试剂盒从不同重量的人类粪便标本中提取总 DNA，然后对细菌 16S rRNA 基因进行 PCR 扩增，并对扩增子进行 DGGE 分离。
无论使用哪种试剂盒，从 10 到 50mg（湿重）的粪便标本中都能获得最大的 DNA 产量，并且获得的 DGGE 图谱相似。然而，试剂盒 FSp 和 FSo 提取的每克干粪便标本中的 DNA 量明显多于试剂盒 M 和 Q，因为它们使用了含珠的裂解基质和剧烈的摇床步骤，并且在其 DGGE 图谱上产生了更多的条带。16S rRNA 基因 PCR 产物的 DGGE 适合捕捉人类肠道微生物群落的图谱，并能够快速比较评估个体间和个体内的差异。
我们得出结论，从 10 到 50mg（湿重）的人类粪便标本中提取高质量 DNA 的试剂盒（含珠的裂解基质和剧烈的摇床），可通过 PCR-DGGE 技术作为细菌群落指纹图谱进行解析。随后，PCR-DGGE 技术可用于研究人类肠道微生物群落的变化。
