Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens.
Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens.
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DOI：
10.1186/1475-2891-9-23
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发表时间：
2010-05-22
影响因子：
5.4
通讯作者：
Nakatsu CH
中科院分区：
文献类型：
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作者：
Ariefdjohan MW;Savaiano DA;Nakatsu CH
主要使用传统微生物学方法研究饮食对肠道微生物群的影响。尽管这些研究对人类肠道微生物区系有了深入的了解，但必须应用新方法来促进科学进步。聚合酶链反应和变性梯度凝胶电泳 (PCR-DGGE) 的独立于培养物的分子指纹分析方法已用于研究各种环境样品中的微生物群落。然而，这些协议必须在应用之前进行优化，以提高下游分析的质量和准确性。在本研究中，评估了四种商业 DNA 提取试剂盒（Mobio Ultra Clean® 粪便 DNA 分离试剂盒，M；QIAamp® DNA Stool Mini 试剂盒，Q；FastDNA® SPIN 试剂盒，FSp；FastDNA® SPIN 土壤试剂盒，FSo）的相对功效。此外，还评估了 PCR-DGGE 技术检测人类肠道细菌指纹图谱差异的可行性。使用四种不同的试剂盒从不同重量的人类粪便样本中提取总 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。无论使用何种试剂盒，使用 10 至 50 mg（湿重）粪便样本可获得最大 DNA 产量，并获得类似的 DGGE 图谱。然而，与 M 和 Q 试剂盒相比，FSp 和 FSo 试剂盒每克干燥粪便样本提取的 DNA 量显着增加，并且在其 DGGE 谱上产生更多条带，因为它们使用了含珠的裂解基质和剧烈的摇动步骤。 16S rRNA基因PCR产物的DGGE适合捕获人类肠道微生物群落的概况，并能够快速比较评估受试者间和受试者内的差异。我们得出的结论是，结合了含有珠子的裂解基质和剧烈摇动的提取试剂盒从人类粪便样本（10至50毫克，湿重）中产生了高质量的DNA，可以使用PCR-DGGE技术将其解析为细菌群落指纹。随后，PCR-DGGE技术可用于研究人类肠道微生物群落的变化。
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® 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.