[ Objective] The aim was to develop a fast and effective DNA extraction method of intestinal microflora, a modified method of chloroform extraction, and to provide the basis for quantitative and qualitative detection....[ Objective] The aim was to develop a fast and effective DNA extraction method of intestinal microflora, a modified method of chloroform extraction, and to provide the basis for quantitative and qualitative detection. [ Method] Through the improvement of conventional DNA extraction method, a rapid and efficient DNA extraction method was developed. Compared with the real-time PCR result of control sample and the result of QIAamp DNA Stool Mini kit, the developed method was verified. [ Result] The DNA yield of the developed method was 100 times as much as that of QIAamp DNA Stool Mini kit. And the real-time PCR result showed that the efficiency of DNA extraction of the developed method was higher than that of the QIAamp DNA Stool Mini kit. [ Conclusion] This modified method is inexpensive, efficient and rapid, and it is suitable for large quantities of feces samples.展开更多
沉积物成分复杂,腐殖酸等物质含量较高,这些杂质在DNA提取过程中不易除去,可能会对后续的PCR扩增等产生抑制作用。因此,高质量DNA的获得是海洋微生物分子生态学研究的基础和前提。本研究采用6种不同的方法,分别提取同一来源海洋沉积物...沉积物成分复杂,腐殖酸等物质含量较高,这些杂质在DNA提取过程中不易除去,可能会对后续的PCR扩增等产生抑制作用。因此,高质量DNA的获得是海洋微生物分子生态学研究的基础和前提。本研究采用6种不同的方法,分别提取同一来源海洋沉积物样品中的微生物基因组DNA。对DNA纯度、得率、16SrRNA基因拷贝数和微生物群落特征等多方面进行比较,结果表明:方法1(QIAamp DNA Stool Mini Kit)、方法2(PowerSoil~DNA Isolation Kit)和方法3(RNA PowerSoil~DNA Elution Accessory Kit)得到的DNA产量较低,纯度却较高,可以直接用于后续分子生物学分析;与方法2相比,方法3得到的细菌16SrRNA基因拷贝数和单位质量DNA中可扩增细菌16SrRNA基因模板量较低;方法4(SDS高盐法)得到的DNA虽然产量高,但杂质含量也高,抑制了后续PCR反应的进行;方法5(方法4得到的DNA经QIAamp DNA Stool Mini Kit纯化)和方法6(方法4得到的DNA经PowerSoil~DNA Isolation Kit纯化),虽然纯度有所提高,但DNA大量损失,且耗时长,花费高。另外,方法2在提取过程中可以最大程度裂解菌体细胞壁,能更好反映微生物群落特征。综合以上结果,方法2(PowerSoil~DNA Isolation Kit)提取海洋沉积物微生物基因组DNA效果最佳。本研究可为海洋微生物分子生态学研究提供一种有效的技术手段。展开更多
文摘[ Objective] The aim was to develop a fast and effective DNA extraction method of intestinal microflora, a modified method of chloroform extraction, and to provide the basis for quantitative and qualitative detection. [ Method] Through the improvement of conventional DNA extraction method, a rapid and efficient DNA extraction method was developed. Compared with the real-time PCR result of control sample and the result of QIAamp DNA Stool Mini kit, the developed method was verified. [ Result] The DNA yield of the developed method was 100 times as much as that of QIAamp DNA Stool Mini kit. And the real-time PCR result showed that the efficiency of DNA extraction of the developed method was higher than that of the QIAamp DNA Stool Mini kit. [ Conclusion] This modified method is inexpensive, efficient and rapid, and it is suitable for large quantities of feces samples.
文摘沉积物成分复杂,腐殖酸等物质含量较高,这些杂质在DNA提取过程中不易除去,可能会对后续的PCR扩增等产生抑制作用。因此,高质量DNA的获得是海洋微生物分子生态学研究的基础和前提。本研究采用6种不同的方法,分别提取同一来源海洋沉积物样品中的微生物基因组DNA。对DNA纯度、得率、16SrRNA基因拷贝数和微生物群落特征等多方面进行比较,结果表明:方法1(QIAamp DNA Stool Mini Kit)、方法2(PowerSoil~DNA Isolation Kit)和方法3(RNA PowerSoil~DNA Elution Accessory Kit)得到的DNA产量较低,纯度却较高,可以直接用于后续分子生物学分析;与方法2相比,方法3得到的细菌16SrRNA基因拷贝数和单位质量DNA中可扩增细菌16SrRNA基因模板量较低;方法4(SDS高盐法)得到的DNA虽然产量高,但杂质含量也高,抑制了后续PCR反应的进行;方法5(方法4得到的DNA经QIAamp DNA Stool Mini Kit纯化)和方法6(方法4得到的DNA经PowerSoil~DNA Isolation Kit纯化),虽然纯度有所提高,但DNA大量损失,且耗时长,花费高。另外,方法2在提取过程中可以最大程度裂解菌体细胞壁,能更好反映微生物群落特征。综合以上结果,方法2(PowerSoil~DNA Isolation Kit)提取海洋沉积物微生物基因组DNA效果最佳。本研究可为海洋微生物分子生态学研究提供一种有效的技术手段。
