用于土壤生物PCR介导检测的直接DNA提取法。
Direct DNA extraction for PCR-mediated assays of soil organisms.
作者信息
Volossiouk T, Robb E J, Nazar R N
机构信息
Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
出版信息
Appl Environ Microbiol. 1995 Nov;61(11):3972-6. doi: 10.1128/aem.61.11.3972-3976.1995.
Abstract
By using the rDNA of a plant wilt pathogen (Verticillium dahliae) as the target sequence, a direct method for the extraction of DNA from soil samples which can be used for PCR-mediated diagnostics without a need for further DNA purification has been developed. The soil organisms are disrupted by grinding in liquid nitrogen with the natural abrasives in soil, and losses due to degradation and adsorption are largely eliminated by the addition of skim milk powder. The DNA from disrupted cells is extracted with sodium dodecyl sulfate-phenol and collected by ethanol precipitation. After suitable dilution, this DNA extract can be assayed directly by PCR amplification technologies. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of specific soil organisms or pathogens on a large-scale basis.
摘要
以一种植物枯萎病原体（大丽轮枝菌）的核糖体DNA（rDNA）作为靶序列，开发了一种从土壤样品中直接提取DNA的方法，该方法可用于无需进一步DNA纯化的PCR介导诊断。通过在液氮中与土壤中的天然研磨剂一起研磨来破坏土壤微生物，通过添加脱脂奶粉可大大消除由于降解和吸附造成的损失。用十二烷基硫酸钠-苯酚从破碎的细胞中提取DNA，并通过乙醇沉淀收集。经过适当稀释后，该DNA提取物可直接通过PCR扩增技术进行检测。该方法快速、成本效益高，并且与合适的内部对照相结合时，可大规模应用于特定土壤微生物或病原体的检测和定量。
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