摘要
目的:提出一种适于微生物多样性分析的青贮饲料中微生物总DNA的提取方法,并评价其效果。方法:间接法抽提样本的总DNA,通过琼脂糖电泳、紫外吸收及PCR分析DNA质量,DGGE评价提取效果,用PCR扩增目的菌株的特定片段来检测提取方法的灵敏度。结果:两个样本DNA的A260/A280值分别为1.99和1.93,A260/A230值分别为2.19和1.90,提取的DNA不需纯化便可直接用于16S rRNA基因的扩增,提取方法灵敏度为3cfu/g,DGGE结果表明提取方法可以涵盖样品中的所有微生物。结论:提取的DNA纯度较高,可直接用于下游分子操作,提取方法灵敏度较高,能全面反映样品中的微生物原貌,可用于免培养法研究青贮饲料中的微生物菌群组成。
Objective：To optimize the DNA isolation for microbial diversity analysis in the silage samples, and the efficiency of introduced extraction method was assessed. Method： The method was based on indirect extraction methods. Extracted DNA was measured by agarose gel electrophoresis, and spectrophotometric analysis. The recovery efficiency was assessed using DGGE, sensitivity was evaluated by detecting target sequence of added microorganisms using PCR. Result： A260/280 of total DNA from two sampled silage was 1.99 and 1.93, respectively, while A260/280 ratio was 2.19 and 1.90, respectively. The DNA obtained could directly be used to PCR amplification without purification, the limit of detection was 3cfu/g. The result of DGGE suggested that introduced method had a well recovery. Conclusion： The method described in this paper indicated high efficiency, recovery and sensitivity, suggesting the procedure could be a reliable method to extract DNA from silage for molecular biology purposes.
出处
《生物技术》
CAS
CSCD
北大核心
2009年第1期29-32,共4页
Biotechnology
基金
国家"863"计划项目资助(2006AA10Z3442006AA10Z321)
