大鼠肠内容物细菌基因组DNA提取方法的比较

Comparison of bacterial genomic DNA extraction methods from Sprague-Dawley rat fecal samples

目的比较两种肠内容物前处理和两种提取方法对清洁级SD大鼠肠内容物细菌基因组DNA提取效率。方法分别选用PBS多次离心漂洗、液氮破细胞两种前处理方法和酚/氯仿抽提、试剂盒过柱法两种提取方法进行组合分析,对4份肠内容物和16份含金黄色葡萄球菌肠内容物进行随机提取。结果大鼠肠内容物细菌基因组DNA含量和纯度测定结果显示,与PBS反复离心相比,液氮研磨前处理能显著提高大鼠肠内容物基因组DNA。荧光定量PCR表明,液氮研磨前处理较PBS反复离心能更好地收集细菌基因组DNA,其Ct值最低。结论研究结果表明,采用液氮研磨试剂盒法在大鼠肠内容物DNA提取中是较为优良的方法,该方法为建立实验动物中微生物的定量PCR检测方法打下了基础。

Objective To compare four methods of bacterial genomic DNA extraction from Sprague-Dawley rat fecal samples.Methods Sixteen fecal samples with 106 cfu S.aureus and 4 fecal samples without S.aureus were equally divided into four groups.Bacterial genomic DNA were extracted by following methods：（1） Several wash by PBS plus phenol/chloroform purification;（2） Liquid nitrogen digestion plus column purification;（3） PBS wash plus column purification,and（4） Liquid nitrogen digestion followed by phenol/chloroform purification.The DNA concentration and A260/A280 were detected.Ct values were also measured by a quantitative PCR.Results Pre-extration by liquid nitrogen improved the DNA concentration and values of A260/A280.The Ct values of Group 2 was the lowest.Conclusion Our results indicate that liquid nitrogen extraction plus column purification is suitable for bacterial genomic DNA extraction in PCR detection of pathogens in laboratory animals.