口腔龈上菌斑5种DNA提取方法的比较

摘要

目的 寻找方便、快捷、经济、获取细菌信息量最多的细菌基因组DNA抽提方法.方法 采用洗涤+冻融+Bead Beater+酚氯仿抽提法(X法)、试剂盒法(Y法)、洗涤+冻融+试剂盒法(Z法)、直接煮沸法(U法)和加Chelex-100后煮沸法(V法)5种DNA抽提方法,分别提取3例慢性牙周炎患者龈上菌斑DNA,检测DNA片段大小和浓度;同时运用变性梯度凝胶电泳分析样本微生物多样性和相似度,从多个角度对5种DNA提取方法 进行评价.结果 X法DNA片段集中于3 000～20 000bp处;Y法和Z法DNA片段均匀分布于500～20 000 bp处;U法无明显DNA片段出现;V法DNA片段主要分布于500～2 000 bp处.Y法、Z法与V法所提DNA浓度较高,X法与U法所提DNA浓度较低.X法、Y法、Z法和V法反应的龈上菌斑微生物多样性无显著差异.X法、Y法和Z法的相似度较高,而V法与X法、Y法、Z法的相似度较低.结论 洗涤+冻融+Bead Beater+酚氯仿抽提法,试剂盒法,洗涤+冻融+试剂盒法,加Chelex-100后煮沸法的DNA提取效果无显著差异;直接煮沸法的DNA提取效果较差.%Objective To find a bacteria DNA extraction method which is convenient,quick,cheap and also can get more bacteria information. Methods To extract supragingival plaque DNA of 3 chronic periodontitis patients with Washing + Freeze-Thawing + Bead Beater + Phenol-Chloroform Extraction ( Method X), DNA Extraction Kit ( Method Y ), Washing + Freeze-Thawing + DNA Extraction Kit (Method Z), Boiling (Method U) and Boiling With Chelex-100 (Method V), and then analysed the length and concentration of DNA, include the microorganism diversity and similarity with Denaturing Gradient Gel Electrophoresis( DG, GE), and assenas these DNA extraction methods from different aspects. Results DNA located 3kbp-20kbp in Method X and 500bp-20kbp in Method Y and Z;there was no significant DNA image in Method U; and in Method V DNA located 500bp-2kbp. The concentration of DNA was high in Method Y、 Z and V, however, it was low in Method X and U. There was no significant difference in the microorganism diversity of supragingival plaque in Method X、Y、Z and V. The similarity was high among Method X、Y and Z,but comparing Method V to Method X、Y and Z, the similarity was low. Conclusions There was no significant difference in Washing + Freeze-Thawing + Bead Beater + Phenol-Chloroform Extraction, DNA Extraction Kit, Washing + Freeze-Thawing + DNA Extraction Kit, and Boiling With Chelex-100. Boiling was not good enough for DNA extraction.