摘要
目的:比较提取高质量艰难梭菌基因组总DNA的方法。方法:采用4种不同方法提取艰难梭菌基因组DNA,比较作为模板用于扩增艰难梭菌看家基因磷酸丙糖异构酶(tpi)基因的优缺点。结果:除沸水浴法,其他3种方法制备的艰难梭菌DNA均能成功用作PCR扩增的模板。CTAB/NaCl法进行提取可有效去除多糖成分,但操作要求较高。碱裂解法操作简便、省时、成本低经济可靠,提取的DNA量及纯度较合适;试剂盒抽提出的产物纯度和产量明显高于CTAB/NACL、碱裂解法(P<0.05),但成本高。结论:四种方法各具优缺点,应根据实验室条件和实验要求进行选择。
Objective： To compare the relative efficiency and quality of extraction of C.difficile DNA using four different methods. Methods： Extract C.difficile genomic DNA with four different methods： immersion method, CTAB degrading solution, Alkali solit method and kit. To amplified the special gene （tpi） of C. difficile by real-time PCR. Results： The products by the three methods are suitable for real-time PCR amplification, but except immersion method. Extraction with CTAB degrading solution could remove the polysaccharide, but the method demands more operating skill. Alkali solit method offered a higher OD260/280 （1.80＋0.06） and this method is simpler and cheaper. The purity and output of DNA extracted by kit were obviously higher （P〈0.05） ,but the method was more amplicated and expensive. Conclusion： The four methods all have advantages and disadvantages of their own, and which method to use should be based on the lab conditions and research demands.
基金
江苏检验检疫局科研项目(2010KJ17)
