8种快速制备细菌基因组DNA方法的效果评价

发布时间：2018-01-10 15:16

本文关键词：8种快速制备细菌基因组DNA方法的效果评价　出处：《现代预防医学》2017年16期 　论文类型：期刊论文

【摘要】：目的探索用于革兰阴/阳性细菌基因组DNA制备的快速、简便、经济方法。方法采用水煮沸法、碱煮沸法、碱-SDS-煮沸法、丙酮-SDS-煮沸法、NP-40-煮沸法、Triton-100-煮沸法、SDS-煮沸法、Triton-100-NP-40-煮沸法制备大肠埃希菌和金黄色葡萄球菌DNA,观察不同方法所得DNA的量、分子大小、纯度以及用其作为模板进行实时荧光定量PCR扩增反应效果。结果与其他方法比较,碱-SDS-煮沸法、丙酮-SDS-煮沸法和SDS-煮沸法得到的DNA量较多、DNA分子较大、纯度较好;碱-SDS-煮沸法上清液DNA含量最大及PCR扩增效果最佳,水煮沸法所得DNA含量显著低于其他方法,差异有统计学意义(P0.05)。各方法煮沸5 min与10 min所得DNA量和PCR检测DNA的循环阈值均无统计学差异(P0.05),煮沸10 min的DNA液中有机杂质增多。结论碱-SDS-煮沸法煮沸5min制备细菌基因组DNA的效果最优,水煮沸法效果最差。
[Abstract]:Objective to explore for gram negative / positive bacterial genomic DNA prepared by rapid, simple, economic method. Methods using water boiling method, alkali boiling method, -SDS- acetone -SDS- alkali boiling method, boiling method, NP-40- boiling method, Triton-100- boiling method, SDS- boiling method, Triton-100-NP-40- boiling preparation of Escherichia coli and Staphylococcus Staphylococcus DNA, observation of DNA from different methods, molecular size, purity and use it as a template for amplification effect of real-time fluorescence quantitative PCR. Results compared with other methods, -SDS- alkali boiling method, DNA large amount of acetone and SDS- -SDS- obtained by boiling boiling method, DNA large molecules, high purity alkali; -SDS- the supernatant DNA content maximum boiling method and PCR amplification effect is the best, the boiling water method DNA is significantly lower than the other methods, the difference was statistically significant (P0.05). The method of boiling 5 min and 10 min of the DNA and PCR. There was no significant difference in the threshold value of DNA for measuring the P0.05 (P0.05). The organic impurities in the DNA liquid boiled 10 min increased. Conclusion the boiling effect of alkali -SDS- boiling on 5min is the best, and the boiling method is the worst.

【作者单位】：成都中医药大学医学技术学院;
【分类号】：R440
【正文快照】： 随着核酸快速鉴定时代的到来,更需要加快和简化核酸制备,包括减少有害化学品使用、减少冷藏、更少制备步骤、更快或更有效的裂解、自动化[1]。核酸制备涉及裂解细胞/生物以释放核酸、核酸与蛋白分离、去除核酸中抑制物等。常用细胞裂解方法有物理法(煮沸、反复冻-融、超声波)

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