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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(P<0.05)。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(P>0.05),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。
Objective To explore a rapid, simple and economical method for genomic DNA preparation of gram negative / positive bacteria. Methods Triton-100-NP-40 was prepared by the methods of water boiling, alkaline boiling, alkali-SDS-boiling, acetone- SDS- boiling, NP- 40- boiling, Triton- 100- boiling, SDS- boiling, - Boiling method for the preparation of Escherichia coli and Staphylococcus aureus DNA, observed the amount of DNA obtained by different methods, molecular size, purity and use as a template for real-time fluorescence quantitative PCR amplification reaction. Results Compared with other methods, the amount of DNA obtained by alkali-SDS-boiling method, acetone-SDS-boiling method and SDS-boiling method was larger, the DNA molecules were larger and the purity was better. The content of DNA was the highest and the PCR amplification was the best. The DNA content of water-boiling method was significantly lower than other methods, the difference was statistically significant (P <0.05). There was no significant difference in the DNA content between 5 min and 10 min boiled and 10 min DNA recycle (P> 0.05). The organic impurities in the DNA solution boiled for 10 min increased. Conclusion Alkali-SDS-Boiling is the best method to prepare bacterial genomic DNA after boiling for 5 minutes, and water-boiling method is the worst.