三种方法提取金黄色葡萄球菌DNA的比较  

Compare three DNA extraction methods for Staphylococcus aureus

作　　者：葛婧[1] 钟雪珊[1] 肖刚[2] 陈佑明[2] 陈清[1] 

机构地区：[1]南方医科大学公共卫生学院,广东广州510515 [2]南方医科大学第三附属医院检验科,广东广州510630

出　　处：《热带医学杂志》2016年第10期1235-1238,1244,共5页Journal of Tropical Medicine

基　　金：国家自然科学基金(81373051)

摘　　要：目的 对三种金黄色葡萄球菌DNA提取方法的效果进行比较,以期获得一种简便、经济的提取方法。方法 采用改良SDS法、NP40法和细菌DNA试剂盒法分别提取同一浓度的金黄色葡萄球菌ATCC25923细菌DNA,利用Nano Drop 2000核酸检测仪测定核酸浓度和纯度,纯度用核酸在波长260 nm处的吸光值与在波长280 nm处的吸光值的比值A260/A280表示,用1%琼脂糖凝胶电泳检测DNA质量;提取不同浓度梯度的金黄色葡萄球菌ATCC25923细菌DNA,PCR扩增16S r DNA片段检测并比较三种方法的灵敏度。结果 三种方法提取金黄色葡萄球菌所得核酸浓度差异有统计学意义（χ^2=6.25,P〈0.05）,NP40法提取浓度较高,改良SDS法较低。试剂盒法提取的DNA纯度较高,A260/A280值在1.8~2.0之间;NP40法提取纯度较差;DNA电泳结果表明SDS法和TIANGEN试剂盒法提取的DNA条带清晰,无拖带现象,NP40法未出现DNA主带,且有拖带现象。所有方法所得DNA经PCR扩增均出现阳性条带。NP40法提取DNA的PCR检测敏感度最高,为1.5×10^6 cfu/ml,改良SDS法敏感度最低,为1.5×10^8 cfu/ml。结论 NP40法提取DNA成本低,耗时少,灵敏度高,适用于金黄色葡萄球菌的快速初筛。Objective To find a simple and economic method of DNA extraction for Staphylococcus aureus by evaluating three different methods. Methods DNAs from the same concentration of Staphylococcus aureus （ATCC 25923） were extracted by three different DNA extraction methods, the modified SDS method, the NP40 method and the commercial bacteria DNA kit method. The concentrations and purities of nucleic acid were detected by using Thermo Scientific NanoDrop 2000. The purities were expressed by the value of A260/A280, which was the ratio of nucleic acid sample absorbance at 260 nm to absorbance at 280 nm. 1% agarose gel electrophoresis was used to detect DNA′s quality. Staphylococcus aureus （ATCC 25923） DNA was extracted by the three methods using different bacteria concentrations , and amplified by PCR using primers for 16SrDNA to compare the sensitivity. Results The differences in the concentrations of nucleic acid extracted by the three methods were statistically significant （χ^2=6.25,P〈0.05）. The yield of the NP40 method was superior, while the modified SDS method was inferior. The kit method was with the better purity of DNA and offered a better value of A 260/A280（1.8~2.0）, while the purity of the NP40 method was inferior. The agarose gel electrophoresis of DNA obtained by the modified SDS method and the kit method demonstrated clear bands without trailing. While the agarose gel electrophoresis of the DNA obtained by NP40 method displayed no main band and there was trailing. Signals were obtained using PCR on the DNA samples extracted from the same concentration of the three methods. The result of electrophoresis showed that high sensitivity of PCR on the DNA extracted by NP40 method, which was 1.5×10^6 cfu/ml. A low sensitivity of 1.5×10^8 cfu/ml was found for the sample obtained by modified SDS method. Conclusion The NP40 method was a low cost, less time-consuming method with high sensitivity, which was appropriate for rapid primary screening of Staphylococcus aureus.

关 键 词：金黄色葡萄球菌 DNA提取 核酸 

分 类 号：R378.11[医药卫生—病原生物学]