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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,A_(260)/A_(280)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 compare the effects of three DNA extraction methods of Staphylococcus aureus in order to obtain a simple and economical extraction method. Methods The bacterial DNA of Staphylococcus aureus ATCC25923 was extracted by modified SDS method, NP40 method and bacterial DNA kit method respectively. The nucleic acid concentration and purity were determined by Nano Drop 2000 nucleic acid detector. A260 / A280, the ratio of the absorbance value at 280 nm and the absorbance at the wavelength of 280 nm. The quality of DNA was detected by 1% agarose gel electrophoresis. The bacterial DNA of Staphylococcus aureus ATCC25923 with different concentration was extracted and the 16S r DNA fragment was amplified by PCR And compare the sensitivity of the three methods. Results The results showed that there was a significant difference in the nucleic acid concentration of S. aureus (χ ~ 2 = 6.25, P <0.05). The concentration of NP40 was higher and the SDS was lower. The purity of the DNA extracted by the kit method was higher, the value of A 260 / A 280 was between 1.8 and 2.0, and the purity of NP40 was poor. The results of DNA electrophoresis showed that the DNA bands extracted by SDS method and TIANGEN kit Clear, no towing phenomenon, NP40 method does not appear the main band of DNA, and the towing phenomenon. All methods of DNA obtained by PCR amplification positive bands. The highest PCR sensitivity of NP40 DNA extraction was 1.5 × 10 ~ 6 cfu / ml, while the lowest sensitivity of modified SDS was 1.5 × 10 ~ 8 cfu / ml. Conclusion The NP40 DNA extraction method has the advantages of low cost, less time consuming and high sensitivity, and is suitable for rapid screening of Staphylococcus aureus.