牛奶中无乳链球菌DNA提取方法的比较

Comparison of different methods for DNA extraction of Streptococcus aga-lactiae in milk

中文关键词:  牛奶  无乳链球菌  DNA提取  紫外分光光度计法  实时荧光PCR

英文关键词:milk  Streptococcus agalactiae  DNA extraction  ultraviolet spectrophotometry  real time fluorescence PCR

中文摘要:

      目的 以牛奶中的致病性无乳链球菌作为研究对象, 对9种常用的细菌核酸提取方法进行比较。方法 选用紫外分光光度计法和实时荧光PCR法对各方法进行系统评估。结果 超声波处理结合天根吸附柱的方法能提取到得率较高、纯度较好的DNA, 结合实时荧光PCR法后对牛奶中无乳链球菌的检测灵敏度能达到90 cfu; 天根吸附柱法提取所得DNA得率稍低, 结合实时荧光PCR法后对牛奶中无乳链球菌的检测灵敏度也能达到90 cfu; 溶菌酶法和超声波破碎提取法用于实时荧光PCR后对牛奶中无乳链球菌的检测灵敏度能达到350 cfu; Chelex-100法更适于纯菌的检测, 该法结合实时荧光PCR法对无乳链球菌的检测灵敏度能达到350 cfu的灵敏度。结论 应根据样品类型、方法要求和检测成本来选择适合的核酸提取方法。

英文摘要:

      Objective To compare the 9 extraction methods of extracting the DNA from Streptococcus agalactiae in milk. Methods The methods were evaluated by using ultraviolet spectrophotometry and real time fluorescence PCR. Results The results showed that the purity and yield of genomic DNA by TianGen adsorption column method with sonication was the most suitable one among the 10 methods. The low detection limit in milk of TianGen adsorption column method and TianGen adsorption column method with sonication were 90 cfu. Enzymolysis method, sonication method and TianGen adsorption column method were fitted for the detection of Streptococcus agalactiae in milk, while Chelex-100 method was fitted for the detection of Streptococcus agalactiae in pure bacteria. The low detection limit in milk of enzymolysis method and sonication method were both of 350 cfu, and the low detection limit in pure bacteria of Chelex-100 method was 350 cfu. Conclusion The appropriate nucleic acid extraction method should be decided according to sample types, methods and testing cost.