五种DNA提取方法对酸奶及菌粉中益生菌DNA提取效果比较
Evaluation of five DNA extraction methods for Probiotic yogurt and powder
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摘要: 针对酸奶及菌粉样品,比较多种样品前处理条件,包括取样量、菌粉溶剂及溶菌酶孵育时间等,最大程度去除样品中的脂肪、蛋白质及多糖,比较五种DNA提取试剂盒对益生菌DNA的提取效果。提取的DNA经Nano Vue Plus测定浓度和纯度,PCR扩增综合评价提取效果。结果显示,酸奶取样量为100 mg时DNA浓度为49.00 ng/μL,A260/A280比值为1.8;采用纯牛奶溶解菌粉更为彻底,0.5 g/m L牛奶溶解菌粉后,提取DNA浓度可达到81.00 ng/μL;溶菌酶最佳孵育时间为1 h,增加孵育时间对细菌裂解没有明显帮助。同时,采用五种试剂盒所提取的DNA浓度均在20 ng/μL以上,A260/A280比值大于1.8或接近1.8,其中,Tiangen试剂盒提取的DNA浓度和纯度综合评价优于其他4种试剂盒。结果表明,实验建立的前处理方法能够有效的去除酸奶及菌粉中的脂肪、蛋白质及多糖,提高DNA提取的浓度及纯度;采用的五种商品化试剂盒均能用于酸奶和菌粉中益生菌DNA快速提取,在步骤、得率及纯度上各有差异,应按照不同的实验要求进行选择。Abstract: Yogurt and Lactic acid bacteria( LAB) powder were chosen as samples.The sampling quantities,solvents of LAB powder and incubation time of lysozyme have been optimized to remove fats,proteins and polysaccharides.Then five commercialized DNA extraction kits were compared on their DNA extraction efficiency after sample pretreatment. The quality and quantity of the extracted DNA were evaluated by Nano Vue Plus and conventional PCR.It is showed that when the sampling quantity of yogurt was 100 mg,the DNA concentration was49.00 ng / μL,and A260/A280 ratio was 1.8. The milk was more suitable as the solvent for LAB powder compared to distilled water,and when the sampling quantity of LAB powder was 0.5 g / m L,the DNA concentration was 81.00 ng / μL.The best incubation time of lysozyme was 1 h,and the expanded incubation time had no significant improvement for cell lysis.The extracted DNA concentrations from five kits were all over 20 ng / μL,and the A260/A280 ratio was greater than or close to 1.8.Among the five kits,the one from Tiangen had the best DNA concentration and purity.It was proved that the established pretreatment method can effectively remove fats,proteins and polysaccharides.Each of the five kits can be used to extract DNA from yogurt and LAB powder,however,the choice should be made based on different experiment requests.
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