| 摘要: |
| 【目的】提高牛奶中牛DNA的分离提取效率，建立稳定的牛奶中牛DNA提取的试剂盒法。【方法】以从牧场中采集的新鲜牛奶为材料，在前期建立的牛奶中牛DNA提取方法基础上，针对消化温度(55,60,65 ℃)、消化时间(10,60,120,180,240 min)、质量分数20%十二烷基硫酸钠（SDS）裂解液用量（20,35,50,65,80 μL）、20 mg/mL蛋白酶K用量(0,10,30,50,70 μL)等重要参数依次进行优化，通过DNA品质测定筛选最佳的DNA提取条件，建立稳定的牛奶中牛DNA提取的试剂盒方法，并用深加工的奶粉和高低温液态奶制品对试剂盒方法进行验证。【结果】 不同消化温度和消化时间从牛奶中提取的总DNA、线粒体DNA（mtDNA）和核DNA（nDNA）的完整性均表现良好，但不同消化温度和消化时间的DNA质量浓度和纯度存在差异，在消化温度为60 ℃、消化时间为10 min、SDS裂解液用量为50 μL、蛋白酶K用量为50 μL时，提取的DNA品质较优，是最佳的DNA提取条件,该条件下DNA平均质量浓度为97 ng/μL，平均纯度为1.44。对优化指标后建立的试剂盒方法进行验证，结果表明该方法不仅适用于生鲜牛奶，而且还适用于液态和固态奶制品。【结论】在所获得的最佳DNA提取条件下，牛奶体细胞裂解充分、完全，所提DNA质量浓度和纯度较高，完整性较好，PCR扩增性能较强。建立的牛奶中牛DNA提取试剂盒法适用范围较广。 |
| 关键词: 牛奶 DNA提取 提取条件优化 试剂盒法 |
| DOI： |
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| 基金项目:陕西省重大科技项目（2019ZDLNY06-05）；西安市科技计划项目（2017050NC/NY007-3）；中央高校基本科研业务费专项(GK201805002) |
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| Establishment of DNA kit method of bovine milk |
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WANG Yifan,QIAO Chunyan,LIU Yongfeng
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| Abstract: |
| 【Objective】A stable kit extraction method was established to improve the separation and extraction efficiency of DNA from bovine milk.【Method】Based on previous established method for DNA extraction from bovine milk, important parameters including incubation temperature,incubation time,amount of sodium dodecyl sulfate (SDS) and proteinase K were optimized.Through DNA quality determination,the optimal conditions were screened to establish a stable kit method for extraction DNA from bovine milk.Then,the kit method was verified with deep processed milk powder and high and low temperature liquid milk products.【Result】The integrity of total DNA,mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) extracted from bovine milk at different incubation temperatures and incubation times was good with different DNA concentration and purity.When incubation temperature was 60 ℃,incubation time was 10 min,amount of SDS was 50 μL,and amount of proteinase K was 50 μL,the quality of the extracted DNA was good and they were the best conditions for DNA extraction. Under these conditions,average DNA concentration and purity were 97 ng/μL and 1.44.The verification of the established kit method showed the method was suitable for fresh milk as well as liquid and solid dairy products.【Conclusion】Under the established optimal DNA extraction conditions,bovine milk somatic cells lysed fully and completely,the DNA concentration and purity were high,and the integrity of DNA and PCR amplification performance were good.The established bovine DNA extraction kit method has a wide range of applications. |
| Key words: bovine milk DNA extraction optimization of extraction conditions kit method |
