Application of an Alkaline and Silica Membrane DNA Extraction Method to Detect Mitochondrial DNA in Foods
Application of an Alkaline and Silica Membrane DNA Extraction Method to Detect Mitochondrial DNA in Foods
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应用碱性硅膜DNA提取法检测食品中线粒体DNA
DOI：
10.1007/s12161-014-0002-9
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发表时间：
2015
影响因子：
2.9
通讯作者：
Shinomiya K.
中科院分区：
文献类型：
--
作者：
Harikai N;Shinomiya K.
为了提高从食品中提取线粒体DNA(MtDNA)的效率，将碱性十二烷基硫酸钠裂解和DNA结合在硅膜上，开发了一种简单的方法--PrepM。PrepM使用新鲜、冷藏和高压灭菌的大鼠肝脏样本进行评估，并与商业总DNA提取试剂盒进行比较。当将PrepM应用于新鲜和冷藏样品时，DNA产量较低，但OD260/280比值接近1.8。细胞色素C氧化酶亚基I(COI)拷贝数(单位重量的线粒体DNA拷贝数)和COI/β-肌动蛋白(线粒体与基因组DNA的比率)均高于总试剂盒。当将PrepM用于高压灭菌时，DNA纯度、产量和单位DNA重量的CoI拷贝数较高，但CoI/β-肌动蛋白的比率较低。然后将PrepM应用于虾样本，这是一种已知的过敏性食物。从冷和高压灭菌的虾样品中获得的数据与从大鼠肝脏样品中获得的数据相似。当将PrepM用于添加虾的食品样品时，DNA纯度较高，但DNA产量和mtDNA水平与总DNA提取试剂盒几乎相同。我们得出结论：PrepM在检测相对新鲜的样品中的mtDNA序列时具有相对的优势，其性能几乎与用于高压灭菌和/或复杂混合物样品的通用总DNA提取试剂盒相同。
To improve the efficiency of mitochondrial DNA (mtDNA) extraction from food, a simple method, PrepM, was developed by effectively combining alkaline sodium dodecyl sulfate lysis and DNA binding to a silica membrane. PrepM was evaluated using fresh, cold, and autoclaved rat liver samples and compared with commercial total DNA extraction kits. When PrepM was applied to fresh and cold samples, the DNA yield was low, but the OD260/280 ratio was close to 1.8. The cytochrome C oxidase subunit I (COI) copy number, which is the number of mtDNA copies, per DNA weight and the ratio of COI to β-actin, which is the ratio of mitochondrial to genomic DNA, were higher with PrepM than with total DNA extraction kits. When PrepM was applied to autoclaved samples, the DNA purity and yield and the COI copy number per DNA weight were relatively high, but the COI to β-actin ratio was low. PrepM was then applied to shrimp samples, a known allergenic food. Data obtained from cold and autoclaved shrimp samples were similar to data obtained from rat liver samples. When PrepM was applied to food samples spiked with shrimp, the DNA purity was high, but the DNA yield and the mtDNA levels were almost same as those obtained with total DNA extraction kits. We concluded that PrepM has a comparative advantage for sensitive polymerase chain reaction detection of mtDNA sequences in relatively fresh samples, and it has almost the same performance as general total DNA extraction kits with autoclaved and/or complex mixture samples.