膜法改进十六烷基三甲基溴化铵法结合实时荧光聚合酶链式反应检测高色素食品DNA

Detection of DNA in highly pigmented foods by membrane-based improved cetyltrimethylammonium bromide combined with real-time fluorescence polymerase chain reaction

中文关键词:  玻璃纤维滤膜  核酸纯化  脱色  实时荧光聚合酶链式反应  高色素食品

英文关键词:glass fiber membrane  nucleic acid purification  decolorization  real-time fluorescence polymerase chain reaction  highly pigmented foods

中文摘要:

      目的 建立膜法改进十六烷基三甲基溴化铵(cetyltrimethylammonium bromide, CTAB)法结合实时荧光聚合酶链式反应(polymerase chain reaction, PCR)检测高色素食品DNA的方法。方法 以黑芝麻、茶籽粕和菜籽粕为样本, 采用0.7 μm玻璃纤维膜结合70%乙醇淋洗液及三羟甲基氨基甲烷-乙二胺四乙酸[tris(hydroxymethyl)aminomethane ethylenediaminetetraacetic acid, TE]缓冲液洗脱3次的优化方案。通过滤膜特异性吸附和选择透过性去除色素, 对比传统CTAB法与膜法改进CTAB法的提取效果。利用实时荧光PCR技术检测内源基因, 评估DNA纯度和扩增效率。结果 改进方法获得的DNA脱色效果显著优于传统CTAB法, 3种样本均成功检测到内源基因, DNA质量浓度均在100 ng/μL以上, 循环阈值(cycle threshold, Ct)在20~25之间, 而传统CTAB法因色素残留导致实时荧光PCR扩增失败。结论 膜法改进CTAB法通过简化纯化步骤、提升脱色效率, 显著提高了高色素食品的DNA提取及检测成功率, 为食品安全检测提供了高效可靠的技术支持。

英文摘要:

      Objective To establish a method for detecting DNA in highly pigmented food samples by membrane-based improved cetyltrimethylammonium bromide (CTAB) combined with real-time fluorescence polymerase chain reaction (PCR). Methods Black sesame, tea seed meal and rapeseed meal were selected as samples. The optimized protocol employed 0.7 μm glass fiber membrane with 70% ethanol as the rinsing solution and 3 rounds of tris(hydroxymethyl)aminomethane ethylenediaminetetraacetic acid (TE) buffer elution. Pigment removal was achieved through membrane-specific adsorption and selective permeability. The extraction efficiency of the modified CTAB method was compared with that of the traditional CTAB method, and DNA purity and amplification efficiency were evaluated via real-time fluorescence PCR targeting endogenous genes. Results The modified method demonstrated significantly superior DNA decolorization effects compared to the traditional CTAB method. Endogenous genes were successfully detected in all 3 kinds of samples, with DNA mass concentrations exceeding 100 ng/μL and cycle threshold (Ct) values ranging between 20 and 25. In contrast, the traditional CTAB method failed to achieve real-time fluorescence PCR amplification due to pigment residue. Conclusion The membrane-optimized CTAB method simplifies purification steps, enhances decolorization efficiency, and significantly improves the extraction and detection success rate of DNA from highly pigmented foods. This approach provides a reliable and efficient technical solution for food safety testing.