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0.7μm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。
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 0.7μm 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.
作者
汤一超
段吴燕
孙思扬
马驰远
夏彪
董永全
TANG Yi-Chao;DUAN Wu-Yan;SUN Si-Yang;MA Chi-Yuan;XIA Biao;DONG Yong-Quan(School of Environmental and Chemistry Engineering,Nanchang Hangkong University,Nanchang 330063,China;Nanchang Customs Technical Center/National Key Laboratory of Tea Oil Testing,Nanchang 330038,China)
出处
《食品安全质量检测学报》
2026年第1期206-217,共12页
Journal of Food Safety and Quality
基金
海关总署科研项目(2024HK015)。
关键词
玻璃纤维膜
核酸纯化
脱色
实时荧光聚合酶链式反应
高色素食品
glass fiber membrane
nucleic acid purification
decolorization
real-time fluorescence polymerase chain reaction
highly pigmented foods
