DNA 提取方法对实时荧光定量 PCR 检测花生过敏原 Ara h 2 基因的比较研究

目前，食物过敏是一个世界性的公共卫生问题，其中花生过敏最为严重。基于DNA的分子生物学检测方法目前已广泛应用于花生过敏原检测，样品DNA的提取质量会显著影响检测的灵敏度及准确率。本研究比较了5种DNA提取方法，包括十六烷基三甲基溴化铵（cetyl trimethyl ammonium bromide，CTAB）法、柱式法及3种基于磁珠纯化技术的DNA快速提取法，对生花生、煮花生、炸花生、烤花生、花生酥和花生酱6种不同加工方式的花生基质样品进行DNA提取，考察了不同方法获得的DNA浓度、纯度指标，并采用实时荧光定量PCR（quantitative real-time PCR, qPCR）对花生过敏原Ara h 2基因进行了检测分析。结果表明，月桂酰肌氨酸钠（Sodium Lauroyl Sarcosine）磁珠法（简称SLS磁珠法）的适用性广、提取率高，对于6种花生基质提取的DNA均能高效检出花生过敏原Ara h 2基因；对于花生含量为0.05%～1.00%的小麦粉二元混合物，SLS磁珠法的DNA提取率总体优于CTAB法，并且能有效提取出与花生共用一条生产线的燕麦片中污染的花生DNA，证实SLS磁珠法提取的实际样品DNA能够满足花生过敏原检测目的。本研究为花生及其制品DNA提取方法提供了参考，特别是磁珠类方法，高效快速，提取质量能够保障后续基于DNA的花生过敏原分子生物学方法检测结果的准确性。

Comparison of DNA Extraction Methods for Detection of Arah 2 Gene in Peanut Allergen by Real time Fluorescence Quantitative PCR

Currently, food allergy is a worldwide public health problem, among which peanut allergy is more serious.?Molecular biological detection methods based on DNA have been widely used in peanut allergen detection, but the quality of sample DNA extraction will significantly affect the sensitivity and accuracy of detection. In this study, five DNA extraction methods were compared, including cetyl trimethyl ammonium bromide (CTAB), column method and three rapid DNA extraction methods based on magnetic beads purification. DNA was extracted from peanut matrix samples of 6 different processing methods: raw peanut, boiled peanut, fried peanut, roasted peanut, peanut crisp and peanut butter. The concentration and purity of DNA obtained by different methods were investigated. Quantitative real-time PCR (qPCR) was used to detect Ara h 2 gene of peanut allergen and the results showed that the Sodium Lauroyl Sarcosine (SLS) magnetic beads method had wide applicability and high extraction rate, and it could efficiently detect the Ara h 2 gene of peanut allergen for the DNA extracted from six peanut substrates. For the wheat flour binary mixture with peanut content of 0.05% ~ 1.00%, the DNA extraction rate of SLS magnetic beads method was generally better than that of CTAB method, and it could effectively extract peanut DNA contaminated by oatmeal which shared the same production line with peanut, which confirmed that the actual sample DNA extracted by SLS magnetic beads method could meet the purpose of peanut allergen detection. This study provides a reference for DNA extraction methods of peanut and its products, especially magnetic beads method, which is efficient and rapid. The extraction quality can ensure the accuracy of subsequent DNA-based molecular biological methods for peanut allergen detection.