基于榛子过敏原2S albumin基因的实时荧光聚合酶链式反应检测方法开发

Development of real-time fluorescent polymerase chain reaction assay based on the gene of Corylus avellana allergen 2S albumin

中文关键词:  榛子过敏原  实时荧光聚合酶链式反应  2S albumin基因  检出限

英文关键词:Corylus avellana allergen  real-time fluorescent polymerase chain reaction  2S albumin gene  limit of detection

中文摘要:

      目的 开发一种基于2S albumin基因的实时荧光聚合酶链式反应检测方法(real-time fluorescent polymerase chain reaction, qPCR)用于检测食品中的榛子成分。方法 从美国国家生物技术信息中心数据库中检索并选取具有高特异性的榛子2S albumin基因序列的DNA片段进行引物探针设计, 建立一种新的用于检测微量榛子成分的实时荧光PCR方法, 并对建立的方法的特异性、扩增效率、检出限和稳健性进行验证。结果 本方法对检测目标呈现出高特异性, 与其余12种非目标样品无交叉反应。扩增效率为99.42%, 相关系数r2为0.9941, 两者均符合实时荧光PCR方法验证指南要求。该方法检出限(limit of detection, LOD)为每个反应5拷贝(2.5 copies/μL), 可检测出食品中含有的微量榛子成分。使用正交实验设计评估该方法具有很好的稳健性(P=0.07), 可转移到其他实验室及常规分析中使用。结论 基于2S albumin基因组分的qPCR检测方法可用于识别食品中潜在的榛子成分, 对于预防榛子过敏及开发减敏脱敏榛子食品研究具有很好的技术支撑。

英文摘要:

      Objective To develop a real-time fluorescent polymerase chain reaction (qPCR) assay based on 2S albumin gene for the detection of Corylus avellana components in food. Methods DNA fragments of the 2S albumin gene with high specificity were extracted from the National Center for Biotechnology Information database and utilized for the primers and probe design. A novel real-time fluorescent PCR assay was developed, designed for the detection of hazelnut components at trace levels. The method’s specificity, amplification efficiency, limit of detection and robustness were verified. Results This method exhibited exceptional specificity for detecting the target and no interaction with the other 12 kinds of non-target samples. The amplification efficiency and correlation coefficient r2 were 99.42% and 0.9941, respectively, both fulfilling the real-time PCR validation guidelines. The limit of detection (LOD) was 5 copies (2.5 copies/μL) per reaction, making it possible to identify trace Corylus avellana components in food. The method’s robustness was assessed using the orthogonal experimental design (P=0.07), guaranteeing its reproducibility in other laboratories and routine analysis. Conclusion The qPCR technique, utilizing the detection of 2S albumin gene components, can effectively detect potential allergens present in Corylus avellana-derived food products. This method provides significant technical support for the prevention of Corylus avellana allergies and the development of immunotherapy-based food research.