基于多重连接依赖探针扩增（MLPA）技术检测加工食品中过敏原成分

Detection of the Allergens in Processed Foods Based on Multiple Ligation-dependent Probe Amplification (MLPA)

摘要:

该研究基于多重连接探针扩增技术（Multiplex Ligation-dependent Probe Amplification，MLPA）同时检测开心果、巴西坚果、芹菜、麸质、夏威夷果、芝麻、榛子、大豆、花生、葵花籽、核桃、腰果、杏仁、芥末等14种植物源性的过敏原成分，针对ITS序列设计特异性杂交探针，样本核酸经95 ℃变性后，与探针进行特异性结合，经连接和PCR扩增反应得到不同大小的目标片段，通过毛细管电泳分析目标片段的有无来判断是否含有待测过敏原成分。利用混合探针体系检测单一模板只能扩增出单一扩增峰，表明探针具有高特异性，检测限结果表明，MLPA扩增最低可检出的DNA质量浓度为1 ng/μL。通过20份实际样本的检测，证明该研究基于MLPA建立的加工食品中过敏原成分的检测方法，具有特异性强，灵敏度高的特点，可以应用于食品安全监管工作。

Abstract:

In this study, multiplex ligation-dependent probe amplification (MLPA) was applied to simultaneously detect 14 plant-derived allergens from pistachio, Brazil nut, celery, gluten, macadamia nut, sesame, hazelnut, soybean, peanut, sunflower seed, walnut, cashew, almond and mustard. Specific hybridization probes were designed for ITS sequences. After the sample nucleic acid was denatured at 95 ℃, specific binding was carried out with the probes, and target fragments of different sizes were obtained through ligation and PCR amplification reaction. The target fragments were analyzed by capillary electrophoresis to determine whether or not the allergens were present. Using the mixed probe system to detect a single template canonly amplifya single amplification peak, indicating that the probe had high specificity. The detection limit results showed that the lowest detectable DNA concentration of MLPA amplification was 1 ng/μL. Through the detection of 20 actual samples, it is proven that the detection method of allergens in processed foods established in this study on the basis of MLPA has the characteristics of high specificity and high sensitivity, and can be applied to food safety supervision.