Construction and application of loop-mediated isothermal amplification method for detection of brazil nut as a food allergen
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摘要: 根据巴西坚果2S白蛋白基因序列,利用设计软件Primer Explorer Version 4设计并筛选了食品过敏原巴西坚果的环介导等温扩增引物,对反应体系和反应条件进行了优化,建立了巴西坚果的环介导等温扩增检测方法,结果判断可采用实时荧光法和荧光染料终点显色法。以澳洲坚果、开心果、碧根果等17种常见坚果来验证方法的特异性;将巴西坚果DNA进行梯度稀释后验证方法的灵敏度;将0.5%、1%和1.5%3个浓度梯度的巴西坚果DNA重复检测20次来验证方法的稳定性。结果表明,本方法能够特异、灵敏、稳定地检测食品中的巴西坚果成分,检测低限为0.5%。此外,对7种市售食品样品的检测结果表明,该方法与食品标签标示的过敏原成分结果吻合率为100%,假阳性率和假阴性率均为0%。Abstract: According to the Bertholletia excels 2S albumin gene sequences, this article designed and screened sets of loop-mediated isothermal amplification (LAMP) primers using a design software named Primer Explorer Version 4, then established the relevant LAMP method for detection of Brazil nut as a food allergen, whose results could be measured by real-time fluorescence or using a fluorescent dyes in the end reaction. The specificity of this method was evaluated by detection of 17 nut samples include macadamia nut, pistachio, pecan, and so on. The sensitivity was tested using DNA gradient dilutions, and the stability was assessed with detection of 0.5%, 1%, 1.5% gradient dilutions of Brazil nut DNA for 20 times. It was indicated that this method had satisfied specificity, sensitivity and stability in the identification of Brazil nut in foods with a LOD of 0.5%. In addition, the detection of 7 market food samples using this method showed the 100% consistency to the labeled information of allergen ingredients with 0% false positive and negative rates.
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