酶促恒温扩增技术快速鉴定食品中牡蛎成分

Rapid Identification for Oysters in Foods by Enzymatic Recombinase Amplification

目的:针对牡蛎建立基于酶促恒温扩增(Enzymatic recombinase amplification,ERA)技术的快速检测方法。方法:基于NCBI上牡蛎线粒体的基因保守序列,通过Primer Premier 5.0软件设计5对ERA特异检测引物,并筛选出最佳引物。优化ERA反应的模板浓度、引物浓度、扩增温度和时间,最终获得最佳的反应体系,并进行灵敏度和特异性的验证,同时分别选取市场上几种牡蛎及其相关产品进行样本验证。结果:最终得出最佳ERA反应温度为39℃,最佳反应时间为20 min,优化后反应体系检测出牡蛎相关食品中的牡蛎成分灵敏度可达800 fg/μL,且特异性良好。结论:建立的检测方法具有操作简单、快速、不依赖于高端设备的优势,较高的特异性和灵敏度,为食品中牡蛎成分的快速、恒温、现场鉴定提供了一种新手段。

Objective:To establish a rapid detection method based on enzymatic recombinase amplification(ERA)technology for oyster.Methods:Based on the conservative gene sequence of oyster mitochondria on NCBI,five pairs of ERA-specific detection primers were designed through Primer Premier 5.0 software,and the best primers were screened.The template concentration,primer concentration,amplification temperature and time of ERA reaction were optimized to obtain the optimized reaction system,and the sensitivity and specificity were verified.Meanwhile,several oysters and their related products on the market were selected for sample verification.Results:The optimal ERA reaction temperature was 39℃and reaction time was 20 min.After optimization,the reaction system can detect oyster components in oyster-related foods with a sensitivity of 800 fg/μL and good specificity.Conclusion:The established method has the advantages of simple and rapid operation,high specificity and sensitivity,and does not rely on high-end equipment.The established detection method has the advantages of simple operation,rapidity and even independent of high-end equipment.The high specificity and sensitivity provided a new method for rapid,constant temperature and on-site identification of oyster components in food.