植物蛋白饮料中榛子成分环介导等温扩增(LAMP)快速检测方法的建立

该研究基于植物蛋白饮料掺杂使假现象普遍，建立一种针对榛子成分为检测目标的环介导等温扩增（Loop-mediated isothermal amplification，LAMP）快速检测技术方法。根据欧洲榛子染色体Ca8（登录号：LR899430.1）基因保守序列（第19517533 ~19519500 bp），设计榛子成分环介导等温扩增检测特异性引物，构建反应体系，并验证方法的特异性、灵敏度和稳定性；以Real-Time PCR（Polymerase Chain Reaction）为对照方法，采用显著性差异检验（McNemar’s检验）方法，通过32种植物蛋白饮料检测结果验证LAMP方法的性能指标和准确性。该研究建立的LAMP方法特异性强，33种植物成分中除了榛子DNA外，其它植物成分DNA均未获得阳性扩增结果；重复性实验稳定性好（扩增Ct值，RSD=5.41%），最低检出限为0.1%（以质量分数计）；通过植物蛋白饮料产品应用性验证，得出方法特异性和灵敏度均为100%，不存在假阳性和假阴性。综上，该研究建立的植物蛋白饮料榛子成分LAMP检测技术，具有操作简单、快速高效（从样品处理到最终结果可在2 h内完成）、准确度高等优点，可应用于植物蛋白饮料榛子成分快速检测。

Establishment of a Loop-mediated Isothermal Amplification (LAMP) Method for the Rapid Detection of Hazelnut Components in Plant-based Protein Beverages

To address the widespread adulteration of plant-based protein beverages, a loop-mediated isothermal amplification (LAMP) method was developed for the rapid detection of hazelnut components. The conserved sequence (No. 19517533~19519500 bp) of chromosome Ca8 (accession number: LR899430.1) in European hazelnut was adopted to design the specific primers for the LAMP detection of hazelnut components. The reaction system was optimized and the specificity, sensitivity, and stability of the LAMP method were verified. Using real-time polymerase chain reaction as the reference method, McNemar''s test was performed to verify the performance indexes and accuracy of the LAMP method for 32 plant-based protein beverages. The LAMP method had strong specificity, and no positive amplification results were obtained for 33 plant ingredients except hazelnut. The reproducibility test was stable (Ct value, RSD=5.41%), and the lower limit of detection was 0.1% (by mass fraction). The results indicated that the LAMP method had a specificity and sensitivity of 100%, with no false positives or false negatives. In conclusion, the LAMP detection method had the advantages of simple operation, fast and efficient detection (<2 h from sample processing to final results), and high accuracy. Hence, it can be applied to the rapid detection of hazelnut components in plant-based protein beverages.