Costa-Ribeiro Ana, Lamas Alexandre, Mora Azucena, Prado Marta, Garrido-Maestu Alejandro
International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal.
Department of Biochemistry, Genetics and Immunology, University of Vigo, 36310, Vigo, Spain.
Curr Res Food Sci. 2024 Mar 7;8:100716. doi: 10.1016/j.crfs.2024.100716. eCollection 2024.
Rapid identification of Shiga toxin-producing , or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problematic as they are consumed raw, and are rich in compounds that inhibit DNA-based detection methods such as qPCR. In the present study a novel method based on Loop-mediated isothermal amplification (LAMP) to overcome the limitations associated with current molecular methods for the detection of STEC in RTE vegetables targeting 1 and 2 genes. In this sense, LAMP demonstrated to be more robust against inhibitory substances in food. In this study, a comprehensive enrichment protocol was combined with four inexpensive DNA extraction protocols. The one based on silica purification enhanced the performance of the method, therefore it was selected for its implementation in the final method. Additionally, three different detection chemistries were compared, namely real-time fluorescence detection, and two end-point colorimetric strategies, one based on the addition of SYBR Green, and the other based on a commercial colorimetric master mix. After optimization, all three chemistries demonstrated suitable for the detection of STEC in spiked RTE salad samples, as it was possible to reach a LOD50 of 0.9, 1.4, and 7.0 CFU/25 g for the real-time, SYBR and CC LAMP assays respectively. All the performance parameters reached values higher than 90 %, when compared to a reference method based on multiplex qPCR. More specifically, the analytical sensitivity was 100, 90.0 and 100 % for real-time, SYBR and CC LAMP respectively, the specificity 100 % for all three assays, and accuracy 100, 96 and 100 %. Finally, a high degree of concordance was also obtained (1, 0.92 and 1 respectively). Considering the current technological advances, the method reported, using any of the three detection strategies, demonstrated suitable for their implementation in decentralized settings, with low equipment resources.
快速鉴定产志贺毒素大肠杆菌（STEC）对于确保食品无害至关重要。STEC已与不同类型食品引发的疫情有关，然而，其中即食（RTE）蔬菜尤其成问题，因为它们是生食的，并且富含抑制基于DNA的检测方法（如qPCR）的化合物。在本研究中，一种基于环介导等温扩增（LAMP）的新方法克服了当前用于检测RTE蔬菜中STEC的分子方法的局限性，该方法针对1个和2个基因。从这个意义上说，LAMP对食品中的抑制物质表现出更强的耐受性。在本研究中，一个全面的富集方案与四种廉价的DNA提取方案相结合。基于硅胶纯化的方案提高了该方法的性能，因此被选用于最终方法中。此外，还比较了三种不同的检测化学方法，即实时荧光检测和两种终点比色策略，一种基于添加SYBR Green，另一种基于商业比色预混液。经过优化，所有三种化学方法都证明适用于检测加标RTE沙拉样品中的STEC，实时、SYBR和CC LAMP检测分别达到0.9、1.4和7.0 CFU/25 g的50%检出限（LOD50）。与基于多重qPCR的参考方法相比，所有性能参数的值均高于90%。更具体地说，实时、SYBR和CC LAMP的分析灵敏度分别为100%、90.0%和100%，所有三种检测方法的特异性均为100%，准确性分别为100%、96%和100%。最后，还获得了高度一致性（分别为1、0.92和1）。考虑到当前的技术进步，所报道的方法，使用三种检测策略中的任何一种，都证明适用于在设备资源较少的分散环境中实施。
