产志贺毒素大肠杆菌的快速鉴定,或STEC,最重要的是确保食品的无害性。然而,STEC与不同类型食品相关的疫情有牵连,其中,即食(RTE)蔬菜特别成问题,因为它们是生吃的,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)的新方法克服了与当前分子方法相关的局限性,该方法用于检测针对stx1和stx2基因的RTE蔬菜中的STEC。在这个意义上,LAMP被证明对食物中的抑制物质更强大。在这项研究中,一个全面的富集方案与四个廉价的DNA提取方案相结合.基于二氧化硅纯化的方法提高了该方法的性能,因此,它被选择用于最终方法的实施。此外,比较了三种不同的检测化学物质,即实时荧光检测,和两个终点比色策略,一个基于SYBRGreen的添加,另一种基于商业比色主混合物。优化后,所有三种化学物质都被证明适合检测加标RTE沙拉样品中的STEC,因为实时可以达到0.9、1.4和7.0CFU/25g的LOD50,分别进行SYBR和CCLAMP测定。所有性能参数均达到高于90%的值,与基于多重qPCR的参考方法相比。更具体地说,实时分析灵敏度为100、90.0和100%,分别为SYBR和CC灯,所有三种检测的特异性为100%,和精度100%,96%和100%。最后,也获得了高度的一致性(分别为1、0.92和1)。考虑到目前的技术进步,报告的方法,使用三种检测策略中的任何一种,证明适合在分散的环境中实施,设备资源低。
Rapid identification of Shiga toxin-producing Escherichia coli, 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 stx1 and stx2 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.