International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal; Health and Environment Research Center, School of Health, Polytechnic Institute of Porto, R. Dr. Roberto Frias 712, 4200-465, Porto, Portugal; Department of Biochemistry, Genetics and Immunology, University of Vigo, 36310, Vigo, Spain.
International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal; College of Pharmacy/School of Veterinary Sciences, University of Santiago de Compostela, Campus Vida, E-15782, Santiago de Compostela, Spain.
Anal Chim Acta. 2023 Aug 1;1267:341357. doi: 10.1016/j.aca.2023.341357. Epub 2023 May 17.
Ready-to-eat products, such as leafy greens, must be carefully controlled as they are directly consumed without any treatment to reduce the presence of potential pathogens. Food industries, especially those that process products with short shelf-life, demand rapid detection of foodborne pathogens such as Shiga Toxin-producing Escherichia coli (STEC). In this sense, molecular methods can fulfill both requirements of turnaround time and consumer safety. The most popular rapid methods are those based on real-time PCR (qPCR) however, vegetables contain inhibitory compounds that may inhibit the amplification reaction thus, there is a need for novel sample preparation protocols.
In the current study, a low-cost sample treatment based on sequential filtration steps was developed. This protocol was combined with covalent organic frameworks (COFs), and compared against a chelating resin, to evaluate their performance by multiplex qPCR targeting the major virulence genes of STEC, namely stx1, stx2, and eae, along with the rfbE for the specific identification of serogroup O157 due to its particularly high incidence, and an Internal Amplification Control to assess reaction inhibition. The optimized sample treatment effectively removed vegetable qPCR inhibitory compounds, and it was possible to detect STEC in spiked ready-to-eat salad samples in one working day, roughly 5 h, with an LOD of 8.7 CFU/25 g with high diagnostic sensitivity and specificity. The method was also assessed in samples with cold-stressed bacteria with good results, further demonstrating its applicability.
It was demonstrated for the first time that COFs are suitable for DNA extraction and purification. In addition to this, due to the tunable nature of these materials, it is envisioned that future modifications in terms of pore size or combination with magnetic materials, will allow to further improve their performance. In addition to this, the rapid and low-cost sample treatment protocol developed demonstrated suitable for the rapid screening of STEC vegetable samples.
即食产品，如叶菜类，由于直接食用且未经任何处理以减少潜在病原体的存在，因此必须进行严格控制。食品行业，尤其是那些加工保质期较短产品的行业，需要快速检测食源性病原体，如产志贺毒素大肠杆菌（STEC）。从这个意义上说，分子方法既可以满足周转时间的要求，也可以满足消费者安全的要求。最流行的快速方法是基于实时 PCR（qPCR）的方法，然而，蔬菜中含有抑制化合物，可能会抑制扩增反应，因此需要新的样品制备方案。
在本研究中，开发了一种基于顺序过滤步骤的低成本样品处理方法。该方案结合了共价有机框架（COFs），并与螯合树脂进行了比较，通过针对 STEC 的主要毒力基因（stx1、stx2 和 eae）以及 rfbE 的多重 qPCR 来评估它们的性能，以鉴定 O157 血清群，因为其发病率特别高，并且使用内部扩增对照来评估反应抑制情况。优化后的样品处理方法可有效去除蔬菜 qPCR 抑制化合物，并且可以在一个工作日内（大约 5 小时）检测到添加到即食沙拉样品中的 STEC，检测限为 8.7 CFU/25 g，具有较高的诊断灵敏度和特异性。该方法还在冷藏应激细菌样品中进行了评估，结果良好，进一步证明了其适用性。
首次证明 COFs 适合用于 DNA 提取和纯化。除此之外，由于这些材料的可调性质，可以设想未来在孔径或与磁性材料的组合方面进行修改，将进一步提高它们的性能。此外，开发的快速、低成本的样品处理方案适用于快速筛选蔬菜中的 STEC 样品。
