Foodborne pathogens are still a significant source of morbidity and mortality worldwide. In addition to this the current methodologies to track these microorganisms cannot cope with the current intensive production systems, thus novel methods are of outmost importance. DNA-based methods have already demonstrated suitable to address this issue, but most of them are targeted methods such as real-time PCR (qPCR), meaning that one will only find what is looking for, thus taking the risk of missing relevant pathogens in a given sample. To overcome this limitation we have developed an easy-to-implement methodology which enables the detection of several pathogens simultaneously by using long-read Next Generation Sequencing (NGS) with MinION. The method was named “semi-targeted” due to the combination of a non-targeted detection method, NGS, with the usage of selective media in order to partially eliminate non-pathogenic interfering bacteria. To this end, we included an enrichment step for the recovery of different pathogens, namely Salmonella Enteritidis and Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7, after DNA extraction and library preparation, the samples were analyzed with MinION implementing the low-cost Flongle Flow Cells. The methodology was successfully evaluated in spiked milk samples with an excellent agreement with the results obtained by qPCR and culture-based methods. The method can provide accurate results after only 2 h of sequencing. Sample multiplexing, along with the lower cost of the Flongle Flow Cells and the reduced price of the MinION platform, make the assay cost-effective that is of importance for the food industry. Starting the method with a classical microbiological approach, the enrichment, the method is easy to implement in testing laboratories, it provides flexibility in terms of potential pathogens to be detected, and the positive results can be easily confirmed following culture-based, or other type, of confirmation procedures.

食源性病原体仍然是全世界发病率和死亡率的重要来源。除此之外，目前追踪这些微生物的方法无法应对目前的集约化生产系统，因此新方法最为重要。基于 DNA 的方法已经证明适合解决这个问题，但其中大多数是靶向方法，例如实时 PCR (qPCR)，这意味着人们只会找到正在寻找的东西，从而冒着丢失相关病原体的风险。给定的样本。为了克服这一限制，我们开发了一种易于实施的方法，通过使用 MinION 的长读长下一代测序 (NGS) 同时检测多种病原体。由于结合了非靶向检测方法 NGS、使用选择性培养基以部分消除非致病性干扰细菌。为此，我们包括了一个用于回收不同病原体的富集步骤，即肠炎沙门氏菌和鼠伤寒杆菌、单核细胞增生李斯特氏菌和大肠杆菌O157:H7，在 DNA 提取和文库制备后，使用 MinION 实施低成本 Flongle Flow Cell 分析样品。该方法在加标牛奶样品中得到成功评估，与 qPCR 和基于培养的方法获得的结果非常吻合。该方法仅需测序 2 小时即可提供准确的结果。样品多路复用以及 Flongle Flow Cell 的较低成本和 MinION 平台的降低价格，使分析具有成本效益，这对食品行业很重要。该方法以经典微生物学方法开始，富集，该方法易于在检测实验室中实施，它在检测潜在病原体方面提供了灵活性，并且可以在基于培养或其他类型后轻松确认阳性结果,