Department of Functional Biology, University of Santiago de Compostela, Santiago de Compostela, Spain.
Food Quality & Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga S/N, 4715-330, Braga, Portugal.
Appl Microbiol Biotechnol. 2024 Nov 12;108(1):510. doi: 10.1007/s00253-024-13318-9.
Listeria monocytogenes is a major foodborne pathogen affecting developing, and developed countries. The analysis of food contact surfaces in food industries is key for better controlling this pathogen. The current study focused on the development, optimization, and evaluation of a rapid and simple method for the detection of L. monocytogenes on stainless steel surfaces, suitable for decentralized setups, taking advantage of Loop-mediated isothermal amplification (LAMP). This was accomplished using a general pre-enrichment broth (TSB), with a simple DNA extraction based on a chelating resin, and final isothermal amplification. Two different detection strategies were tested, real-time fluorescence and naked-eye colorimetric, which were evaluated after 5, 7, and 24 h of pre-enrichment. Regardless the detection chemistry selected, after 5-7 h of pre-enrichment, 10-10 CFU/cm were needed to obtain a positive result, while after 24 h, it was possible to detect concentrations below 10 CFU/cm. Within each given time, all the performance parameters calculated, relative sensitivity, specificity, and accuracy, reached values higher than 80-90%; likewise, a Cohen's k of concordance with a culture-based approach higher than 0.8. Overall, the most sensitive assay can be performed in roughly 25 h. This time-to-result outperforms commercial kits with the added value of specifically detecting L. monocytogenes instead of Listeria spp. KEY POINTS: • Real-time fluorescence and naked-eye colorimetric, were compared for the novel assay. • An LOD50 of 3.4 CFU/cm and 4.2 CFU/cm was calculated for the two assays. • Three pre-enrichment times were compared providing 24 h better results.
李斯特菌 monocytogenes 是一种主要的食源性致病菌，影响着发展中国家和发达国家。对食品工业中食品接触面的分析是更好地控制这种病原体的关键。本研究旨在开发、优化和评估一种快速简便的方法，利用环介导等温扩增 (LAMP) 检测不锈钢表面的李斯特菌 monocytogenes，适用于分散式设置。这是通过使用普通预增菌肉汤 (TSB) 完成的，采用基于螯合树脂的简单 DNA 提取方法和最终等温扩增。测试了两种不同的检测策略，实时荧光和肉眼比色，在预增菌 5、7 和 24 小时后进行了评估。无论选择哪种检测化学方法，在预增菌 5-7 小时后，需要 10-10 CFU/cm 才能获得阳性结果，而在 24 小时后，可以检测到浓度低于 10 CFU/cm 的李斯特菌 monocytogenes。在每个给定的时间内，计算出的所有性能参数，相对灵敏度、特异性和准确性，均达到 80-90%以上；同样，与基于培养的方法相比，Cohen's k 值的一致性也高于 0.8。总体而言，最敏感的检测可以在大约 25 小时内完成。与商业试剂盒相比，这种结果时间具有更高的价值，因为它可以特异性检测李斯特菌 monocytogenes 而不是李斯特菌属。关键点：• 比较了实时荧光和肉眼比色两种新型检测方法。• 两种检测方法的 LOD50 分别为 3.4 CFU/cm 和 4.2 CFU/cm。• 比较了三种预增菌时间，24 小时的结果更好。
