Laboratory of Applied Micro and Nanotechnology (LAMINATE), DTU-Bioengineering (Department of Biotechnology and Biomedicine), Technical University of Denmark, Kgs. Lyngby, Denmark.
Biolabchip group, DTU-Bioengineering (Department of Biotechnology and Biomedicine), Technical University of Denmark, Kgs. Lyngby, Denmark.
Microb Biotechnol. 2023 Nov;16(11):2105-2113. doi: 10.1111/1751-7915.14343. Epub 2023 Sep 30.
Sensitive detection of pathogens in livestock farms is an integral part of the One Health Action Plan of the European Union (EU). Ensuring this requires on-site testing devices that are compatible with complex matrices such as primary production samples. Among all, faeces are considered the most challenging matrix type that makes it difficult to identify pathogens because of complexity in sample preparation for molecular testing. We have developed a loop-mediated isothermal amplification (LAMP) based veterinary point-of-care (POC) device (VETPOD) and adapted it to detect Salmonella enterica in primary production samples. Three different sampling methods (semi-wet chicken faeces, boot socks collection and dust samples from poultry shed) were iteratively tested to assess their nature of complexity and possibility for adapting them as suitable sampling methods for on-site testing. During the study, the sample preparation method that included a two-step centrifugation combined with washing of the enriched Salmonella cells was found crucial in eliminating amplification inhibitors originating from the faecal matrices. A total of 90 samples were tested that included 60 samples for sensitivity study and 30 samples for relative level of detection (RLOD, a level of detection in comparison to ISO 6579:1 reference method). Overall, the VETPOD had a sensitivity of 90%, 84.62% and 81.82% for boot sock, faecal and dust samples, respectively. The RLOD was 2.23 CFU/25 g which was found to be 1.33 times higher than the ISO 6579:1. Performing with an excellent agreement with ISO 6579:1, the VETPOD proved as a promising alternative to detect Salmonella spp. in primary production and animal husbandry samples.
在欧盟（EU）的“同一健康”行动计划中，对农场中的病原体进行敏感检测是其中不可或缺的一部分。要做到这一点，需要现场检测设备与初级生产样本等复杂基质相兼容。在所有基质中，粪便被认为是最具挑战性的基质类型，由于分子检测的样品制备复杂，因此很难识别病原体。我们开发了一种基于环介导等温扩增（LAMP）的兽医即时检测（POC）设备（VETPOD），并对其进行了适应性改造，以检测初级生产样本中的沙门氏菌。三种不同的采样方法（半湿鸡粪、靴袜采集和禽舍灰尘样本）经过反复测试，以评估其复杂性本质和将其作为现场检测的合适采样方法的可能性。在研究过程中，发现包括两步离心和富集沙门氏菌细胞洗涤的样品制备方法对于消除来自粪便基质的扩增抑制剂至关重要。总共测试了 90 个样本，其中 60 个样本用于敏感性研究，30 个样本用于相对检测水平（RLOD，与 ISO 6579:1 参考方法相比的检测水平）。总的来说，VETPOD 对靴袜、粪便和灰尘样本的灵敏度分别为 90%、84.62%和 81.82%。RLOD 为 2.23 CFU/25 g，比 ISO 6579:1 高 1.33 倍。VETPOD 与 ISO 6579:1 具有极好的一致性，证明它是一种有前途的替代方法，可以检测初级生产和畜牧业样本中的沙门氏菌属。
