Lin Min, Dan Hanhong, Guan Jiewen
Canadian Food Inspection Agency, Ottawa Laboratory Fallowfield, Ottawa, Ontario, Canada.
Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
Microbiol Spectr. 2025 Apr;13(4):e0157724. doi: 10.1128/spectrum.01577-24. Epub 2025 Feb 25.
, a rod-shaped Gram-positive bacterium widely distributed in nature, can contaminate foods and represents a foodborne pathogen of public health significance causing a high mortality rate of 20%-30%. Rapid and reliable identification of foods and food-processing environments contaminated with is a crucial step in implementing effective intervention strategies to ensure food safety and limit the transmission of bacteria to humans. This study designed and refined a practical workflow to streamline and accelerate the detection of a low level of present in ground beef. The workflow coupled an abbreviated 5 h culture enrichment in PALCAM liquid medium with physical separation (filtration and centrifugation) to preprocess enrichment samples. Specific capture was achieved using magnetic separation with a bacteriophage endolysin-derived cell wall-binding domain in a Hyglos capture kit. Molecular detection was performed using a MicroSEQ RTi-PCR detection kit combined with a nested PCR strategy. Preprocessing of enrichment culture samples using a multi-stage filtration system constructed for the study or commercially available BagFilter Pull-up filter bags, in conjunction with centrifugation, enabled the recovery of ~30 colony-forming units (CFUs) from the enrichment culture of a 25 g ground beef sample artificially contaminated with 1 CFU of . Integration of magnetic separation into the workflow for capturing cells specifically from preprocessed samples and further cleaning up the samples yielded bacterial counts similar to those obtained by direct plating of preprocessed samples. The RTi-PCR-based molecular detection method integrated into the workflow was capable of detecting pure cultures of as low as 12.5 CFUs. Evaluation of the workflow using artificially ground beef demonstrated the consistent detection of within an 8 h workday in a 25 g sample unit containing the cell count as low as 2 CFU following a 5 h culture enrichment.
Consuming foods contaminated with the bacterial pathogen can lead to the development of human listeriosis, a severe and life-threatening foodborne illness. Timely detection of present at a low level in foods and food processing environments is a necessary measure to prevent the spread of the -associated illness. This study designed and evaluated a multi-step workflow for testing in artificially contaminated food samples. The workflow was composed of a short 5 h culture enrichment, filtration-based sample preprocessing, magnetic separation, a single-tube nested RTi-PCR, and culture plating. It allowed to be detected within 8 h from a 25 g ground beef sample containing the target cells as low as 2 colony-forming units, significantly improving and streamlining the detection methods for this important foodborne pathogen.
[细菌名称]是一种杆状革兰氏阳性菌，在自然界广泛分布，可污染食品，是一种具有公共卫生意义的食源性病原体，致死率高达20%-30%。快速、可靠地鉴定被[细菌名称]污染的食品及食品加工环境，是实施有效干预策略以确保食品安全并限制细菌传播给人类的关键一步。本研究设计并完善了一种实用的工作流程，以简化和加速对碎牛肉中低水平[细菌名称]的检测。该工作流程将在PALCAM液体培养基中进行的5小时简化培养富集与物理分离（过滤和离心）相结合，对富集样品进行预处理。使用Hyglos捕获试剂盒中源自噬菌体溶菌酶的细胞壁结合结构域进行磁分离，实现特异性捕获。采用MicroSEQ RTi-PCR检测试剂盒结合巢式PCR策略进行分子检测。使用为本研究构建的多级过滤系统或市售的BagFilter Pull-up过滤袋结合离心对富集培养样品进行预处理，能够从人工接种1个[细菌名称]CFU的25克碎牛肉样品的富集培养物中回收约30个菌落形成单位（CFU）。将磁分离整合到工作流程中，用于从预处理样品中特异性捕获[细菌名称]细胞并进一步净化样品，得到的细菌计数与直接接种预处理样品获得的计数相似。整合到工作流程中的基于RTi-PCR的分子检测方法能够检测低至12.5 CFU的[细菌名称]纯培养物。使用人工碎牛肉对该工作流程进行评估表明，在5小时培养富集后，在一个工作日8小时内能够一致地检测出25克样品单位中低至2 CFU细胞计数的[细菌名称]。
食用被细菌病原体[细菌名称]污染的食品可导致人类李斯特菌病，这是一种严重且危及生命的食源性疾病。及时检测食品和食品加工环境中低水平存在的[细菌名称]是预防与[细菌名称]相关疾病传播的必要措施。本研究设计并评估了一种用于检测人工污染食品样品中[细菌名称]的多步骤工作流程。该工作流程由5小时的短时间培养富集、基于过滤的样品预处理、磁分离、单管巢式RTi-PCR和培养平板计数组成。它能够在8小时内从含有低至2个菌落形成单位目标细胞的25克碎牛肉样品中检测出[细菌名称]，显著改进并简化了对这种重要食源性病原体的检测方法。
