获得安全和营养的食物对于维持生命和支持身体健康至关重要。食用被病原体污染的食物会导致从腹泻到癌症等严重疾病。许多食源性感染可导致长期损害甚至死亡。因此，早期检测食源性病原体（例如致病性大肠杆菌菌株）对于公共安全至关重要。检测这些细菌的常规方法基于在选择性培养基上培养并遵循标准生化鉴定。尽管这些方法很准确，但它们很耗时。基于 PCR 的病原体检测依赖于精密设备和专业技术人员，而在资源有限的地区很难找到这些设备和专业技术人员。而 CRISPR 技术对于识别病原菌更具特异性和敏感性，因为它采用了针对特定 DNA 序列的可编程 CRISPR-Cas 系统，最大限度地减少了非特异性结合和交叉反应性。在该项目中，开发了一种基于 CRISPR-Cas12a 传感的稳健检测方法，该方法快速、灵敏且特异，可检测从田纳西州 17 个农场的成年山羊粪便样本中收集的致病性大肠杆菌分离株。检测反应包含致病区域的扩增PCR产物、报告探针、Cas12a酶和针对三个致病基因-stx1、stx2和hlyA的crRNA。与病原菌的 CRISPR 反应在紫外线激发下会发出荧光。为了评估该测定的检测灵敏度和特异性，将其结果与基于 PCR 的检测测定进行比较。对于相同的样品，两种方法都得到相似的结果。该技术非常精确、高灵敏度、快速、成本效益高且易于使用，并且可以轻松克服现有检测方法的局限性。该项目可以产生一种多功能检测方法，可轻松适应对人类健康以及动植物生产构成大规模生物安全威胁的疾病的检测和监测中的快速响应。版权所有 © 2024 Rathore、Basnet、Kilonzo-Nthenge 、杜梅尼奥、亚德加里和塔赫里。

Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes-stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.Copyright © 2024 Rathore, Basnet, Kilonzo-Nthenge, Dumenyo, Yadegari and Taheri.