PDF(Pubmed)
Abstract：
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.
摘要：
获得安全和营养的食物对于维持生命和支持健康至关重要。食用被病原体污染的食物会导致从腹泻到癌症的严重疾病。许多食源性感染可导致长期损害甚至死亡。因此，早期检测食源性致病菌如致病性大肠杆菌菌株对公共安全至关重要。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。尽管他们的准确性,这些方法是耗时的。基于PCR的病原体检测依赖于复杂的设备和专业的技术人员，这些设备和技术人员在资源有限的地区很难找到。而CRISPR技术对于鉴别致病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，最小化非特异性结合和交叉反应性。在这个项目中,提出了一种基于CRISPR-Cas12a传感的鲁棒检测方法,这是快速的，对从田纳西州17个农场的成年山羊的粪便样品中收集的致病性大肠杆菌分离株具有敏感性和特异性。检测反应中含有致病区PCR扩增产物,记者探测,Cas12a酶，和对三个致病基因stx1，stx2和hlyA具有特异性的crRNA。当在UV光下激发时,与致病菌的CRISPR反应发射荧光。为了评估该测定法的检测灵敏度和特异性，将其结果与基于PCR的检测方法进行比较。两种方法对于相同的样品产生相似的结果。这项技术非常精确，高度敏感,快,成本有效，易于使用，并且可以容易地克服现有检测方法的局限性。该项目可以产生一种通用的检测方法，该方法易于适应快速检测和监测对人类健康构成大规模生物安全威胁的疾病，植物和动物生产。
