Lanzhou University of Technology, School of Life Science and Engineering, Lanzhou, China.
The Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Key Laboratory of Agricultural Genetics and Breeding, Shanghai, China.
Anal Bioanal Chem. 2024 Jun;416(15):3509-3518. doi: 10.1007/s00216-024-05301-0. Epub 2024 Apr 22.
Escherichia coli O157:H7 (E. coli O157:H7) is a foodborne pathogenic microorganism that is commonly found in the environment and poses a significant threat to human health, public safety, and economic stability worldwide. Thus, early detection is essential for E. coli O157:H7 control. In recent years, a series of E. coli O157:H7 detection methods have been developed, but the sensitivity and portability of the methods still need improvement. Therefore, in this study, a rapid and efficient testing platform based on the CRISPR/Cas12a cleavage reaction was constructed. Through the integration of recombinant polymerase amplification and lateral flow chromatography, we established a dual-interpretation-mode detection platform based on CRISPR/Cas12a-derived fluorescence and lateral flow chromatography for the detection of E. coli O157:H7. For the fluorescence detection method, the limits of detection (LODs) of genomic DNA and E. coli O157:H7 were 1.8 fg/µL and 2.4 CFU/mL, respectively, within 40 min. Conversely, for the lateral flow detection method, LODs of 1.8 fg/µL and 2.4 × 10 CFU/mL were achieved for genomic DNA and E. coli O157:H7, respectively, within 45 min. This detection strategy offered higher sensitivity and lower equipment requirements than industry standards. In conclusion, the established platform showed excellent specificity and strong universality. Modifying the target gene and its primers can broaden the platform's applicability to detect various other foodborne pathogens.
产志贺毒素大肠杆菌 O157:H7（Escherichia coli O157:H7）是一种食源性病原体，普遍存在于环境中，对全球人类健康、公共安全和经济稳定构成重大威胁。因此，早期检测对于控制产志贺毒素大肠杆菌 O157:H7 至关重要。近年来，已经开发出一系列产志贺毒素大肠杆菌 O157:H7 检测方法，但这些方法的灵敏度和便携性仍有待提高。因此，在本研究中，构建了一种基于 CRISPR/Cas12a 切割反应的快速高效检测平台。通过重组聚合酶扩增和侧向流动层析的整合，我们建立了基于 CRISPR/Cas12a 衍生荧光和侧向流动层析的双解释模式检测平台，用于检测产志贺毒素大肠杆菌 O157:H7。对于荧光检测方法，基因组 DNA 和产志贺毒素大肠杆菌 O157:H7 的检测限（LOD）分别为 1.8 fg/μL 和 2.4 CFU/mL，在 40 分钟内完成。相反，对于侧向流动检测方法，基因组 DNA 和产志贺毒素大肠杆菌 O157:H7 的 LOD 分别为 1.8 fg/μL 和 2.4×10 CFU/mL，在 45 分钟内完成。该检测策略的灵敏度高于行业标准，设备要求较低。总之，所建立的平台具有出色的特异性和强大的通用性。修改靶基因及其引物可以拓宽平台的适用性，以检测各种其他食源性病原体。
