Shi Cong, Tan Huimin, Yu Zhou, Li Weilin, Man Yan, Zhang Qinghai
Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, School of Public Health, Guizhou Medical University, No. 6 Ankang Road, Guian New Area, Guiyang 561113, China.
Institute of Quality Standard and Testing Technology, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China.
Foods. 2025 May 26;14(11):1892. doi: 10.3390/foods14111892.
The rapid and ultrasensitive detection of holds strategic significance for food safety surveillance and public health protection systems. This study innovatively developed a label-free biosensing platform based on the synergistic integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a and the fluorescent deoxyribozyme Aurora for the efficient detection of foodborne Salmonella. The detection mechanism operates through a molecular cascade reaction: target-activated Cas12a protein specifically degrades Aurora deoxyribozyme via its trans-cleavage activity, thereby abolishing the enzyme's catalytic capability to convert 4-methylumbelliferyl phosphate (4-MUP) into the highly fluorescent product 4-methylumbelliferone (4-MU). This cascade ultimately enables quantitative target analysis through fluorescence signal attenuation. Following systematic optimization of critical reaction parameters, the biosensing system demonstrated exceptional analytical performance: a detection limit of 1.29 CFU/mL with excellent linearity (R = 0.992) spanning six orders of magnitude (1.65 × 10-10 CFU/mL), along with high specificity against multiple interfering bacterial strains. Spike-and-recovery tests in complex food matrices (milk, chicken, and lettuce) yielded recoveries of 90.91-99.40% (RSD = 3.55-4.72%), confirming robust practical applicability. Notably, the platform design allows flexible detection of other pathogens through simple replacement of CRISPR guide sequences.
对食源性病原体进行快速、超灵敏检测对食品安全监测和公共卫生保护系统具有战略意义。本研究创新性地开发了一种基于成簇规律间隔短回文重复序列（CRISPR）-Cas12a与荧光脱氧核酶Aurora协同整合的无标记生物传感平台，用于高效检测食源性沙门氏菌。检测机制通过分子级联反应进行：靶标激活的Cas12a蛋白通过其反式切割活性特异性降解Aurora脱氧核酶，从而消除该酶将磷酸4-甲基伞形酮（4-MUP）转化为高荧光产物4-甲基伞形酮（4-MU）的催化能力。这种级联最终通过荧光信号衰减实现对靶标的定量分析。在对关键反应参数进行系统优化后，该生物传感系统展现出卓越的分析性能：检测限为1.29 CFU/mL，在六个数量级（1.65×10-10 CFU/mL）范围内具有出色的线性（R = 0.992），同时对多种干扰细菌菌株具有高特异性。在复杂食品基质（牛奶、鸡肉和生菜）中的加标回收试验回收率为90.91-99.40%（相对标准偏差=3.55-4.72%），证实了其强大的实际适用性。值得注意的是，该平台设计允许通过简单替换CRISPR引导序列来灵活检测其他病原体。
