Guo Yiqing, Li Chen, Guo Wang, Zhang Xinai, Wang Li, Zhang Wen, Zou Xiaobo, Sun Zongbao
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China.
School of Pharmacy, Jiangsu University, Zhenjiang 212013, China.
J Agric Food Chem. 2024 Oct 2. doi: 10.1021/acs.jafc.4c07308.
() is a prevalent foodborne pathogen that poses significant challenges to food safety. Herein, a sensitive and specific electrochemical biosensor based on RPA-CRISPR/Cas12a is developed for evaluating . In the presence of , the extracted target DNA fragments are efficiently amplified by recombinase polymerase amplification (RPA). The designed crRNA, binding to Cas12a, effectively recognizes the target fragment cleaving hpDNA. The signal molecule of hpDNA is cleaved from the sensing interface, resulting in a reduction of current response. Under optimal experimental conditions, the developed electrochemical biosensor exhibits remarkable sensitivity in detecting . The linear range for quantifying in pure culture is 1.04 × 10-1.04 × 10 CFU/mL, with a detection limit as low as 3 CFU/mL. In addition, the biosensor enables the accurate and sensitive detection of in milk within a linear range of 1.07 × 10-1.07 × 10 CFU/mL. The electrochemical biosensor enhances anti-interference capability owing to the specific amplification of RPA primers and the single-base recognition ability of crRNA. The RPA-CRISPR/Cas12a biosensor exhibits exceptional anti-interference capability, precision, and sensitivity, thereby establishing a robust foundation for real-time monitoring of microbial contamination.
（）是一种普遍存在的食源性病原体，对食品安全构成重大挑战。在此，开发了一种基于重组酶聚合酶扩增（RPA）-CRISPR/Cas12a的灵敏且特异的电化学生物传感器用于评估（）。在（）存在的情况下，提取的目标DNA片段通过重组酶聚合酶扩增（RPA）得到有效扩增。设计的与Cas12a结合的crRNA有效识别切割发夹型DNA（hpDNA）的目标片段。hpDNA的信号分子从传感界面被切割，导致电流响应降低。在最佳实验条件下，所开发的电化学生物传感器在检测（）时表现出显著的灵敏度。纯培养物中定量（）的线性范围为1.04×10 - 1.04×10 CFU/mL，检测限低至3 CFU/mL。此外，该生物传感器能够在1.07×10 - 1.07×10 CFU/mL的线性范围内准确灵敏地检测牛奶中的（）。由于RPA引物的特异性扩增和crRNA的单碱基识别能力，电化学生物传感器增强了抗干扰能力。RPA - CRISPR/Cas12a生物传感器表现出卓越的抗干扰能力、精度和灵敏度，从而为微生物污染的实时监测奠定了坚实基础。
