Yu Chen, Liu Yang, Zhang Wen, Yao Xiaoli
Xiang'an Hospital of Xiamen University, No. 2000, East Xiang'an Road, Xiang'an District, Xiamen City, Fujian Province, 361100, China.
Anal Methods. 2025 Jan 2;17(2):284-290. doi: 10.1039/d4ay01728e.
(, PA) is a rod-shaped Gram-negative opportunistic bacterium capable of causing nosocomial infections during nursing, such as burn wound infections and meningitis. However, sensitive and rapid PA detection remains a huge challenge. Herein, a new fluorescent biosensor was developed for the sensitive detection of PA using a three-way junction (TWJ) probe for specific identification and CRISPR/Cas14a1 for enhanced exponential amplification. The interaction between PA and its aptamer on a DNA TWJ structure probe triggered the migration of the double-stranded DNA branch, inducing DNA polymerase/endonuclease assisted chain displacement and the generation of single-stranded DNA sequences. The amplification products have the ability to activate CRISPR/Cas14a1, resulting in effective -cleavage and the subsequent release of fluorescence from the reporter probes. Under optimized conditions, the proposed biosensor was able to detect a wide range of bacterial concentrations, ranging from 10 to 10 cfu mL within 30 min. The limit of detection, which is the lowest concentration that can be reliably detected, was determined to be 3.4 cfu mL according to the 3 rule. The results of the recovery test suggest that the biosensor shows significant potential for clinical applications. The established biosensor utilizing the TWJ probe generated multiple isothermal exponential amplification and the CRISPR/Cas14a1 biosensor is an excellent platform for rapidly detecting pathogenic bacteria in postoperative infection.
铜绿假单胞菌（PA）是一种杆状革兰氏阴性机会致病菌，能够在护理期间引起医院感染，如烧伤创面感染和脑膜炎。然而，灵敏快速地检测PA仍然是一个巨大的挑战。在此，开发了一种新型荧光生物传感器，用于灵敏检测PA，该传感器使用三链交界处（TWJ）探针进行特异性识别，并利用CRISPR/Cas14a1进行增强指数扩增。PA与其适配体在DNA TWJ结构探针上的相互作用触发了双链DNA分支的迁移，诱导DNA聚合酶/核酸内切酶辅助链置换并产生单链DNA序列。扩增产物能够激活CRISPR/Cas14a1，导致有效切割并随后从报告探针释放荧光。在优化条件下，所提出的生物传感器能够在30分钟内检测10至10 cfu/mL范围内的多种细菌浓度。根据3规则，检测限（即可可靠检测的最低浓度）确定为3.4 cfu/mL。回收率测试结果表明，该生物传感器在临床应用中具有显著潜力。利用TWJ探针建立的生物传感器产生了多个等温指数扩增，CRISPR/Cas14a1生物传感器是快速检测术后感染中病原菌的优秀平台。
