Department of Gastroenterology, People's Hospital Of Chongqing Liang Jiang New Area, No. 199 Renxing Road, Renhe Street, Yubei District, Chongqing, 401147, China.
Biotechnol Lett. 2024 Nov 25;47(1):4. doi: 10.1007/s10529-024-03550-8.
Evaluating the methicillin resistance of Staphylococcus aureus (S. aureus) is highly important for adapting nursing strategies. Nevertheless, the identification of methicillin-resistant S. aureus (MRSA) that is both sensitive and reliable continues to pose a significant obstacle. This study describes a method for detecting MRSA using a combination of fixed rolling circle amplification (RCA) and the exonuclease-iii (Exo-iii) assisted CRISPR-Cas12a system for signal amplification. When MRSA is present, the interaction between the "b" chain in the capture probe and MRSA allows the "a" chain to be exposed. This "a" chain acts as a primer to initiate the fixed RCA process. The H probe, which includes the crRNA segment, forms a bond with the RCA product and then releases the crRNA segment with the aid of Exo-iii. The Cas12a protein, when combined with the crRNA, generates an activated CRISPR-Cas12a system that cleaves the "Reporter" probe, resulting in the production of fluorescent signals. Furthermore, this fluorescent test has been utilized for the examination of clinical samples with a satisfactory rate of retrieval. Based on the elegant design, the proposed method exhibited a low detection limit of 4.6 cfu/mL, while maintaining a high specificity for MRSA even from a mixture of several interfering bacteria. Due to its cost-effectiveness, simplicity, and adaptability, the sensing system shows potential as a platform for detecting MRSA and evaluating postoperative nursing for stomach cancer patients.
评估金黄色葡萄球菌（S. aureus）的耐甲氧西林情况对于制定护理策略至关重要。然而，鉴定既敏感又可靠的耐甲氧西林金黄色葡萄球菌（MRSA）仍然是一个重大挑战。本研究描述了一种使用固定滚环扩增（RCA）和外切酶-iii（Exo-iii）辅助 CRISPR-Cas12a 系统进行信号放大来检测 MRSA 的方法。当存在 MRSA 时，捕获探针中的“b”链与 MRSA 的相互作用使“a”链暴露出来。这条“a”链充当引物启动固定 RCA 过程。包含 crRNA 片段的 H 探针与 RCA 产物结合，然后在 Exo-iii 的帮助下释放 crRNA 片段。Cas12a 蛋白与 crRNA 结合后，产生激活的 CRISPR-Cas12a 系统，切割“Reporter”探针，产生荧光信号。此外，该荧光检测法已用于临床样本的检测，具有令人满意的检出率。基于巧妙的设计，该方法表现出低至 4.6 cfu/mL 的检测限，并且对 MRSA 具有很高的特异性，即使在几种干扰细菌的混合物中也是如此。由于其具有成本效益、简单性和适应性，该传感系统有望成为检测 MRSA 和评估胃癌患者术后护理的平台。
