Zhou Yuqing, Kang Hanli, Zhang Xiaoguang, Ji Yalu, Han Wenyu, Wang Bin, Gu Jingmin
State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun, 130062, China.
College of Food Science and Engineering, Jilin University, Changchun, 130062, China.
Anal Chim Acta. 2025 Sep 22;1368:344300. doi: 10.1016/j.aca.2025.344300. Epub 2025 Jun 11.
Staphylococcus aureus (S. aureus) is one of the most important pathogens that causes foodborne illnesses. The development of new methods for the early detection and surveillance of S. aureus is key to preventing and controlling the occurrence of foodborne diseases.
In this study, the C54A mutation of LysGH15 (LysGH15-C54A) of the S. aureus bacteriophage lysin was fused with enhanced green fluorescent protein (EGFP) to construct the recombinant protein LysGH15-C54A-EGFP. A fluorescence (FL) detection protocol using LysGH15-C54A-EGFP as the FL signal carrier and LysGH15-C54A coupled with immunomagnetic beads (LysGH15-C54A-IMBs) as the isolating element and capturing agent for the detection of S. aureus was established on the basis of the high specificity and efficiency of LysGH15-C54A in recognizing S. aureus. LysGH15-C54A-IMBs were able to specifically separate and capture S. aureus, and LysGH15-C54A-EGFP was used to combine the captured surface vacancies of S. aureus to achieve FL detection of S. aureus using two-site recognition. When S. aureus was detected in the concentration range of 10-10 CFU/mL, the FL method showed good linearity with a detection limit of 85 CFU/mL, and the whole process lasted less than 1 h. The procedure was successfully applied to quantify S. aureus in fresh milk samples, and the recovery values ranged from 93.09 % to 101.04 %.
Based on these results, we believe that LysGH15-C54A-based FL detection method hold great potential for rapid, accurate, and specific diagnosis of S. aureus, and it has good prospects in practical applications.
金黄色葡萄球菌是引起食源性疾病的最重要病原体之一。开发金黄色葡萄球菌早期检测和监测的新方法是预防和控制食源性疾病发生的关键。
在本研究中，将金黄色葡萄球菌噬菌体溶素的LysGH15（LysGH15-C54A）的C54A突变体与增强型绿色荧光蛋白（EGFP）融合，构建重组蛋白LysGH15-C54A-EGFP。基于LysGH15-C54A识别金黄色葡萄球菌的高特异性和高效性，建立了以LysGH15-C54A-EGFP为荧光信号载体，LysGH15-C54A偶联免疫磁珠（LysGH15-C54A-IMBs）为分离元件和捕获剂的荧光（FL）检测方法用于检测金黄色葡萄球菌。LysGH15-C54A-IMBs能够特异性分离和捕获金黄色葡萄球菌，LysGH15-C54A-EGFP用于结合捕获的金黄色葡萄球菌表面空位，通过两点识别实现金黄色葡萄球菌的荧光检测。当检测金黄色葡萄球菌的浓度范围为10-10 CFU/mL时，荧光法显示出良好的线性，检测限为85 CFU/mL，整个过程持续时间不到1小时。该方法成功应用于鲜牛奶样品中金黄色葡萄球菌的定量分析，回收率在93.09%至101.04%之间。
基于这些结果，我们认为基于LysGH15-C54A的荧光检测方法在金黄色葡萄球菌的快速、准确和特异性诊断方面具有巨大潜力，在实际应用中具有良好的前景。
