Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Wuhan, 430070, China; College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
Jingzhou Institute for Food and Drug Control, Jingzhou, 434000, China.
Anal Chim Acta. 2024 Nov 15;1329:343248. doi: 10.1016/j.aca.2024.343248. Epub 2024 Sep 17.
Staphylococcus aureus is a significant foodborne pathogen, commonly detected in milk and meat products. Conventional detection methods have limited sensitivity and specificity, which are time-consuming and susceptible to background interference from complex samples, and cannot effectively distinguish live and dead bacteria.
Herein, we developed a novel adenosine triphosphate (ATP) bioluminescence method coupled with magnetic separation, which is based on phage-encoded endolysin LysSA163-CBD (CBD 163) for rapid and specific detection of viable Staphylococcus aureus. The expressed protein (CBD 163) was derived from the phage LSA2301 and was successfully expressed in Escherichia coli BL21 following an induction period of 4 h at 37 °C, with a molecular weight approximating 40 kDa. The optimal conditions for CBD-magnetic beads (cMBs) to capture S. aureus cells were determined to be 100 μL/mL cMBs at 25 °C for 30 min. The viable S. aureus cells were disrupted by the Cetyl trimethyl ammonium bromide (CTAB) to release intracellular ATP. Then, the ATP reacted with the firefly luciferase and D-Luciferin-based bioluminescence (BL) reagents solution to generate intensive BL signal. The CBD-magnetic separation-ATP bioluminescence (cMS-BL) assay was able to quickly detect viable S. aureus via ATP bioluminescence in 45 min, with a detection range from 5 × 10 to 5 × 10 CFU/mL and limit of detection (LOD) of 190 CFU/mL. Additionally, the cMS-BL method exhibited high specificity and anti-interference ability, which has been successfully applied to quantify S. aureus cells in crayfish-tail, chicken, and skim milk.
These results demonstrate the potential of CBD 163 as a versatile and robust biorecognition element for rapid and specific detection of viable S. aureus in food matrices. The proposed phage-derived bacteria-binding proteins-based protocol for BL detection shows various advantages, including high sensitivity, simple operation, and the capability to distinguish live bacteria, providing a strategy for designing high-quality biorecognition element toward foodborne pathogens.
金黄色葡萄球菌是一种重要的食源性致病菌，常存在于牛奶和肉制品中。传统的检测方法灵敏度和特异性有限，耗时且易受复杂样品背景干扰，无法有效区分活菌和死菌。
本研究建立了一种新型的基于噬菌体编码的溶菌酶 LysSA163-CBD（CBD163）的三磷酸腺苷（ATP）生物发光法，用于快速、特异性检测金黄色葡萄球菌的活菌。该表达蛋白（CBD163）来源于噬菌体 LSA2301，在 37°C 诱导 4 小时后成功在大肠杆菌 BL21 中表达，分子量约为 40kDa。确定 CBD 磁珠（cMBs）捕获金黄色葡萄球菌细胞的最佳条件为 25°C 下 30min、cMBs 的浓度为 100μL/mL。细胞用十六烷基三甲基溴化铵（CTAB）破坏后，细胞内的 ATP 被释放出来。然后，ATP 与萤火虫荧光素和 D-荧光素酶基生物发光（BL）试剂反应，产生强烈的 BL 信号。CBD 磁分离-ATP 生物发光（cMS-BL）检测法可通过 45min 内 ATP 的生物发光快速检测活菌，检测范围为 5×10 到 5×10 CFU/mL，检测限（LOD）为 190CFU/mL。此外，该 cMS-BL 方法具有较高的特异性和抗干扰能力，已成功应用于定量小龙虾尾、鸡肉和脱脂乳中的金黄色葡萄球菌细胞。
这些结果表明，CBD163 作为一种多功能、稳健的生物识别元件，可用于快速、特异性检测食品基质中的活菌金黄色葡萄球菌。该噬菌体衍生的细菌结合蛋白 BL 检测方法具有灵敏度高、操作简单、能够区分活菌等优点，为设计针对食源性致病菌的高质量生物识别元件提供了一种策略。
