School of Food and Biological Engineering, Chengdu University, Chengdu, 610106, China; Engineering Research Center of Sichuan-Xizang Traditional Medicinal Plant, Chengdu University, Chengdu, 610106, China.
School of Food and Biological Engineering, Chengdu University, Chengdu, 610106, China; Engineering Research Center of Sichuan-Xizang Traditional Medicinal Plant, Chengdu University, Chengdu, 610106, China.
Biosens Bioelectron. 2024 Dec 15;266:116727. doi: 10.1016/j.bios.2024.116727. Epub 2024 Aug 30.
The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 10 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.
从各种样本中分离和鉴定致病菌对于控制与细菌感染相关的健康问题至关重要。传统方法，如平板计数和聚合酶链反应方法，往往耗时且依赖于特定仪器，严重限制了这些病原体的有效鉴定。在本研究中，我们利用金黄色葡萄球菌噬菌体 80α（80α）内溶素细胞壁结合（CBD）结构域对宿主细菌的特异性进行分离。酰胺酶 3-CBD 缀合磁珠能够成功地从牛奶、血液和唾液中分离出低至 1×10 CFU/mL 的金黄色葡萄球菌细胞。与商用细菌基因组 DNA 提取试剂盒相比，80α 内溶素的细胞壁水解活性平均将基因组 DNA 的提取效率提高了 12.7 倍。然后，重组酶聚合酶扩增（RPA）用于在 37°C 下从提取的 DNA 中扩增金黄色葡萄球菌的 nuc 基因，30 分钟。RPA 产物激活 Cas12a 内切酶活性，切割荧光标记的 ssDNA 探针。然后，我们将产生的信号转换为荧光读数，可通过肉眼或带有超高灵敏度的便携式自组装仪器进行检测。从分离到鉴定的整个过程可以在 2 小时内完成。本研究中开发的方法简单且灵敏，在金黄色葡萄球菌检测中具有很大的应用价值，尤其是在资源供应有限的地区。
