基于二氧化硅荧光纳米颗粒与核酸染料SYBRGreenⅠ的双色显微成像技术用于E.coliO157:H7的检测

摘要

将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合,发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157:H7的检测.采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157:H7抗体进行修饰,基于抗体-抗原相互作用实现了其对目标大肠杆菌O157:H7的特异性标记；同时以核酸染料SYBR Green Ⅰ对细菌进行染色,将细菌和纳米颗粒团聚体区分开,实现了对大肠杆菌O157:H7的双色标记,并通过激光共聚焦显微镜进行免分离的荧光成像检测.结果表明,该方法可用于缓冲溶液体系和混合细菌样品中目标大肠杆菌O157:H7的特异性检测,在仅含5％目标菌的混合样品中仍能观察到具有明显黄色荧光的大肠杆菌O157:H7,且整个检测步骤包括样品预处理可在3h内完成.该方法则具有较好的灵敏度,可检出2.6×103 Cell/mL的目标细菌样品.若采用针对其它病原菌细胞壁抗原的特异性抗体,则有望发展成为一种通用技术用于多种病原菌的快速和灵敏检测.%A novel two-color immunofluorescent microscopic imaging technique for the rapid and sensitive detection of E. Coli O157: H7 was developed by fluorescent silica nanoparticles and SYBR Green I . Based on antigen-antibody interaction, the anti-E. Coli 0157: H7 antibody conjugated RuBpy-doped silica nanoparticles were used to recognize E. Coli 0157: H7 specifically. The bacteria sample was then stained with a nucleic acid dye SYBR Green I and imaged with confocal microscope. The separation-free detection of E. Coli 0157: H7 was successfully carried out in buffer and bacterial mixture, respectively. The whole detection process could be finished within 3 h, and the detection limit for E. Coli 0157: H7 was 2.6 × 103Cell/mL. Considering the antibodies for various bacteria, this proposed method might be promising for rapid and sensitive detection of other bacteria.