紫胶红素对食源性致病菌“活体染色”机理及新型双向免疫层析技术研究

Later Flow Immunochromatography Assay（LFIA） has been widely used in the field of human，animal，plant medicine and food safety, which can detect various targets quickly, accurately and visually. The improvement of detection performance is seriously affected by antibody labels and antibody performance. Gold nanoparticles and other materials are mostly used to achieve the "visualization", and the.function of carrying antibodys is only being forced to. This technology can be completely getting rid of the dependence on the label materials if we"colored” the target and get the visible results. On the basis of previous studies, the bacteria which ingests “Lac Dyer Red” becomes "red bacteria" by adding natural dyes“Lac Dyer Red” to the bacteria enrichment broth. And then we use the method of Laser Scanning Confocal Microscopy（LSCM），Flow Cytometry(FCM) and other techniques to evaluate the toxicity of bacteria, DNA damage and other toxicological; And we use transmission electron microscopy (TEM), the sucrose density gradient centrifugation and other methods to study the mechanism of “Lac Dyer Red”staining. Finally,we will design the immunochromatographic test strip which is composed by the detection line and the control line and between them be connected by the absorbent pads. A new immunochromatographic technique is set up via actual detection and optimizing conditions to develop, and this technique only need one antibody and get "visualization" result without using any nano materials. The technology is a significant innovation in detection field, and not only used for rapid detection of bacteria but also detection of other multiple targets.

广泛运用于人体和动植物医学、食品安全领域，可快速、准确、可视化检测各种靶标的侧向免疫层析技术，其检测性能的提升遭遇抗体和抗体标记材料的制约。纳米金等抗体标记材料的主要功能是实现检测结果的“可视化”，如果对检测靶标进行“染色”，则可完全摆脱免疫层析技术对抗体标记材料的依赖。基于此理念，在前期研究的基础上，通过在细菌増菌过程中添加天然染料紫胶红素，使细菌摄入紫胶红素变成“红色细菌”。使用激光共聚焦显微成像、流式细胞术等手段，就紫胶红素对细菌的毒性进行评估，通过透射电镜、蔗糖密度梯度离心等手段研究紫胶红素染色机理，依据以上研究优化紫胶红素加入浓度、染色条件等参数，确保紫胶红素对细菌活力无影响。另外，设计由检测区和质控区组成，中间用吸水垫连接的双向免疫层析试纸，该新型免疫层析技术无需纳米金等材料标记、只需一株抗体、结果肉眼可见，在増菌过程中同步对细菌进行染色，适合于食源性致病菌的快速检测。

广泛运用于人体和动植物医学、食品安全领域，可快速、准确、可视化检测各种靶标的侧向免疫层析技术，其检测性能的提升遭遇抗体和抗体标记材料的制约。纳米金等抗体标记材料的主要功能是实现检测结果的“可视化”，如果对检测靶标进行“染色”，则可完全摆脱免疫层析技术对抗体标记材料的依赖。本研究采用在食源性致病菌増菌过程中，添加天然染料紫胶红素（Laccaic Acid，LA） 使得细菌“有色化”，以实现LFICA检测结果的“可视化”。通过确定紫胶红素媒染食源性致病菌的媒染剂和媒染方式；设计了由检测区和质控区组成，中间用吸水垫连接的双向免疫层析试纸条，测定了此双向免疫层析试纸条的灵敏度和特异性，并进行了模拟带菌检测。结果表明，紫胶红素染色试纸条对大肠杆菌O157:H7的灵敏度为106 CFU/mL，与传统胶体金试纸条检测灵敏度一致，特异性和稳定性良好；对模拟带菌的食品成功检出大肠杆菌O157:H7，食品样品前增菌至10 h就可快速检测，相比国标法前增菌的时间缩短了约14h。该新型免疫层析技术无需纳米金等材料标记、只需一株抗体、结果肉眼可见，且具有高灵敏度、高准确性和高稳定性，推动了免疫层析分析技术在食源性致病菌快速检测中的应用。.项目完成了各项任务指标，发表第一标注科研论文7篇，其中中文核心期刊2篇，SCI论文5篇，申请相关专利10项，培养青年老师3名，博士后1名，硕博士研究生7名。