基于大体系下(50 mL)纳米免疫磁珠高效富集单核增生李斯特菌的方法学研究

项目名称： 基于大体系下(50 mL)纳米免疫磁珠高效富集单核增生李斯特菌的方法学研究

项目编号： No.31271863

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 熊勇华

作者单位： 南昌大学

项目金额： 80万元

中文摘要： 免疫磁分离技术是食源性致病菌快速筛查技术的重要组成部分之一，该技术可高效捕获、浓缩增菌液中目标菌，提高致病菌检测灵敏度。现有的静态免疫磁分离方法因分离体积小（1～1.5 mL）而致富集效率低。增大磁分离体积是提高磁富集效率的有效有段。实现50 mL与1～1.5 mL分离体积相同的磁捕获效率，理论分析免疫磁珠用量需是小体积下的数十倍，因此按现有技术方案，大体积免疫磁分离方法的操作成本太高。本项目拟以Lm菌为分离模型，利用纳米免疫磁珠（100～200 nm）为磁捕获载体，从提高抗原/抗体反应效率，增加细菌表面纳米磁珠的结合数量等免疫学角度入手探讨降低大体积下免疫磁珠用量的理论依据，并结合数学建模的思想通过实验测试提出解决适合于纳米磁细菌快速分离的磁场设计方案，其最终目的为建立一套50 mL体积纳米免疫磁珠高效富集Lm以至其他食源性致病菌的免疫磁分离方法奠定理论基础。

中文关键词： 单增李斯特菌；免疫磁分离；信号放大；多重PCR；动态光散射

英文摘要： Immunomagnetic separation (IMS) has become one of the most important techniques in foodborne pathogen rapid screening process. IMS is efficient in target bacteria enrichment from complicated food samples, and thus improve the detection sensitivity. The capture efficiency of existing static immunomagnetic separation methods are high, however, the enrichment factor of these methods are low owing to the small processing ability itself (1-1.5 mL). Thus, it is essential to enlarge the sample volume in order to reach higher enrichment factor. Theoretically, the amount of magnetic beads will increase 10 times when increase the sample volume from 1-1.5 mL to 50 mL, which will cause a significant increase of the direct cost. In this proposal, Listeria monocytogenes and nano-magnetic beads with diameter of 100 and 200 nm will be employed to set up the separation model. The theoretical basis of decreasing the amount of immunomagnetic beads in 50 mL system will be discussed by increasing the antibody binding capacity and the number of nano-magnetic beads on the surface of L. monocytogenes. The characterization of magnetic field for nano-magnetic bacteria rapid separation in a large volume will be determined by cell tracking velocimetry measurement combined with mathematical modeling analysis of magnetic separation. Taken to

英文关键词： listeria monocytogenes；immunomagnetic separation；signal amplification；multiplex PCR；dynamic light scattering