荧光免疫层析法结合免疫磁珠分离技术快速检测单增李斯特菌

Rapid Detection of Listeria monocytogenes Using a Fluorescence Immunochromatographic Assay Combined with Immunomagnetic Bead Separation

摘要:

为建立单增李斯特菌简单快速、灵敏度高和特异性强的检测方法，本研究以抗单增李斯特菌单克隆抗体偶联磁珠制备免疫磁珠；以羧基荧光微球标记的抗单增李斯特菌多克隆抗体及鼠IgG为标记抗体，抗单增李斯特菌多克隆抗体和羊抗鼠二抗分别作为检测线和质控线制备荧光免疫层析试纸条。将免疫磁珠分离与荧光免疫层析法相结合应用于单增李斯特菌的现场快速检测中。结果表明：荧光免疫层析试纸条对纯培养单增李斯特菌的检测限为4×105 CFU/mL，联合检测方法10倍、100倍浓缩时，检测限分别为4×104 CFU/mL和1×104 CFU/mL。联合检测体系特异性较好，与实验室保存的10株细菌无交叉反应。人工污染样本检测限为1×104 CFU/mL，同纯培养物相比检测灵敏度并没有降低。本方法的建立对于食品中单增李斯特菌的现场快速检测具有重要意义。

Abstract:

In order to establish a simple, rapid, sensitive, and specific method to detect Listeria monocytogenes, in this study, immunomagnetic beads were prepared by coupling anti-L. monocytogenes monoclonal antibody with magnetic beads. The fluorescence immunochromatographic strips were composed of anti-L. monocytogenes polyclonal antibody and mouse IgG marked by fluorescent microspheres as the detection antibody, anti-L. monocytogenes polyclonal antibody as the test line, and the goat anti-mouse IgG secondary antibody as the control line. Immunomagnetic separation was combined with a fluorescence immunochromatographic assay, and this method was applied to rapidly detect L. monocytogenes. The results showed that the detection limit of fluorescence immunochromatographic strips for pure cultures was 4 × 105 CFU/mL. For the joint detection method using samples concentrated 10- and 100-fold, the detection limits of pure culture samples were 4 × 104 CFU/mL and 1 × 104 CFU/mL, respectively. The joint detection method showed good specificity, and no cross-reactivity of the 10 strains kept in the laboratory was observed. The detection limit of artificially contaminated samples was also 1 × 104 CFU/mL and was not reduced compared with pure cultures. This method is of great value for rapid on-site detection of L. monocytogenes in food products.