免疫磁性糊精微球的制备及快速分离检测单增李斯特菌

研究免疫磁性糊精微球的制备及在快速分离检测单增李斯特菌中的应用。将磁性糊精微球由环氧氯丙烷活化后,与抗体交联制得免疫磁性糊精微球,通过单因素和正交试验对合成条件进行优化。再利用合成好的免疫磁性糊精微球捕获单増李斯特菌,在外加磁场的作用下,富集免疫磁性糊精微球,提取细菌DNA,经过PCR扩增后,进行琼脂糖凝胶电泳。结果表明:活化反应的最佳条件,环氧氯丙烷用量0.4 mL/g,NaOH浓度2.0 mol/L,最佳反应时间5 h;制备免疫磁性糊精微球的最优合成条件:抗体质量浓度1.0 mg/mL,反应温度28℃,反应时间2.5 h;捕获单增李斯特菌时,免疫磁性糊精微球的最佳用量100μL、最佳捕获时间1 h。通过研究得出免疫磁性糊精微球可以快速捕获单增李斯特菌,并能扩增出特异性产物,检出限达到每mL菌液中10个单增李斯特菌。

Rapid Isolation and Detection of Listeria monocytogenes in Vitro by Immunomagnetic Dextrin Microspheres

To investigate the application of immunomagnetic dextrin microspheres for rapid isolation and detection of Listeria monocytogenes.The magnetic dextrin microspheres were activated by epichlorohydrin and then crosslinked with antibodies to prepare immunomagnetic dextrin microspheres.The synthesis conditions were optimized by single factor and orthogonal tests.To capture a single extended Listeria monocytogenes using immunomagnetic microspheres synthesized cyclodextrin,under external magnetic field,the magnetic enrichment of immune bacterial DNA extraction,dextrin microspheres,amplified by PCR,agarose gel electrophoresis.The results show that the best condition is the activation reaction of epichlorohydrin dosage is 0.4 mL/g,the concentration of NaOH is 2.0 mol/L,the reaction time is 5 h;the optimal synthesis conditions of preparation of immunomagnetic microspheres is antibody dextrin mass concentration is 1.0 mg/mL,the reaction temperature is 28℃,the reaction time is 2.5 h;capture Listeria bacteria the best dosage of immunomagnetic microspheres,dextrin as the best of 100μL,the capture time is 1 h.The results showed that the immunomagnetic dextrin microspheres could effectively and rapidly capture the single Listeria strain and amplify the specific products.The detection limit could reach 10 single Listeria strains per mL of bacteria.