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目的采用纳米免疫磁珠分离副溶血性弧菌,建立副溶血性弧菌环介导等温扩增检测方法。方法采用副溶血性弧菌单克隆抗体,制备纳米免疫磁珠,特异性吸附副溶血性弧菌,结合环介导等温扩增技术,建立副溶血性弧菌快速检测方法。结果副溶血性弧菌纳米免疫磁珠在菌体浓度为103cfu/ml水平时,对副溶血性弧菌的捕获率达到74%。免疫磁分离结合环介导等温扩增技术,在纯培养、无需增菌情况下,检测灵敏度达到140 cfu/ml增菌液;通过对134株副溶血性弧菌和74株非目标菌的测试,环介导等温扩增技术具有良好的特异性;食品基质添加试验中,在增菌时间缩短至8 h的条件下,其检测限为2 cfu/25 g样品。结论副溶血性弧菌免疫纳米磁珠结合环介导等温扩增技术,有效缩短了增菌时间,适用于副溶血性弧菌的快速检测。
Objective To isolate Vibrio parahaemolyticus (Vibrio parahaemolyticus) by nano-immunomagnetic beads and establish a loop-mediated Isothermal Amplification Assay (Vibrio parahaemolyticus). Methods Vibrio parahaemolyticus monoclonal antibody was used to prepare nano-immunomagnetic beads for the specific adsorption of Vibrio parahaemolyticus. The rapid detection of Vibrio parahaemolyticus was established by ring-mediated isothermal amplification. Results Vibrio parahaemolyticus nanocomposite magnetic beads reached 74% of Vibrio parahaemolyticus when the concentration of bacteria was 103cfu / ml. Immunomagnetic separation combined with ring-mediated isothermal amplification, in pure culture, without increasing bacteria, the detection sensitivity of 140 cfu / ml enrichment broth; by 134 strains of Vibrio parahaemolyticus and 74 non-target bacteria test , And the ring-mediated isothermal amplification was of good specificity. The limit of detection (LOD) was 2 cfu / 25 g when the enrichment time was shortened to 8 h in the food matrix addition test. Conclusion Vibrio parahaemolyticus immunized with nano-magnetic beads and ring-mediated isothermal amplification technique effectively shorten the time of enrichment and is suitable for the rapid detection of Vibrio parahaemolyticus.