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目的建立一种快速且质优价廉的检测副溶血性弧菌的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法。方法制备副溶血性弧菌跨膜转录调控蛋白toxR多克隆抗体,鉴定纯化后,进行磁珠包被制成免疫磁珠。利用免疫磁珠分离法和环介导等温扩增联用完成对副溶血性弧菌的检测,扩增产物经SYBR Green I染色肉眼观察和电泳鉴定。将副溶血性弧菌菌液作一系列稀释后,运用LAMP法进行敏感性检测,利用副溶血性弧菌和非副溶血性弧菌来验证LAMP方法的特异性。结果LAMP检测副溶血性弧菌的检测下限为10~5 CFU/ml。副溶血性弧菌出现LAMP特征性扩增反应,非副溶血性弧菌未出现LAMP扩增。结论 LAMP检测方法为快速诊断试剂盒打下实验基础,适合日常监测,可满足快速检测的需要。
Objective To establish a rapid and inexpensive loop-mediated isothermal amplification (LAMP) assay for the detection of Vibrio parahaemolyticus. Methods Polyclonal antibody against toxR of transmembrane transcriptional regulatory protein of Vibrio parahaemolyticus was prepared, identified and purified, and then magnetic beads were coated to form immunomagnetic beads. The detection of Vibrio parahaemolyticus was completed by immunomagnetic bead separation and ring-mediated isothermal amplification. The amplified products were visualized by SYBR Green I staining and electrophoresed. Vibrio parahaemolyticus was diluted to a series of dilutions, then LAMP assay was used to detect the sensitivity. Vibrio parahaemolyticus and non-Vibrio parahaemolyticus were used to verify the specificity of LAMP assay. Results The detection limit of LAMP for Vibrio parahaemolyticus was 10 ~ 5 CFU / ml. Vibrio parahaemolyticus LAMP characteristic amplification reaction, non-Vibrio parahaemolyticus did not appear LAMP amplification. Conclusion The LAMP detection method provides the experimental basis for rapid diagnostic kit, suitable for routine monitoring, to meet the needs of rapid detection.