表面活性剂脱氧胆酸钠在单增李斯特菌死活细胞EMA-PCR鉴别中的应用

摘要

通过脱氧胆酸钠(sodium deoxycholate,SD)结合染料叠氮溴乙锭(ethidium bromide monoazide,EMA)与PCR反应体系相结合,建立纯培养条件下区分单增李斯特菌死活细胞的方法,即SD-EMA-PCR鉴别单增李斯特菌死活细胞检测法.结果表明:向浓度为2.0×108 CFU/mL的单增李斯特死菌悬液中加入终浓度为1.5 mg/mL的表面活性剂脱氧胆酸钠后,EMA激活光解最佳曝光时间为25 min.SD-EMA-PCR检测体系中,对死菌细胞DNA的PCR扩增抑制的EMA最小浓度为1.6 μg/mL；而对活菌细胞DNA的PCR扩增不抑制的EMA最大浓度为9 μg/mL,活菌细胞的最低检出限为20 CFU/mL.SD-EMA-PCR检测法能够减小EMA-PCR漏检死菌细胞给检测结果带来假阳性的可能,降低了检测的成本,为单增李斯特菌的快速检测提供了一种新的有效途径.%Through the treatment of the sodium deoxycholate to the cells,Ethidium bromide monoazide was utilized to selectively allow the PCR amplification of a targeted DNA sequence in viable but not dead cells of Listeria monocytogenes,namely SD-EMA-PCR.The optimal concentration of sodium deoxycholate incorporating into the PCR system could enhance the discrimination of viable and dead cells.The results showed that:with the treatment of the Sodium deoxycholate,the optimized light exposure time to achieve cross-linking of DNA by the EMA in dead cells and to photolyse the free EMA in solution was at least 25 min,and the maximum concentration of EMA which did not inhibit the PCR amplification of DNA extracted from viable cells was 9 μg/mL.but the minimum concentration of EMA which completely inhibited the PCR amplification of DNA extracted from dead cells was 1.6 μg/mL; a detection limit of the assay for the viable cells was 20 CFU/mL.SD-EMA-PCR could minimize the possibility of the false-positive by the way of EMA-PCR and reduce the cost of detection.It is a new efficient approach for the rapid detection of the isolate of Listeria monocytogenes.