EMA-LAMP方法快速鉴别检测单增李斯特菌

Rapid Detection and Discrimination Viable Cell of Listeria monocytogenes by EMA-LAMP Assay

DOI：10.3969/j.issn.1673-1689.2012.09.009

中文关键词: 单增李斯特菌 EMA-LAMP 快速鉴别检测 死活细胞

英文关键词: Listeria monocytogenes EMA-LAMP rapid detection and identification viable and dead cells

基金项目:辽宁省自然科学基金项目(201102196);沈阳市大型科学仪器设备共享服务专项项目(090009)

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中文摘要:

作者建立一种将叠氮溴化乙锭(ethidium bromide monoazide,EMA)结合环介导等温扩增(loop-mediated isothermal amplification,LAMP)的新分析方法(EMA-LAMP),快速鉴别检测单增李斯特菌。通过对单增李斯特菌hly基因的六个区域设计4条LAMP特异性引物,EMA-LAMP在63℃下反应1 h,检测单增李斯特菌的死活细胞。结果表明,在浓度为2.0×108CFU/mL的单增李斯特死菌悬液中,使EMA激活光解进入死细胞中且与DNA结合的最佳曝光时间至少为20 min,不抑制活菌细胞DNA的LAMP扩增的最大EMA质量浓度为10μg/mL,而抑制死菌细胞DNA的LAMP扩增的最小EMA质量浓度为4.0μg/mL;活菌细胞的检出限为20 CFU/mL。

英文摘要:

Viable cells of Listeria monocytogenes could be selectively discriminated from dead cells by applying Ethidium bromide monoazide(EMA) cooperated into loop-mediated isothermal amplification(LAMP) assay.The EMA-LAMP method was used for the rapid detection and identification of foodborne pathogene Listeria monocytogenes.The amplification can be obtained in an hour under the isothermal condition at 63°C by designing four LAMP specifically primers from the six different sequences on the hly gene.The EMA-LAMP assay discriminated the viable and dead cells of Listeria monocytogenes.The result showed that 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 20 min,and the use of 10 μg/ml or less of EMA did not inhibit the LAMP amplification of DNA derived from viable cells,but the minimum concentration of EMA to completely inhibit the LAMP amplification of DNA derived from dead cells was 4.0 μg/ml;a detection limit of the assay for the viable cells was 20 CFU/ml.

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