EMA结合实时荧光PCR方法检测单核细胞增生李斯特氏菌

Detection of Live Listeria monocytogenes by EMA-qPCR

建立叠氮溴化乙锭(EMA)结合实时荧光PCR(qPCR)方法检测单核细胞增生李斯特氏菌(Listeria monocytogenes)活菌。以李斯特溶血素O(LLO)基因hly设计引物、TaqMan探针,用李斯特属典型菌、沙门氏菌等56株致病菌株验证特异性,不同质量浓度EMA处理进行qPCR检测。尝试脱氧胆酸钠溶液(SD)强化抑制效果,73份不同的人工污染食品、环境样本(卤鸡肉、牛奶、肉馅、垃圾渗滤液)测试实用性。结果表明,引物探针准确检测L.monocytogenes,对其他菌株无特异性扩增。EMA最适质量浓度2.5μg/mL,经过15 min光激活与死菌DNA共价结合明显抑制了扩增,方法检出限为150 CFU/mL。SD处理L.monocytogenes活菌Ct值增加。与传统培养法比较,EMA-qPCR方法检测100%准确,操作简单、省时高效,在食品、环境方面应用前景广阔。

An ethidium monoazide in combination with real-time PCR method(EMA-PCR)was newly developed for selective detection of live Listeria monocytogenes cells from dead cells.The primers and TaqMan probes were designed using the hly gene which encoded listeriolysin O(LLO).The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of Listeria monocytogenes strains and other pathogenic bacteria strains(n=56).A qPCR assay was detected with EMA of different concentrations.Sodium deoxycholate solution(SD)was used to strengthen the inhibitory effect.In addition,73 various artificial contaminated food and environmental samples(braised chicken,milk,minced meat and garbage leachate)were investigated for L.monocytogenes.The primer probe accurately detected L.monocytogenes without specific amplification for other strains.EMA could penetrate dead cells resulting in the covalent links of DNA upon 15 min light exposure and therefore inhibition of amplification under an optimal concentration of 2.5μg/mL with a detection limit of 150 CFU/mL.The Ct value of live Lm increased under SD treatment.The diagnostic accuracy of EMA-qPCR method was up to 100%compared to the traditional culture method.This method with simple operation,time-saving and high efficiency has a promising application on accurate microbiological monitoring of food safety and environmental source.