EMA-PCR方法检测饮用水中3种食源性致病菌活菌研究

发布时间：2019-05-20 12:18

【摘要】：目的将EMA(Ethidium Monoazide Bromide)选择渗透性与PCR技术相结合,建立有效快速检测饮用水中3种食源性致病菌活菌EMA-PCR方法。方法以鼠伤寒沙门菌inv A、肠出血性大肠杆菌O157:H7 rfb E、铜绿假单胞菌opr I基因为PCR检测靶基因,纯培养物提取模板进行PCR,进行EMA使用浓度及灵敏度试验。结果 EMA浓度不大于10μg/ml对活菌DNA扩增没有明显抑制,终浓度为1μg/ml EMA能有效抑制死菌扩增;对鼠伤寒沙门菌、肠出血性大肠杆菌O157:H7、铜绿假单胞菌的EMA-PCR灵敏度分别为1.2×104CFU/ml、2×105CFU/ml、3×104CFU/ml;用10μg/ml EMA处理饮用水水样,可同时检测出饮用水中含有的3种食源性致病菌活菌。结论 EMA-PCR方法可一次检测饮用水中3种食源性致病菌活菌,能避免PCR检测可能造成假阳性结果。
[Abstract]:Objective to establish an effective and rapid EMA-PCR method for the detection of three kinds of food-borne pathogenic bacteria in drinking water by combining EMA (Ethidium Monoazide Bromide) selective permeability with PCR technique. Methods Salmonella typhimurium inv A, enterohemorrhagic Escherichia coli O157:H7 rfb E and Pseudomonas aeruginosa opr I gene were used as target genes for PCR detection. PCR, was extracted from pure culture template for EMA concentration and sensitivity test. Results when the concentration of EMA was not more than 10 渭 g / ml, the DNA amplification of living bacteria was not significantly inhibited, but the final concentration of 1 渭 g / ml EMA could effectively inhibit the amplification of dead bacteria. The EMA-PCR sensitivity of Salmonella typhimurium, enterohemorrhagic Escherichia coli O157 and Pseudomonas aeruginosa were 1.2 脳 10 ~ 4 CFU / ml, 2 脳 10 ~ 5 CFU / ml, 3脳104CFU / ml;, respectively. Three kinds of living bacteria containing food-borne pathogenic bacteria in drinking water can be detected at the same time when 10 渭 g / ml EMA is used to treat drinking water samples. Conclusion EMA-PCR method can detect three kinds of living bacteria of food-borne pathogenic bacteria in drinking water at one time, which can avoid the false positive results of PCR detection.
【作者单位】：南京医科大学;江苏省昆山市疾病预防控制中心;江苏省苏州市疾病预防控制中心;
【基金】：苏州市饮用水安全与水性疾病监测公共服务平台2012年开放课题(SZPT2012002)
【分类号】：R155.5

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