EMA-PCR方法检测饮用水中3种食源性致病菌活菌研究  

Detection of 3 live food-borne pathogenic bacteria in drinking water by EMA-PCR

作　　者：陆星羽 张宏斌[2] 张梦寒[3] 陆巧荣[2] 

机构地区：[1]南京医科大学,江苏南京210029 [2]江苏省昆山市疾病预防控制中心,江苏昆山215300 [3]江苏省苏州市疾病预防控制中心,江苏苏州215004

出　　处：《现代预防医学》2015年第10期1845-1849,1885,共6页Modern Preventive Medicine

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)

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。Objective This study aimed to establish an effective and rapid method for detecting food-borne pathogenic bacteria in drinking water by combining Ethidium Monoazide Bromide（EMA） with PCR（EMA-PCR）. Methods Genes inv A, rfb E and opr I were used as the targets for PCR detection of Salmonella typhimurium, Escherichia Coli O157： H7 and Pseudomonas aeruginosa.PCR amplifications were carried out by utilizing their pure isolates as the templates. EMA concentration optimization and Sensitivity test were carried out. Results When EMA concentration was no more than 10μg/ml, EMA has no obvious inhibitory effect against the amplification of DNA derived from viable bacteria. The PCR amplification of DNA derived from dead bacteria can be effectively inhibited by EMA of 1μg/ml. The EMA-PCR sensitivity to the detection of S. typhimurium, E. Coli O157：H7 and P. aeruginosa were 1.2×10^4CFU/ml, 2×10^5CFU/ml and 3×10^4CFU/ml, respectively. Three live food-borne pathogenic bacteria were detected in the drinking water treated by 10 μg/ml EMA at the same time. Conclusion EMA-PCR can effectively detect three live food-borne pathogenic bacteria in the drinking water in one time and avoid false positive result of the PCR detection.

关 键 词：食源性致病菌 EMA-PCR 活菌检测 

分 类 号：R155.5[医药卫生—营养与食品卫生学]