EMA和qPCR结合检测食品中活性肠出血性大肠杆菌O157:H7方法研究  

Detection of active E. coli O157: H7 in food combining EMA and qPCR

作　　者：潘素华 欧阳小明[2] 苏伟霞 陶爱林[2] PAN Su- hua;OU Yang Xiao- ming;SU Wei -xia;Tao Ai -lin(Panyu Customs,Guangzhou,Guangdong 511400,China)

机构地区：[1]番禺海关,广东广州511400 [2]广州医科大学第二附属医院,广东广州510260

出　　处：《现代预防医学》2018年第15期2809-2813,共5页Modern Preventive Medicine

基　　金：广州番禺区科技计划项目(2013-Z03-63);广东检验检疫局科技计划项目(2014GDK74)

摘　　要：目的将叠氮溴化乙锭(EMA)与双重荧光PCR技术结合,建立EMA荧光PCR方法,快速检测食品中活性大肠杆菌O157:H7。方法在已建立大肠杆菌O157:H7双重荧光PCR方法的基础上,优化EMA作用浓度、时间;观察EMA对活菌扩增的影响;利用模拟样品和现行检测方法做比较。结果 30μg/ml的EMA与细菌作用、曝光各15 min,可抑制4×10~6CFU/ml O157:H7死菌DNA的扩增;O157:H7活菌浓度>4×10~5CFU/ml时,EMA对活菌扩增有一定影响,离心可以降低其影响;活菌数/死菌数≥20%时,可以检出活菌;当活菌数量足够大时,死菌的存在不影响活菌的检出;EMA荧光PCR方法检测模拟样品,结果与现行的细菌分离培养金标准SN/T 0973-2010始终保持一致。结论建立的EMA荧光PCR方法可准确鉴定活态大肠杆菌0157:H7,避免死菌DNA造成的假阳性,在食品检测中具有一定的应用价值。Objective To establish an EMA - qPCR - based method for rapid identification of active Escherichia Coli O157 ： H7 in food by combining Ethidium Monoazide Bromide （EMA） and dual real - time PCR. Methods Based on dual real - time PCR method,EMA concentration and reaction time were optimized. The effect of the EMA on proliferation of living cells was observed and the established system compared with current detection methods by simulated samples. Results 30 μg/mL EMA and bacteria that reacted and exposed for 15 minutes respectively could suppress DNA amplification of 4 × 10^6 CFU/mL dead Escherichia Coli O157： H7. When the concentration of O157：H7 was greater than 4 × 10^5 CFU/mL, EMA could affect DNA amplification of live Escherichia Coli, which could be eliminated upon centrifugation. Live Escherichia Coli O157：H7 became detectable when live/dead bacterial count was equal or greater than 20%, and when the quantity of live Escherichia Coli was large enough, the presence of dead cells did not seem to affect the experimental results. The results of EMA qPCR method to the simulated samples were consistent with those of SN/T 0973 - 2010 standard. Conclusion The EMA qPCR method can accurately distinguish dead Escherichia Coli O157：H7 from live ones, thereby avoiding false positive detection. It has a certain application value in food inspection.

关 键 词：EMA 活性大肠杆菌O157:H7 双重荧光PCR 

分 类 号：R117[医药卫生—公共卫生与预防医学]