PMA-qPCR方法快速检测活性E.coli O157:H7  

Rapid Detection of Live E. coli O157:H7 by PMA-qPCR Method

作　　者：李聪聪[1] 余以刚[1] 邱杨[2] 李美玲[1] 肖性龙[1] 吴晖[1] 

机构地区：[1]华南理工大学轻工与食品学院,广东广州510640 [2]东莞出入境检验检疫局,广东东莞523072

出　　处：《食品科学》2012年第22期217-220,共4页Food Science

基　　金：广东省自然科学基金项目(9151064101000070);东莞市高等院校科研机构和医疗卫生单位科技计划项目(201074);广东省科技计划项目(2011B020314004);华南理工大学中央高校基本科研业务费项目(2011ZM0101);华南理工大学百步梯攀登计划研究项目

摘　　要：建立将叠氮溴化丙锭(PMA)与定量PCR(qPCR)技术结合,用于检测热灭活菌背景条件下的大肠杆菌活菌的方法。结果表明:5min以上的强烈光照可以使PMA与DNA共价交联,同时完全钝化游离的PMA以避免"假阴性"结果;抑制死菌DNA扩增的最低PMA质量浓度为10μg/mL,不抑制活菌DNA扩增的最高PMA质量浓度为20μg/mL。当活菌/总菌大于1%时,经PMA预处理,可以消除热灭活菌DNA的干扰,实现对活菌的定量检测。In this work, a method to detect live E. coliO157：H7wasestablishedusingpropidiummonoazide（PMA）coupled with quantitative polymerase chain reaction （qPCR）. The minimum inhibitory concentration against DNA amplification from dead bacteria was 10 ~tg/mL, and PMA did not inhibit DNA application from live bacteria at concentrations equal to or lower than 20 ~tg/mL. Covalent cross-linking of PMA with DNA was induced by strong light illumination for 5 min and meanwhile, free PMA was completely inactivated, resulting in the avoidance of false negative results. When the live to dead bacteria ratio was more than 1%, PMA pretreatment allowed the elimination of DNA interference from thermally inactivated bacteria and therefore live bacteria could be quantified.

关 键 词：叠氮溴化丙锭 定量PCR 活菌 检测 

分 类 号：TS207.4[轻工技术与工程—食品科学]