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Position: Home > Articles > Rapid Detection of Live E. coli O157:H7 by PMA-qPCR Method FOOD SCIENCE 2012,33 (22) 217-220

PMA-qPCR方法快速检测活性E.coli O157:H7

作  者:
李聪聪;余以刚;邱杨;李美玲;肖性龙;吴晖
单  位:
华南理工大学轻工与食品学院;东莞出入境检验检疫局
关键词:
叠氮溴化丙锭;定量PCR;活菌;检测
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。
译  名:
Rapid Detection of Live E. coli O157:H7 by PMA-qPCR Method
作  者:
LI Cong-cong1,YU Yi-gang1,QIU Yang2,LI Mei-ling1,XIAO Xing-long1,*,WU Hui1 College of Light Industry and Food Sciences,South China University of Technology,Guangzhou 510640,China;2.Dongguan Entry-Exit Inspection and Quarantine Bureau,Dongguan 523072,China)
关键词:
propidium monoazide(PMA);quantitative polymerase chain reaction(qPCR);live bacteria;detection
摘  要:
In this work,a method to detect live E.coli O157:H7 was established using propidium monoazide(PMA) coupled with quantitative polymerase chain reaction(qPCR).The minimum inhibitory concentration against DNA amplification from dead bacteria was 10 μg/mL,and PMA did not inhibit DNA application from live bacteria at concentrations equal to or lower than 20 μg/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.

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