中国畜牧兽医

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肠出血性大肠埃希菌O157∶H7实时荧光定量PCR快速检测方法的建立

李丹丹1,徐义刚2,王绥家1,高慎阳3,李一经4   

  1. 1.海南出入境检验检疫局检验检疫技术中心,海南海口  5703112.黑龙江出入境检验检疫局检验检疫技术中心,黑龙江哈尔滨  1500013.辽宁医学院畜牧兽医学院,辽宁锦州  1210014.东北农业大学动医学院,黑龙江哈尔滨  150030
  • 收稿日期:2014-01-07 出版日期:2014-07-20 发布日期:2014-08-21
  • 通讯作者: 徐义刚。E-mail:108074182@qq.com
  • 作者简介:李丹丹(1979—),女,黑龙江人,博士,高级兽医师,研究方向:微生物学与免疫学。
  • 基金资助:

    海南省自然科学基金项目(310106);国家质检总局科技项目(2013IK031);国家质检总局科技项目(2012IK157);海南省应用技术研究与开发专项项目(ZDXM20130025)。

Development of Real-time Quantitative PCR Method for Rapid Detection of Enterohemorrhagic Escherichia coli O157∶H7

LI Dan-dan1,XU Yi-gang2,WANG Sui-jia1,GAO Shen-yang3,LI Yi-jing4   

  1. 1.Animal Quarantine Laboratory Inspection & Quarantine Technology Center of Hainan Entry-exit Inspection & Quarantine Bureau,Haikou 570311,China;2.Animal Quarantine Laboratory Inspection & Quarantine Technology Center of Heilongjiang Entry-exit Inspection & Quarantine Bureau,Harbin 150001,China;3.Department of Animal Husbandry & Veterinary Medicine,Liaoning Medical University,Jinzhou 121001,China;4.College of Veterinary Medicine, Northeast Agricultural University,Harbin 150030,China
  • Received:2014-01-07 Online:2014-07-20 Published:2014-08-21

摘要: 本试验针对肠出血性大肠埃希菌(enterohemorrhagic Escherichia coli,EHEC)O157∶H7高度保守的rfbE基因序列设计合成引物及TaqMan探针,通过优化反应条件,建立了检测EHEC O157∶H7实时荧光定量PCR方法。结果显示,该法灵敏度约为7.3 CFU/mL,对310份肉类、蛋、奶及其制品和动物腹泻物、人工污染样品等进行检测,共检出阳性样本20份,与采用AOAC标准检测结果的符合率为100%。表明建立的实时荧光定量PCR方法操作简便、特异性强、灵敏度高,具有良好的实用性。

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Abstract: A Real-time quantitative PCR method was developed for the rapid detection of enterohemorrhagic Escherichia coli (EHEC) O157∶H7 based on the primers and TaqMan probes which were designed for the conservative domain of rfbE gene of EHEC O157∶H7.The results showed that the sensitivity of Real-time PCR was 7.3 CFU/mL.20 from 310 samples of meat,egg,milk and its products,animal diarrhea materials and artificial contamination samples were positive detected by Real-time PCR assay,which was in accordance with the testing result according to AOAC standard.The results indicated that Real-time PCR assay was a sensitive,rapid and simple tool.

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