应用DPO-PCR技术检测肠出血性大肠杆菌O157∶H7

doi:10.7506/spkx1002-6630-201408032

食品科学

应用DPO-PCR技术检测肠出血性大肠杆菌O157∶H7

徐义刚1，李丹丹2，崔丽春3，刘忠梅1，李苏龙1

1.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 150001；2.海南出入境检验检疫局检验检疫技术中心，
海南海口 570125；3.东北林业大学研究生院，黑龙江哈尔滨 150040

出版日期:2014-04-25 发布日期:2014-05-13
基金资助:
国家质检总局科技计划项目（2012IK157）；质检公益性行业科研专项（201310126）

Detection of Enterohemorrhagic Escherichia coli O157:H7 by DPO-PCR Method

XU Yi-gang1, LI Dan-dan2, CUI Li-chun3, LIU Zhong-mei1, LI Su-long1

1. Technical Center of Inpsection and Quarantine, Heilongjiang Entry-Exit Inspection and Quarantine Bureau, Harbin 150001, China;
2. Technical Center of Inpsection and Quarantine, Hainan Entry-Exit Inspection and Quarantine Bureau, Haikou 570125, China;
3. School of Graduate, Northeast Forestry University, Harbin 150040, China

Online:2014-04-25 Published:2014-05-13

摘要/Abstract

摘要：

利用双启动寡核苷酸引物（dual-priming oligonucleotide，DPO）聚合酶链式反应（polymerase chain reaction，PCR）技术检测肠出血性大肠杆菌O157∶H7。根据DPO引物设计原则，以肠出血性大肠杆菌O157∶H7 rfbE 基因为靶基因设计一对DPO引物，经过反应体系的优化，建立了肠出血性大肠杆菌O157∶H7 DPO-PCR检测方法，其检测灵敏度约为94 CFU/mL。与常规PCR方法相比，所建立的DPO-PCR方法对退火温度不敏感，在引物设计和实验过程中不需要对引物及其退火温度反复优化，同时基于DPO引物的特殊结构又增强了其特异性。DPO-PCR方法设计简易、特异性强，为致病性微生物的快速准确检测提供了新途径。

关键词: 肠出血性大肠杆菌O157∶H7, rfbE基因, 双启动寡核苷酸引物聚合酶链式反应

Abstract:

The aim of this study was to develop a method for the detection of enterohemorrhagic Escherichia coli O157:H7
(EHEC O157:H7) using dual-priming oligonucleotide (DPO) PCR technique. Based on DPO primer principle, a pair of
DPO primers was designed using the rfbE gene of EHEC O157:H7 as the target. Following the optimization of the reaction
system, the DPO-PCR method for the detection of EHEC O157:H7 was successfully developed with a sensitivity of
94 CFU/mL. Compared to the conventional PCR method, the DPO-PCR method was not sensitive to annealing temperature.
Thus, it was not required to repeatedly optimize primer and its annealing temperature during primer design and testing.
Moreover, the specific structure of DPO primers improved the specificity of conventional PCR method. Therefore, the
DPO-PCR method provides a new way for rapid and accurate detection of pathogenic microorganisms.

Key words: Escherichia coli O157:H7 (EHEC O157:H7), rfbE gene, dual-priming oligonucleotide polymerase chain reaction (DPO-PCR) method

中图分类号:

TS207.3

徐义刚1，李丹丹2，崔丽春3，刘忠梅1，李苏龙1. 应用DPO-PCR技术检测肠出血性大肠杆菌O157∶H7[J]. 食品科学, doi: 10.7506/spkx1002-6630-201408032.

XU Yi-gang1, LI Dan-dan2, CUI Li-chun3, LIU Zhong-mei1, LI Su-long1. Detection of Enterohemorrhagic Escherichia coli O157:H7 by DPO-PCR Method[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201408032.

应用DPO-PCR技术检测肠出血性大肠杆菌O157∶H7

Detection of Enterohemorrhagic Escherichia coli O157:H7 by DPO-PCR Method

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