| 姜君1 王鹏志2 刘利成2 吴伟立1 陈唯军1,2,3△.肠出血性大肠杆菌(O157：H7)的特异性荧光探针检测方法的建立[J].,2012,12(12):2201-2204 |
| 肠出血性大肠杆菌(O157：H7)的特异性荧光探针检测方法的建立 |
| Establishment of Specific Fluorescent Quantitative PCR Assay forDetection of the Enterohemorrhagic Escherichia coli O157：H7* |
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| DOI： |
| 中文关键词: 大肠杆菌0157：H7 荧光定量PCR（real-time PCR） Taqman 探针 rfbE |
| 英文关键词: Escherichia coli O157 Real-time PCR TaqMan probe RfbE |
| 基金项目:“十一五”国家科技支撑计划项目(2008BAK41B03)；传染病专项（2008ZX10004-103） |
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| 中文摘要: |
| 目的：建立一种real-time PCR，快速准确检测肠出血性大肠杆菌O157：H7。方法：以肠出血性大肠杆菌0157：H7 rfbE 为待检
靶基因，设计一对引物和一条Taqman 探针，探针5' 端用FAM 基团标记，3' 端用TAMRA 标记。通过重组质粒的构建，建立并优
化了大肠杆菌0157：H7 的荧光定量PCR 检测方法。结果：在人工污染样本无需富集的情况下，检测的最低DNA 浓度是10 拷贝/
反应（3CFU/mL）；特异性检测实验中，0157 菌株检测结果均为rfbE 阳性，而非0157:H7 菌株检测结果均为阴性；重复性实验中，
批内、批间变异系数均小于3％。结论：实验结果显示此荧光定量PCR 方法特异性、灵敏度高，重复性好，可对分离的可疑大肠杆
菌0157:H7 菌株进行快速鉴定。 |
| 英文摘要: |
| Objective: To develop a specific real-time PCR assay for the detection of Escherichia Coli O157:H7 in food. Methods:
Probes and primers for a TaqMan quantitative PCR were designed and synthesized according to the conserved gene sequence rfbE of
Enterohemorrhagic Escherichia coli (EHEC) O157: H7, available in GenBank. The rfbE probe was 5' end labeled with FAM and 3' end
labeled with TAMRA. Then reaction parameters were optimized to develop a TaqMan-quantitative PCR assay. Results: The real-time
PCR assay was applied to samples artificially contaminated by Escherichia Coli O157: H7，the detection limits of the sensitivity assays
were 10 copies/reaction (3CFU/mL) of DNA. The qualitative consensus PCR assay indicated all Escherichia coil O157:H7 were found
rfbE positive and did not detect DNA from non-O157:H7 isolates. In the duplicated experiment，coeficients of variation intra-assay and
inter-assay over the dynamic range of the TaqMan probe assays were lower than 3%. Conclusion: This study shows that the real-time
PCR is a repeatable, specific, sensitive and rapid method for the detection of EHEC O157: H7. |
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