MGB-TaqMan探针实时荧光定量在快速检测产肠毒素大肠杆菌中的价值  

Detection of enterotoxigenic Escherichia coli by real-time polymerase chain reaction by using MGB- TaqMan probe technique

作　　者：代娟[1,2] 李玉峰[1] 袁粒星[3] 杨潇[1] 

机构地区：[1]西华大学生物工程学院,成都610039 [2]成都医学院医学检验系 [3]四川大学华西医学院

出　　处：《中华预防医学杂志》2008年第2期103-106,共4页Chinese Journal of Preventive Medicine

基　　金：四川省科技厅项目（110520503）

摘　　要：目的采用MGB-TaqMan探针建立产耐热肠毒素大肠埃希菌实时荧光定量PCR检测方法。方法针对编码大肠埃希菌耐热肠毒素I基因片段设计引物、MGB探针，采用外标法，绘制标准曲线，进行实时荧光定量PCR检测。评价该方法的特异性，灵敏度，准确度，检测范围，重复性及稳定性等。做阴性对照，排除假阳性，采用UNG酶抗污染。结果实验结果表明引物、探针特异性良好，该方法的灵敏度可达到每反应3DNA拷贝，特异性强，检测范围在10^0～10^9拷贝每反应，重复性及稳定性较好，整个过程只需要2h。结论该方法能快速、灵敏、特异检出产耐热肠毒素大肠埃希菌，对控制由耐热肠毒素引起的腹泻性疾病具有重要意义。Objective To develop a real-time polymerase chain reaction （PCR） based on TaqMan technology by using a new MGB probe for detecting enterotoxigenic Escherichia coli （ETEC） in paper. Methods Primers and MGB probe were designed in the ecoding region of heat-stable toxin of ETEC. Real-time PCR detected ETEC by using the exterior standard method with protracting standard curves. The specificity, sensitivity, accuracy, stability of real-time PCR system was evaluated. An internal negative antithesis was added to the real-time PCR system in order to get rid of the false positive of system. Using UNG enzyme expelled the contamination of PCR reaction. Results Primers and MGB probe were suited to the Real-time PCR. The assay showed that the method was quick, special,sensitive and stable. The real-time PCR system could detect ETEC in a large scale. The assay might be finished in two hour. Conclusion These observations suggested that real-time PCR based on MGB probe should be an excellent candidate for a standard ETEC detection method.

关 键 词：大肠杆菌 分子探针 聚合酶链反应 

分 类 号：R686[医药卫生—骨科学]