【摘要】 针对鸽肠杆菌的传统检测方法检测周期长的问题,笔者建立了快速检测鸽肠杆菌的环介导等温扩增(LAMP)法。笔者以肠杆菌属毒力标志基因(iss基因)的保守区域设计4条特异性LAMP引物,分别进行了反应条件的优化、灵敏度试验、特异性试验、限制性内切酶试验。结果显示,其最优的体系中甜菜碱浓度为1.6 mol/L,Mg SO4浓度为2.5 mmol/L,内外引物浓度比FIP∶BIP∶F3∶B3=3∶3∶1∶1。将所有反应混合物置于63℃恒温反应40 min就能完成对iss基因的扩增,肉眼可观察检验结果。该方法具有良好的特异性,灵敏度是PCR的1000倍。这是一种快速、特异、灵敏的肠杆菌属iss基因LAMP检测方法,其适用于基层和现场检测。更多还原

【Abstract】 The detection period of Enterobacter from the pigeons using traditional testing methods is relatively long. Therefore a kind of rapid detection method, namely a loop-mediated isothermal amplification(LAMP) technology detection Enterobacter, was developed to address this problem. A set of four LAMP primers were respectively designed based on the target sequences of iss genes. Optimal conditions, sensitivity, specificity and Restriction endonuclease test were performed. In results, the optimal system is used the 1.6 mol/L betaine, 2.5 mmol/L MgSO4, FIP∶BIP∶F3∶B3 is 3∶3∶1∶1,and the optimal conditions is keep the system in the 63℃ within 40 minutes and the amplification results were visualized. This process was developed for the rapid detection of iss gene. The sensitivity of LAMP assay was 1000 times higher than PCR. Therefore, the rapid and simple assay—LAMP is a potential useful technique for iss gene detection of Enterobacter in field condition.更多还原