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目的将一种新颖的核酸扩增技术——环介导等温扩增技术(LAMP)应用于小肠结肠炎耶尔森菌的快速检测。方法针对小肠结肠炎耶尔森氏菌毒素基因yruI/yru R设计5条特异性引物,进行LAMP扩增,对扩增反应进行优化。并考核方法的敏感性和特异性。结果最佳反应时间为60min,反应温度为60℃。对8种相关肠道细菌进行LAMP扩增,仅小肠结肠炎耶尔森菌得到阳性扩增结果,证明引物具有很高的特异性。小肠结肠炎耶尔森菌基因组DNA和纯培养物的检测灵敏度分别约为74fg和43cfu/ml。对模拟食品样品进行直接检测,检测限为76cfu/g。结论本方法检测小肠结肠炎耶尔森菌特异性强、灵敏度高,并且操作简便、检测成本低,1.5h即可完成,有望发展成为快速检测小肠结肠炎耶尔森菌的有效手段。
Objective To apply a novel nucleic acid amplification technique called loop-mediated isothermal amplification (LAMP) to the rapid detection of Yersinia enterocolitica. Methods Five specific primers were designed according to yruI / yru R gene of Yersinia enterocolitica, amplified by LAMP, and the amplification reaction was optimized. And assess the sensitivity and specificity of the method. The results showed that the best reaction time was 60min and the reaction temperature was 60 ℃. LAMP amplification was performed on eight kinds of related intestinal bacteria, and only Yersinia enterocolitica positive amplification results, indicating that the primers have high specificity. The detection sensitivity of Yersinia enterocolitica genomic DNA and pure culture was about 74 and 43 cfu / ml, respectively. Direct testing of simulated food samples with a detection limit of 76 cfu / g. Conclusion The method is sensitive and specific for detecting Yersinia enterocolitica. The method is easy to operate and has low detection cost and can be completed within 1.5h. It is expected to be an effective method for rapid detection of Yersinia enterocolitica.