快速检测NDM-1基因的环介导恒温扩增技术的建立与评价

Establishment of loop-mediated isothermal amplification technique for rapid detection of NDM-1 gene

摘要:

基于DNA环介导恒温扩增技术 (Loop-mediated isothermal amplification，LAMP)，探索建立一种应用于NDM-1基因 (New Metallo-β-Lactamase-1 Gene，NDM-1) 的快速检测方法，以适应临床实验室等的检测需求。利用LAMP技术，以NDM-1基因为靶序列，设计4组LAMP引物，并筛选最优引物组，建立LAMP反应体系与条件，进行灵敏度和特异性实验。结果表明整个检测过程仅需1 h，即可通过肉眼直接目测实验结果。在灵敏度试验中，NDM-1基因的最低检测限为6 拷贝/反应。在特异性实验中，以4株病原菌 (肺炎克雷伯氏菌、大肠埃希氏菌、金黄色葡萄球菌、肺炎链球菌) 以及肠道菌群元基因组DNA、土壤菌群元基因组DNA为模板对NDM-1基因进行检测，结果显示均没有发生非特异性扩增反应。文中建立的LAMP检测方法能够快速检测NDM-1基因，且可直接观察到实验结果，实现了检测结果的可视化。具有操作简单安全、检测灵敏度高、特异性高的特点，能够满足基层实验室、应急检测或现场监测等方面的使用需求，具有良好的应用价值。

Abstract:

We established a rapid detection method of New Delhi Metallo-β-Lactamase Gene (NDM-1) based on Loop-mediated Isothermal Amplification (LAMP). With the application of LAMP, we designed four sets of LAMP premiers, using NDM-1 gene as the target sequence, and selected the set of optimal primers. Meanwhile, we established optimal reaction systems and conditions to carry out the sensitivity and specificity experiments. The experiment results showed that the whole detection process took only one hour and could be observed visually. In the experiment of sensitivity, NDM-1 gene had a detection limit of 6 copies in each reaction. In the experiment of specificity, we detected NDM-1 gene in 4 pathogen strains (Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae), and the total DNA from intestinal microbes and the total DNA from soil microbes. We had not detected the amplification reactions. The detection method established could rapidly detect NDM-1 gene and visualize the experiment result. The method is easy to operate and has high sensitivity and specificity and thus has great application value in basic research laboratories, emergent detection and spot detection.