论文部分内容阅读
目的采用切刻内切酶介导恒温扩增技术(nicking enzyme mediated isothermal amplification,NEMA)扩增蜡样芽孢杆菌质粒,考察缓冲体系、温度、离子浓度等条件对扩增效率的影响。方法通过独特的引物设计,采用NEMA技术扩增蜡样芽孢杆菌质粒,通过系列实验优化了缓冲体系、温度和离子浓度等条件,采用琼脂糖凝胶电泳观察扩增结果。结果通过设计不同长度片段的扩增产物,证明采用NEMA技术进行恒温扩增引物设计的正确性。扩增反应中,缓冲体系的组成、扩增温度、体系镁离子及二甲基亚砜(DMSO)的浓度会影响切刻内切酶恒温扩增效率,而海藻糖添加与否在本实验中与扩增效率无关。在最优条件下,NEMA恒温扩增方法对于蜡样芽孢杆菌质粒的灵敏度达到1.7 pmol/L。结论 NEMA可以用来进行较长片段的扩增,有望成为一种常规的恒温扩增技术,应用到战时或者应急条件下的快速核酸诊断中。
OBJECTIVE: To amplify the plasmid of Bacillus cereus by nicking enzyme mediated isothermal amplification (NEMA), and investigate the influence of buffer system, temperature and ion concentration on the amplification efficiency. Methods A unique primer design was used to amplify the plasmid of Bacillus cereus using NEMA. The conditions of buffer system, temperature and ion concentration were optimized by a series of experiments. The amplification results were observed by agarose gel electrophoresis. Results The design of primers for constant temperature amplification by NEMA was proved by designing amplification products of different length fragments. In the amplification reaction, the composition of the buffer system, the amplification temperature, the concentration of magnesium ions and dimethylsulfoxide (DMSO) in the system will affect the constant temperature amplification efficiency of the nicking endonuclease, and whether or not trehalose is added in the experiment Has nothing to do with the efficiency of amplification. Under optimal conditions, the sensitivity of the NEMA isothermal amplification method to Bacillus cereus was 1.7 pmol / L. Conclusion NEMA can be used for amplification of longer fragments and is expected to become a routine constant temperature amplification technique for rapid nucleic acid diagnosis in wartime or emergency situations.