可视化LAMP技术快速检测新冠病毒的方法

Rapid Detection of SARS - CoV - 2 by Visual LAMP Technology

根据时代需要,利用环介导等温扩增技术(loop-mediated isothermal amplifcation,LAMP)对新冠病毒进行快速检测,以研发出新冠病毒检测试剂盒。通过NCBI公布的新冠基因组序列分析,获得新冠病毒特异性N蛋白基因的序列;根据该序列信息,在线设计的几对环介导等温扩增引物以供筛选,并利用新冠病毒全基因的反转录质粒,同时通过大量的实验尝试,得到较为稳定的环介导等温扩增体系,再加入具有逆转录活性的Bst3.0 DNA聚合酶,使体系可扩增RNA模板,最后通过加入适量染料,完成反应结果的可视化。通过不断的实验优化,得到了较为稳定的扩增体系如下:2×Lamp Master Mix 12.5μL,Bst 3.0 DNA聚合酶0.5μL,内引物FIP、BIP(10μM)各2μL、外引物F3、B3(10μM)各0.5μL、模板1μL、dd H_2O 7μL,该体系在65℃下水浴加热1 h即可,同时在体系中加入MnCl_(2)溶液(15 m M)1μL、钙黄绿素(500μM)3μL,使反应结果肉眼可断,加入模板的体系变为绿色,而未加模板的体系为棕黄色。结论:本实验研究的可视化LAMP检测新冠病毒的方法可用于对广大人民群众进行新冠病毒核酸检测。

Rapid and sensitive detection of new coronavirus is very important for interrupting the spread of the COVID-19 We aim to developing a simple,rapid and visual loop-mediated isothermal amplification(LAMP)assay for SARS-CoV-2 detection based on its N gene and develop a COVID-19 diagnosis kit.SARS-CoV-2 viruses are relatively conserved with low sequence divergence and we obtained the specific sequence of N protein of the new coronavirus.Through this sequence,we designed several pairs of loop mediated isothermal amplification primers online,and used SARS-CoV-2(2019-nCoV)cDNA clone expression plasmid as template for amplification.In our amplification system,Bst 3.0 DNA polymerase with reverse transcription activity was added to amplify RNA template.For convenient use,a visual detection version of the SARS-CoV-2 assay was developed by adding appropriate amount of dye.Through continuous experiments and optimization,a LAMP reaction system was set up,containing:2×LAMP Master Mix 12.5μL;Bst 3.0 DNA polymerase 0.5μL;The inter primer FIP and BIP(10μM)each 2μL;The outer primers F3 and B3(10μM)each 0.5μL;Template 1μL;ddH_(2)O_(7)μL;The reaction was performed at 65℃and heated in water bath for 1 hour.For visual detection,1μL MnCl2 solution(15 mM)and 3μL Calcein(500μM)were used.The system with template becomes green,while the system without template turns brown.Conclusion:We developed a new,simple and visual LAMP assay for the fast and accurate detection of SARS-CoV-2 viruses.