Sequence specific visual detection of LAMP reactions by addition of cationic polymers.
Sequence specific visual detection of LAMP reactions by addition of cationic polymers.
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DOI：
10.1186/1472-6750-6-3
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发表时间：
2006-01-10
影响因子：
3.5
通讯作者：
Notomi T
中科院分区：
文献类型：
--
作者：
Mori Y;Hirano T;Notomi T
开发实用的基因即时检测装置（g-POCT装置）需要创新的检测方法，用于在不使用昂贵设备的情况下证明基因扩增反应的结果。我们研究了一种新的方法，通过在环介导等温扩增（LAMP）的扩增子中加入阳离子聚合物，利用沉淀反应进行微量核酸的序列特异性视觉检测。制备了不同荧光染料标记的寡聚DNA探针，用于多种核酸模板的扩增，并在探针存在下进行LAMP反应。在LAMP反应完成时，加入最佳量的低分子量聚乙烯亚胺（PEI），导致不溶性LAMP扩增子-PEI复合物沉淀。将与LAMP产物杂交的荧光标记的Oligo DNA探针掺入沉淀中，并且沉淀发射对应于扩增的核酸模板的荧光。在常规的UV照明器上，发射的荧光的颜色可以通过肉眼容易地检测。微量核酸模板的存在或不存在可以通过目视评估LAMP扩增子-PEI复合物沉淀的颜色以简单的方式检测。我们的结论是，这种检测方法可以促进小型和简单的g-POCT设备的开发。
Development of a practical gene point-of-care testing device (g-POCT device) requires innovative detection methods for demonstrating the results of the gene amplification reaction without the use of expensive equipment. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using precipitation reaction by addition of cationic polymers to amplicons of Loop mediated isothermal Amplification (LAMP). Oligo DNA probes labeled with different fluorescent dyes were prepared for multiple nucleic acid templates, and the templates were amplified by the LAMP reactions under the existence of the probes. At completion of the LAMP reaction, an optimal amount of low molecular weight polyethylenimine (PEI) was added, resulting in the precipitation of the insoluble LAMP amplicon-PEI complex. The fluorescently labeled Oligo DNA probes hybridized to the LAMP product were incorporated into the precipitation, and the precipitate emitted fluorescence corresponding to the amplified nucleic acid templates. The color of emitted fluorescence can be detected easily by naked eye on a conventional UV illuminator. The presence or absence of minute amount of nucleic acid templates could be detected in a simple manner through visual assessment for the color of the LAMP amplicon-PEI complex precipitate. We conclude that this detection method may facilitate development of small and simple g-POCT device.
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