Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes
Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes
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DOI：
10.1016/j.ab.2014.12.016
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发表时间：
2015-03-15
影响因子：
2.9
通讯作者：
Jikihara, Takaaki
中科院分区：
文献类型：
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作者：
Miyamoto, Shigehiko;Sano, Sotaro;Jikihara, Takaaki
由于环介导等温扩增（LAMP）可以在等温条件下扩增大量 DNA，因此其在简单基因检测中的应用引起了相当大的关注。 LAMP反应呈阳性是通过副产物引起的浑浊或向反应溶液中添加金属变色指示剂后的颜色变化来指示，但这些方法都有一定的局限性。无色结晶紫(LCV)是结晶紫(CV)经亚硫酸钠处理后获得的无色染料，被用作检测LAMP的新比色方法。 LCV 通过与双链 DNA (dsDNA) 接触重新转化为 CV。因此，LAMP的阳性反应通过颜色从无色变为紫色来指示。该测定足够灵敏，可以检测 LAMP 产品，dsDNA 的检测限为 7.1 ng/μl。它对 dsDNA 也具有高度选择性，并且未观察到对单链 DNA 和脱氧核苷酸三磷酸 (dNTP) 的干扰。 LCV有利于直接比色检测主产物而不是LAMP反应的副产物；因此，该方法可以在各种反应条件下使用，例如在溶液中添加焦磷酸酶的条件。这种使用 LCV 的比色 LAMP 检测方法因其简单性而适用于护理点基因检测。 (C) 2015 Elsevier Inc. 保留所有权利。
Because loop-mediated isothermal amplification (LAMP) can amplify substantial amounts of DNA under isothermal conditions, its applications for simple genetic testing have attracted considerable attention. A positive LAMP reaction is indicated by the turbidity caused by by-products or by the color change after adding a metallochromic indicator to the reaction solution, but these methods have certain limitations. Leuco crystal violet (LCV), a colorless dye obtained after sodium sulfite treatment of crystal violet (CV), was used as a new colorimetric method for detecting LAMP. LCV is reconverted into CV through contact with double-stranded DNA (dsDNA). Therefore, the positive reaction of LAMP is indicated by color change from colorless to violet. The assay is sensitive enough to detect LAMP products, with a detection limit of 7.1 ng/mu l for dsDNA. It is also highly selective to dsDNA, and interference with single-stranded DNA and deoxynucleotide triphosphates (dNTPs) is not observed. LCV facilitates direct colorimetric detection of the main product rather than a by-product of the LAMP reaction; therefore, this method can be used under various reaction conditions such as those with added pyrophosphatase in solution. This colorimetric LAMP detection method using LCV is useful for point-of-care genetic testing given its simplicity. (C) 2015 Elsevier Inc. All rights reserved.