Miyamoto Shigehiko, Sano Sotaro, Takahashi Koji, Jikihara Takaaki
Medical Device Development Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan.
Medical Device Development Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan.
Anal Biochem. 2015 Mar 15;473:28-33. doi: 10.1016/j.ab.2014.12.016. Epub 2015 Jan 7.
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/μl 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.
由于环介导等温扩增（LAMP）能够在等温条件下大量扩增DNA，其在简单基因检测中的应用备受关注。LAMP阳性反应可通过副产物引起的浊度或向反应溶液中添加金属显色指示剂后的颜色变化来指示，但这些方法存在一定局限性。亮结晶紫（LCV）是经亚硫酸钠处理结晶紫（CV）后得到的无色染料，被用作检测LAMP的新比色法。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。该检测方法灵敏度足以检测LAMP产物，对dsDNA的检测限为7.1 ng/μl。它对dsDNA也具有高度选择性，未观察到对单链DNA和三磷酸脱氧核苷酸（dNTPs）的干扰。LCV有助于直接比色检测LAMP反应的主要产物而非副产物；因此，该方法可在各种反应条件下使用，如溶液中添加焦磷酸酶的条件。鉴于其简单性，这种使用LCV的比色LAMP检测方法对于即时基因检测很有用。
