Colorimetric Detection of Loop-Mediated Isothermal Amplification Reaction by Using Hydroxy Naphthol Blue

TLDR

LAMP amplifies DNA without thermal cycling, producing magnesium pyrophosphate whose presence is normally detected by turbidity or dye‑induced color change, though such dyes have limitations. The authors developed a new colorimetric LAMP assay using hydroxy naphthol blue (HNB) as a metal‑indicator dye. The assay was performed in 96‑well microplates and monitored spectrophotometrically at 650 nm. Adding 120 µM HNB did not impede amplification, and a violet‑to‑sky‑blue color shift reliably signals a positive reaction, making the method suitable for high‑throughput DNA and RNA detection.

Abstract

Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct—namely, magnesium pyrophosphate—without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 µM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.