Halochromism of rosolic acid: a pH-sensitive colorimetric dye combined with a smartphone technique for quantification of DNA in molecular diagnostics

Infectious foodborne pathogens are responsible for serious illnesses and socioeconomic losses worldwide, making early detection crucial to control excessive damage. Colorimetric loop-mediated isothermal amplification (LAMP) assays are a promising molecular detection technique that produces visually discernible color changes. However, factors such as color blindness, age, and sex can cause individuals to perceive colors differently, limiting the effectiveness of colorimetric assays. To address this, quantitative chromatic analysis was employed to reliably distinguish colors and analyze their digital images. We developed a red-green-blue (RGB) channel-based method using a smartphone application (color picker) to quantify colorimetric LAMP amplicons. Two genes, <i>hlyA</i> of <i>Listeria monocytogenes</i> (<i>L. monocytogenes</i>) and <i>esp</i> of <i>Enterococcus faecium</i> (<i>E. faecium</i>), were investigated using LAMP, the most widely used isothermal amplification technique. In this study, the pH indicator "rosolic acid (RA)" was introduced to identify weakly buffered LAMP amplicons based on the colorimetric shift from red to yellow, and the color was quantified using the R/(R + G + B) computation. The limit of quantification was as low as 10 fg μL^-1 for <i>L. monocytogenes</i> and 1 fg μL^-1 for <i>E. faecium</i>. Furthermore, the sensitivity of the proposed method was comparable to that of the traditional LAMP assay, which uses phenol red as a pH indicator. The practical application of the RA-based colorimetric LAMP assay was demonstrated by detecting the blaOXA-23-like carbapenemase gene of <i>A. baumannii</i> in saliva. Thus, the RA-mediated colorimetric LAMP assay, combined with the RGB-based quantitative approach using a smartphone APP, holds significant potential in molecular diagnostics for detecting foodborne pathogens.