Fenton-like reaction coupled with loop-mediated isothermal amplification for colorimetric detection of pathogens

초록

The increasing threat of infectious pathogens has been driving research efforts toward developing point-of-care testing (POCT) to discover effective strategies to prevent the spread of infectious pathogens. In this study, we developed a colorimetric pathogen detection method using a Fenton-like reaction coupled with loop-mediated isothermal amplification (LAMP) for POCT. The reaction between CuSO4 and H2O2, known as a Fenton-like reaction, generates hydroxyl radicals. These radicals can degrade azo linkages in amaranth, a chromogenic molecule, turning the red color of amaranth colorless. However, in the presence of LAMP amplicons, DNA competitively reacts with hydroxyl radicals, thereby reducing the effect of hydroxyl radicals on amaranth and maintaining its innate red color. The Fenton-like reaction was used to detect Enterococcus faecium and Listeria monocytogenes. The detection limit for Listeria monocytogenes DNA was 10-5 ng/mu L (approximate to 3 copies/mu L), with a linear correlation in the range of 10-5-100 ng/mu L (R2 = 0.9907). This work reports, for the first time, the use of an amaranth-based Fenton-like reaction coupled with LAMP for colorimetric pathogen detection. Unlike conventional LAMP colorimetric assays, this strategy exploits the competitive reaction of hydroxyl radicals with DNA amplicons to protect the azo dye (amaranth) from decolorization, creating a simple and visually distinguishable signal. The colorimetric detection established by coupling Fenton-like reaction with LAMP amplification provides a highly sensitive, specific, and user-friendly procedure for detecting pathogens in low-resource settings.

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