Towards on-site detection of gluten-containing cereals with a portable and miniaturized prototype combining isothermal DNA amplification and naked eye detection

Abstract

Miniaturization enables the development of portable devices for rapid on-site DNA analysis, providing cheaper and more convenient systems for target DNA detection in resource-limited settings. Miniaturized devices for DNA-based analysis are highly promising analytical tools for different aims, in particular for food quality and safety analysis in the different points of the food value chain DNA amplification is a key step for the development of highly specific and sensitive detection platforms. Although PCR remains the most widely adopted DNA amplification technique, interest for isothermal DNA amplification approaches, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), has grown exponentially when considering miniaturization. In this work, a portable and miniaturized prototype has been developed to perform isothermal DNA amplification by LAMP and RPA, containing an integrated heating system, and including naked-eye detection, therefore not requiring any additional equipment for the amplification reactions. Prototype performance was evaluated and successfully demonstrated for these amplification techniques, with different detection chemistries being explored. As a proof-of-concept, performance of the integrated heating system was compared to conventional equipment for the detection of gluten-containing cereals in food products, showing similar results in terms of amplification time, specificity, and sensitivity. Amplification by RPA achieved the lowest detectable concentration, presenting a limit of detection of 0.0180 ng µL-1, being more sensitive than LAMP by one order of magnitude, and enabling amplification in less than 20 min.