DsDNA/ssDNA-switchable isothermal colorimetric biosensor based on a universal primer and λ exonuclease

Abstract

Herein, a dsDNA/ssDNA-switchable isothermal colorimetric biosensor was established. In our design, a versatile linker domain invades into recombinase polymerase amplification (RPA) amplicons via a reverse primer with an additional linker. To activate effective amplification, a universal primer (U-P) is introduced to address the impaired efficiency in the invasion process. After billions of dsDNA amplicons with the universal linker accumulate, λ exonuclease acts as a converter and mediates the transformation from dsDNA to ssDNA for launching efficient DNA self-assembly without signal leak. The newly formed G-rich sequences in the ladder-like assembly fold into G-quadruplex DNAzyme for visual signal. A model pathogen (Salmonella spp.) and a model plant (MON810 maize) were detected by our sensor with excellent sensitivity and selectivity, demonstrating the universality. The linearity of Salmonella spp. was y (A450) = 0.1571x (the logarithm of concentrations) + 0.4452 (R2 = 0.9903) from 101 cfu/mL to108 cfu/mL with the detection limit of 3 cfu/mL; and the visible semi-quantified detection limit of MON810 maize was 0.1 %. Meanwhile, a recovery and a selective experiment were performed in yogurt starter with Salmonella spp.. Generally, this study provides a dsDNA/ssDNA-switchable colorimetric strategy based on a universal primer and λ exonuclease. A project of compositive suitcase, constituted by a DNA crude extract kit, an isothermal colorimetric kit and a portable spectrophotometer, was developed for versatile detection of different targets for a mobile laboratory. This shows great promise for on-site bioassays in food safety, environmental monitoring and clinical diagnosis.