Figure 6.

Schematic presentation of the working principle of a biosensor based on RPA amplification to detect plant pathogens. The DNA extracted from a(n) (infected) plant sample is subjected to RPA amplification. The reverse primer is labelled with biotin, while the forward primer contains a 5’ addition complementary to oligonucleotide probes that are bound to gold particles. Should the sample contain the target pathogen (indicated by (+) in the figure), amplification will occur, resulting in amplicons labeled with a biotin label on one end and a DNA sequence complementary to the probes on the gold particles on the other end. The amplified product is incubated together with streptavidin magnetic beads and gold nanoparticles coated with capture probes, and will form a complex if amplification of the target sequence occurred. If the sample contains no target DNA (indicated by(-)), no labeled amplicons are formed. After magnetic separation of the complex, the complex is dissociated by heat treatment, resulting in the release of the gold nanoparticles. The gold nanoparticles will be deposited on an electrode surface, and through differential pulse voltammetry a characteristic signal is obtained, which indicates the presence of a pathogen (adapted from Lau et al., 2017).