On-Gold Recombinase Polymerase Primer Elongation for Electrochemical Detection of Bacterial Genome: Mechanism Insights and Influencing Factors

The detection of bacterial pathogens may be accomplished through their genome material. With that aim, current methods usually rely on polymerase chain reaction (PCR) amplifications, which are time consuming and require specialist equipment. Here, we optimized an on-chip isothermal recombinase polymerase amplification (RPA) with chronoamperometric detection, which could be run with simple equipment. A short thiol-modified oligonucleotide, complementary to a fragment of bipA gene of Salmonella spp., is chemisorbed onto the gold chip surface together with p-aminothiophenol as a blocking agent. Remarkably, we demonstrate that, in the presence of Salmonella genome, this oligonucleotide, acting as a primer in RPA, is directly elongated on the surface, copying the genome and giving rise to an on-surface enzyme-tagged dsDNA. After 30 min of isothermal amplification, the chronoamperometric signal, related to one of the products of the enzyme reaction, increased with the amount of Salmonella genome, which allowed the detection of 10(5) copies. The results of this study may be of general utility in the design of sensors for detecting other bacteria.