Recombinase polymerase amplification in combination with electrochemical readout for sensitive and specific detection...

As part of the ongoing evolution towards personalized anticancer therapy, mutation screening is becoming increasingly important and, therefore, also alternative detection strategies that allow for fast genetic diagnostics at the point of care. In the case of breast cancer, detecting cancer-associated point mutations in the PIK3CA gene is of particular importance for treatment decisions. We developed a recombinase polymerase amplification assay combined with an enzyme-linked electrochemical assay on multi-channel screen-printed gold sensors for specific and highly sensitive detection of three PIK3CA point mutations (H1047R, E545K, and E542K). Recombinase polymerase amplification (RPA) of the target sequences was optimized and characterized with a real-time RPA assay. Comparison with real-time PCR reveals that RPA is slightly inferior in terms of efficiency and sensitivity. However, the desired target DNA is successfully amplified at initial concentrations down to 100 copies μL^-1. For electrochemical readout, biotinylated dCTP is used to label the target DNA during RPA. Single-stranded target DNA is produced with either asymmetric RPA or symmetric RPA followed by lambda exonuclease digestion. Characterization of the two different approaches in terms of sensitivity results in comparable detection limits (229 copies μL^-1 and 224 copies μL^-1, respectively), though RPA followed by lambda exonuclease digestion yields significantly higher currents. Finally, this method, together with a designed wild-type blocking oligo that inhibits binding of the wild-type target DNA during probe-target hybridization, allows for detecting the PIK3CA point mutations H1047R, E545K, and E542K in the presence of wild-type target DNA when the proportion of mutant target DNA is >20%.