Recombinase polymerase amplification in combination with electrochemical readout for sensitive and specific detection of PIK3CA point mutations

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