B-228 Optimization of Recombinase Polymerase Amplification (RPA) Highlights the Importance of Enzyme Consistency for Reproducible Results

Background With novel viruses arising on a routine basis globally, the need for fast, easy to use diagnostic methods that can respond quickly to the demands of the infectious disease community is critical to tracking and containing new outbreaks. As a response, the use of isothermal amplification in point of care (POC) and field-based molecular diagnostic (MDx) testing platforms has grown dramatically over the past decade. An increasingly popular isothermal method is Recombinase Polymerase Amplification (RPA); however, users of the method have reported inconsistent amplification and fluctuating positive signal response times. Here we present research evaluating the roles of individual enzymes in amplification efficiency. Methods Using newly developed individual enzyme activity assays and a standardized RPA reaction, a range of enzyme concentrations and buffer components were evaluated to attempt to eliminate currently experienced lot-to-lot variations in performance. Our work focused on T4 UvsX DNA Recombinase, T4 Gene 32 Protein, Bsu Polymerase, and T4 UvsY Protein as the most significant contributors to overall variability. Shifts in final time to result were used to measure amplification robustness. Results While the RPA assay itself exhibited consistent accuracy and precision, amplification efficiency proved sensitive to changes in individual enzyme quality and concentration. The new methods for accurately measuring single enzyme activity enabled tighter control of the amount of each enzyme added to RPA reactions, which in turn reduced variability. Controlling concentrations of T4 UvsX DNA Recombinase proved especially important in achieving consistent results. Conclusion Although many RPA users have previously reported a high degree of variability, data presented here show that reproducible results can be achieved with rigorous control of enzyme inputs and quality. Given RPA’s numerous advantages over the more common Loop-Mediated Isothermal Amplification (LAMP) method - including simpler primer design, lower required temperature, and shorter amplification time - the improved consistency observed means that RPA can now be more widely adopted in POC or field-based MDx applications.