Nucleic acid quantification with amplicon yield in recombinase polymerase amplification

Abstract

AbstractAmplification-based qPCR provides accurate and sensitive nucleic acid quantification. However, the requirement of temperature cycling and real-time monitoring limits its translation to different settings. Here, we adapted isothermal Recombinase Polymerase Amplification (RPA) reaction to develop a semi-quantitative method that relies on final amplicon yield to estimate initial target nucleic acid copy number. To achieve this, we developed a phenomenological model that captures the essential RPA dynamics. We identified reaction conditions that constrained the reaction yield corresponding to the starting DNA template concentration. We validated these predictions experimentally and show that the amplicon yields at the end of the RPA reaction correlates well to the starting DNA concentration while reducing non-specific amplification robustly. We demonstrate this approach termed here as quantitative endpoint RPA (qeRPA) to detect DNA over five log orders with detection limit of 100 molecules. Using a linear regression model with normalized endpoint intensity (NEI) standard curve, we estimate viral load from the serum of dengue-infected patients with comparable performance to qPCR. Hence, qeRPA can be employed for robust and sensitive nucleic acid estimation at close to room temperature without real-time monitoring and can be beneficial for field-deployment in limited-resource settings.