Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Abstract

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest. Video LinkThe video component of this article can be found at

Factors influencing Recombinase polymerase amplification (RPA) assay outcomes at point of care

“…In this study we demonstrate that adherence to this mix step is vital to the assay performance, particularly in samples with very low concentrations of target sequence. When mixing was not performed during incubation in the Twista, both the intensity of detection stripes on ICS and real time fluorescent signal were noticeably impacted, which has implications for use of RPA for both qualitative and quantitative assays [24]. …”

Cross-subtype detection of HIV-1 using reverse transcription and recombinase polymerase amplification

“…Sequence variation within the primer binding sites did appear to have an impact on time to detection and the final signal intensity across subtypes. This could have implications for ongoing efforts to use RPA for quantitative assays (Crannell et al, 2015;Crannell et al, 2014b). While we observed some linearity within dilution series for each HIV-1 subtype, quantitation across subtypes appeared less reliable (Figure S2).…”

Recombinase Polymerase Amplification for Diagnostic Applications

“…RPA showed minimal requirements for sample preparation and multiplexing was feasible. Hence, integrating assay control was successfully applied to RPA tests (17,66 ). Despite RPA showing comparable clinical performances to PCR and LAMP, it also shows some differences.…”