Crannell, Zachary A.Rohrman, BrittanyRichards-Kortum, Rebecca2017-05-222017-05-222015Crannell, Zachary A., Rohrman, Brittany and Richards-Kortum, Rebecca. "Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control." <i>Journal of Visualized Experiments,</i> 97, (2015) MyJoVE Corp.: http://dx.doi.org/10.3791/52620.https://hdl.handle.net/1911/94335It was recently demonstrated that recombinaseﾠpolymeraseﾠamplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sampleﾠconcentrationﾠusing aﾠstandard curveﾠIn 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.engArticle is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.Journal articlegeneticsrecombinase polymerase amplificationisothermal amplificationquantitativediagnosticHIV-1viral loadhttp://dx.doi.org/10.3791/52620