Editing Recombinase Polymerase Amplification (section)

== Introduction ==
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Quantitative nucleic acid amplification is an important technique for detection of environmental, foodborne, and water-borne pathogens as well as for clinical diagnostics. Real-time quantitative [[polymerase chain reaction]] (qPCR) is the gold standard method for sensitive, specific, and quantitative detection of nucleic acids, e.g., for HIV-1 viral load testing, detection of bacterial pathogens, and screening for many other organisms1–3. During real-time qPCR, [[primers]] [[amplify]] pathogen DNA in cycles, and a fluorescent signal is generated that is proportional to the amount of amplified DNA in the sample at each cycle. A sample containing an unknown concentration of pathogen DNA may be quantified using a standard curve that relates the initial DNA concentration of standard samples and the time at which the fluorescent signal reaches a certain threshold (i.e., the cycle threshold, or CT).

Because real-time [[qPCR]] requires expensive thermal cycling equipment and several hours to receive results, alternative isothermal amplification techniques, such as recombinase polymerase amplification (RPA), have been developed4. These platforms generally provide results faster and amplify [[nucleic acids]] at a lower, single temperature, which may be accomplished with less expensive, simpler equipment. RPA, which is particularly attractive for point-of-care applications, amplifies DNA in minutes, requires a low amplification temperature (37 °C), and remains active in the presence of impurities5,6. RPA assays have been developed for a wide range of applications, including food analysis, pathogen detection, cancer drug screening, and detection of biothreat agents7–12. However, use of RPA for quantification of nucleic acids has been limited13,14.

In previous work, it was shown that real-time quantitative RPA (qRPA) is feasible15. Here, a more detailed protocol is provided for using real-time quantitative RPA to quantify unknown samples using a standard curve, a method that is analogous to quantification using qPCR. This protocol describes how to perform an RPA reaction on a thermal cycler to detect HIV-1 DNA as a proof-of-concept, as well as how to develop an internal positive control (IPC) to ensure the system is functioning properly. Data collection using a thermal cycler or microscope and data analysis for constructing a standard curve using training data is also detailed. Finally, the method for quantifying unknown samples using the standard curve with a custom script is demonstrated. This qRPA technique enables quantification of samples with unknown [[concentrations]] and has many advantages over traditional real-time qPCR.
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