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Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 104 copies of HIV-1 DNA in a background of 0–14 µg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 µg when 106 copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads. [ABSTRACT FROM AUTHOR]

Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic adds in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes realtime fluorescence data to quantify HIV-1 DNA We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic adds and may be adapted for use in single-sample point-of-care diagnostic systems. [ABSTRACT FROM AUTHOR]

Diarrheal diseases cause more morbidity and mortality around the world than human immunodeficiency virus (HIV), malaria, or tuberculosis. Given that effective treatment of persistent diarrheal illness requires knowledge of the causative organism, diagnostic tests are of paramount importance. The protozoan parasites of the genus Cryptosporidium are increasingly recognized to be responsible for a significant portion of diarrhea morbidity. We present a novel nucleic acid test to detect the presence of Cryptosporidium species in DNA extracted from stool samples. The assay uses the isothermal amplification technique recombinase polymerase amplification (RPA) to amplify trace amounts of pathogen DNA extracted from stool to detectable levels in 30 min; products are then detected visually on simple lateral flow strips. The RPA-based Cryptosporidium assay (RPAC assay) was developed and optimized using DNA from human stool samples spiked with pathogen. It was then tested using DNA extracted from the stool of infected mice where it correctly identified the presence or absence of 27 out of 28 stool samples. It was finally tested using DNA extracted from the stool of infected patients where it correctly identified the presence or absence of 21 out of 21 stool samples. The assay was integrated into a foldable, paper and plastic device that enables DNA amplification with only the use of pipets, pipet tips, and a heater. The performance of the integrated assay is comparable to or better than polymerase chain reaction (PCR), without requiring the use of thermal cycling equipment. This platform can easily be adapted to detect DNA from multiple pathogens. [ABSTRACT FROM AUTHOR]