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

Simple item page
dc.citation.articleNumbere52620en_US
dc.citation.journalTitleJournal of Visualized Experimentsen_US
dc.citation.volumeNumber97en_US
dc.contributor.authorCrannell, Zachary A.en_US
dc.contributor.authorRohrman, Brittanyen_US
dc.contributor.authorRichards-Kortum, Rebeccaen_US
dc.date.accessioned2017-05-22T18:57:18Zen_US
dc.date.available2017-05-22T18:57:18Zen_US
dc.date.issued2015en_US
dc.description.abstractIt 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.en_US
dc.identifier.citationCrannell, 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.en_US
dc.identifier.doihttp://dx.doi.org/10.3791/52620en_US
dc.identifier.urihttps://hdl.handle.net/1911/94335en_US
dc.language.isoengen_US
dc.publisherMyJoVE Corp.en_US
dc.rightsArticle 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.en_US
dc.subject.keywordgeneticsen_US
dc.subject.keywordrecombinase polymerase amplificationen_US
dc.subject.keywordisothermal amplificationen_US
dc.subject.keywordquantitativeen_US
dc.subject.keyworddiagnosticen_US
dc.subject.keywordHIV-1en_US
dc.subject.keywordviral loaden_US
dc.titleDevelopment of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Controlen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpublisher versionen_US
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Quantitative-Recombinase.pdf
Size:
892.01 KB
Format:
Adobe Portable Document Format
Description:
Download
Collections
Faculty Publications
Bioengineering Publications