Inhibition of Recombinase Polymerase Amplification by Background DNA: A Lateral Flow-Based Method for Enriching Target DNA

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dc.citation.firstpage1963en_US
dc.citation.issueNumber3en_US
dc.citation.journalTitleAnalytical Chemistryen_US
dc.citation.lastpage1967en_US
dc.citation.volumeNumber87en_US
dc.contributor.authorRohrman, Brittanyen_US
dc.contributor.authorRichards-Kortum, Rebeccaen_US
dc.date.accessioned2015-03-19T15:07:13Zen_US
dc.date.available2015-03-19T15:07:13Zen_US
dc.date.issued2015en_US
dc.description.abstractRecombinase 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.en_US
dc.identifier.citationRohrman, Brittany and Richards-Kortum, Rebecca. "Inhibition of Recombinase Polymerase Amplification by Background DNA: A Lateral Flow-Based Method for Enriching Target DNA." <i>Analytical Chemistry,</i> 87, no. 3 (2015) American Chemical Society: 1963-1967. http://dx.doi.org/10.1021/ac504365v.en_US
dc.identifier.doihttp://dx.doi.org/10.1021/ac504365ven_US
dc.identifier.urihttps://hdl.handle.net/1911/79391en_US
dc.language.isoengen_US
dc.publisherAmerican Chemical Societyen_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.titleInhibition of Recombinase Polymerase Amplification by Background DNA: A Lateral Flow-Based Method for Enriching Target DNAen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpublisher versionen_US
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