Development of Recombinase Polymerase Amplification (RPA) Assays to Diagnose Infectious Diseases

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dc.contributor.advisorRichards-Kortum, Rebecca Raeen_US
dc.contributor.committeeMemberBennett, Georgeen_US
dc.contributor.committeeMemberTkaczyk, Tomasz Sen_US
dc.contributor.committeeMemberWhite, Arthur Cen_US
dc.creatorCrannell, Zacharyen_US
dc.date.accessioned2016-01-07T17:28:02Zen_US
dc.date.available2016-01-07T17:28:02Zen_US
dc.date.created2015-12en_US
dc.date.issued2015-06-24en_US
dc.date.submittedDecember 2015en_US
dc.date.updated2016-01-07T17:28:02Zen_US
dc.description.abstractThis thesis describes the development of Recombinase Polymerase Amplification (RPA) assays that can be used to improve access to diarrheal diagnostics and thereby reduce the number of preventable deaths that occur each year due to persistent diarrhea. In low-resource settings (LRS), where the majority of the almost 1.5 million annual diarrheal deaths occur, a major obstacle to receiving life-saving treatment is the inability to identify the specific cause of diarrhea. Diagnosis in LRS is usually done via stool smear microscopy, which fails to identify the cause of diarrhea up to half of the time. The widely considered gold standard diagnostic method is Polymerase Chain Reaction (PCR), which detects trace amounts of pathogen DNA from stool samples. While highly sensitive, PCR requires highly trained technicians and access to expensive thermal cycling equipment, restricting its use to centralized reference laboratories. The RPA diagnostics presented here amplifies trace amounts of pathogen DNA (much like PCR), but unlike PCR do not require the use of expensive thermal cycling equipment and can function at low temperatures, alleviating the need for any external heating equipment. RPA-based diagnostics and sample preparation protocols that are appropriate for low resource settings were developed to detect Cryptosporidium, Giardia, and Entamoeba, three of the leading causes of diarrhea. The three diagnostic assays were individually characterized on the benchtop where they demonstrated limits-of-detection and specificities comparable to the gold standard of PCR. The assays were further characterized in field studies using clinical samples where they demonstrated sensitivity and specificity nearly equivalent to that of the gold standard PCR. The three individual assays were then integrated into a multiplexed test designed to simultaneously amplify and detect DNA from Cryptosporidium, Giardia, and Entamoeba. This test was also characterized on the benchtop and in pre-clinical studies. All of the assays presented here are read using lateral flow strips that can easily be used in the field. This work demonstrates for the first time that multiplex RPA results can be read with lateral flow strips. By modifying the DNA primers, this diagnostic platform could be adapted to diagnose a broad variety of infectious diseases.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.citationCrannell, Zachary. "Development of Recombinase Polymerase Amplification (RPA) Assays to Diagnose Infectious Diseases." (2015) Diss., Rice University. <a href="https://hdl.handle.net/1911/87760">https://hdl.handle.net/1911/87760</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/87760en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectRecombinase Polymerase Amplificationen_US
dc.subjectdiagnosticen_US
dc.titleDevelopment of Recombinase Polymerase Amplification (RPA) Assays to Diagnose Infectious Diseasesen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBioengineeringen_US
thesis.degree.disciplineEngineeringen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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