The Development of a Low-Resource Appropriate Diagnostic for the Detection of Malaria DNA

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dc.contributor.advisorRichards-Kortum, Rebecca Raeen_US
dc.contributor.committeeMemberDrezek, Rebekah Aen_US
dc.contributor.committeeMemberHafner, Jason Hen_US
dc.contributor.committeeMemberStorthz, Karenen_US
dc.creatorCordray, Michael Scotten_US
dc.date.accessioned2016-01-07T17:25:24Zen_US
dc.date.available2016-01-07T17:25:24Zen_US
dc.date.created2015-05en_US
dc.date.issued2015-03-11en_US
dc.date.submittedMay 2015en_US
dc.date.updated2016-01-07T17:25:24Zen_US
dc.description.abstractDespite recent progress, malaria remains one of the most serious global health threats, especially since it disproportionately affects low-resource areas. One of the keys to management of the disease is access to effective diagnostics. Nucleic acid tests (NATs) are the most sensitive and specific type of diagnostic for malaria, but require too much infrastructure, training, and cost to be widespread in many malaria affected regions. The aim of this thesis was to develop a NAT for malaria that can detect 50 copies/µL or less of target DNA in blood and then package that assay into an easy to use and self-contained system. An assay was investigated to detect small amounts of target DNA using gold nanoparticle (AuNP) aggregation. A method was developed to quantify the results using spectroscopy and found a limit of detection (LOD) of 150 amoles of target DNA and a linear dynamic range that spans 150 amoles – 15fmoles. The conditions of the assay were optimized and a novel method of measuring the assay results was developed, eliminating the need for heating and reducing the time to result to 10 minutes (from 2 hours). These changes also dropped the LOD to 50 amoles (approximately 107 copies/µL) and increased the linear dynamic range of the assay to 50 amoles - 500 fmoles. Since the LOD of the AuNP assay was not low enough to detect clinically relevant amounts of target on its own, I investigated recombinase polymerase amplification (RPA) and a lateral flow dipstick assay to detect the product. Blood was found to inhibit the RPA reaction and protocols were developed to minimize this inhibition. A LOD of 15 copies/µL of target DNA spiked into blood was found. Finally, a paper and plastic platform was developed to carry out the RPA reaction and lateral flow detection of the amplified products. A novel set of RPA primers were designed which target a sequence found in all of the species of malaria which infect humans. Testing these primers in the paper and plastic device I found an LOD of 5 copies/µL in aqueous solution, and 200 copies/µL in blood.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.citationCordray, Michael Scott. "The Development of a Low-Resource Appropriate Diagnostic for the Detection of Malaria DNA." (2015) Diss., Rice University. <a href="https://hdl.handle.net/1911/87758">https://hdl.handle.net/1911/87758</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/87758en_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.subjectnucleic acid testen_US
dc.subjectmalaria diagnosticen_US
dc.subjectRPAen_US
dc.subjectpaper microfluidicsen_US
dc.subjectgold nanoparticlesen_US
dc.subjectisothermal amplificationen_US
dc.subjectpoint of care diagnosticen_US
dc.titleThe Development of a Low-Resource Appropriate Diagnostic for the Detection of Malaria DNAen_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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