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

Despite 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