Fluorescence Detection of DNA Amplification in Porous Media for Point-of-Care Diagnostics

Thesis (Master's)--University of Washington, 2015The translation of sophisticated, laboratory-developed methods for disease diagnosis into devices appropriate for locations with limited infrastructure—whether that is a military setting, resource-poor country, or a home—requires the development of rapid, inexpensive, robust, and semi-autonomous assays. One common diagnostic testing format that addresses these requirements is the lateral flow test (LFT). These tests use modified porous substrates to automate the delivery of samples via wicking, which enables low-cost and equipment-free analysis. These tests also utilize the rehydration of reagents from dry storage in the substrate, which enables multi-step and intervention-free assays. Typically, dry reagents in LFTs are stored with sugars that preserve reagent functionality. However, the formation of concentrated sugar solutions in porous media during drying can lead to non-uniform rehydration of reagents stored by this method. Additionally, in many diagnostic assays, the uniform mixing of targets with multiple reagents at precise concentrations is critical to performance. This is especially true for LFT-based isothermal nucleic acid amplification tests (iNAATs), for which the non-uniform rehydration of reagents causes a drastic reduction in assay performance. Since iNAATs produce results more rapidly and with increased sensitivity than traditional culture-based methods, their use in the LFT format represents an ideal diagnostic test for use in low-resource settings (LRS). Therefore, further study and development of iNAAT reagent rehydration in porous media is needed to enable improved LFT-based iNAATs for disease diagnosis in LRS. To this end, we have developed a novel, quantitative, fluorescence-based method for the observation and analysis of isothermal DNA amplification from dry reagents in porous media. Here, we describe this method and accompanying analytical tools to assess variations in reagent composition, imbibition time and target input levels with direct utility for performance quantification of a real LFT-based iNAAT