Development of a Self-Contained Nucleic Acid Extraction Device to Enable Diagnostic Testing in Resource-Limited Settings

Abstract

Nucleic acid extraction is a significant roadblock to the widespread use of molecular detection strategies such as PCR in resource-limited settings. This thesis explores the potential to simplify nucleic acid extractions by utilizing the surface tension forces acting on fluids arrayed in a length of small-diameter tubing. On this scale, processing solutions required for nucleic acid extraction remained separated by surface tension air valves. Surface functionalized magnetic beads loaded with captured nucleic acids were transported through the valves using an external magnet to wash and elute purified nucleic acids in pre-arrayed solutions. Viral RNA was extracted from respiratory syncytial virus (RSV)-infected cell lysates using this self-contained approach. The extraction recovered ~10% of spiked RSV RNA from cell lysate samples and was comparable to a laboratory-based RNA extraction kit. This format was adapted to extract tuberculosis DNA from human urine samples. A transfer pipette was prepared with lyophilized magnetic beads and DNA-silica binding buffer to interface urine samples with the extraction tubing. This design recovered ~50% of spiked tuberculosis DNA from urine samples with a limit of detection of 77 copies/uL. Magnetic bead processing was automated by wrapping the tubing around a circular cassette, effectively reducing the tubing footprint from a 30 cm length to a compact 12.5 cm disk. A motor rotated the cassette past a fixed magnet to transfer the magnetic beads through the tubing. This automated design effectively recovered nucleic acid and protein biomarkers from patient sample surrogates. The extracted biomarkers were detectable at clinically-relevant low levels of infection for influenza, tuberculosis, malaria, and E. coli. The final study evaluated the importance of magnetic bead surface functionality on the efficacy, simplicity, and speed of nucleic acid extraction. Silica-coated and oligo (dT) functionalized magnetic beads were useful for a wide range of applications due to their simplicity and rapid extraction time, while RSV-specific probe functionalized beads required a significant tradeoff between RNA recovery and total extraction time. Together, the combination of functionalized magnetic beads and surface tension valves has enabled the development of an effective biomarker extraction device suitable for use in resource-limited settings.