Development of nucleic acid detection methods for object provenance and viral diagnostics

Abstract

Starting with the discovery of DNA structure, advances in molecular and systems biology in the past few decades have propelled nucleic acid amplification and detection technologies with far-reaching applications including diagnostics for healthcare to food industries, such as disease prognosis and surveillance during a global pandemic, or object provenance to determine locations of food-borne illness outbreaks. Many of these technologies require extensive instrumentation and infrastructure, restricting access in low-resource environments; therefore, recent efforts have been directed towards developing isothermal nucleic acid amplification methods amenable to field-deployable applications. In this thesis, we used isothermal nucleic acid detection method to develop field-deployable systems in two areas: object provenance and viral diagnostics. First, determining the location history of an object is a fundamental challenge for human health, commerce, and food safety. We leveraged location-specific barcoded microbial spores to create a system that can determine object provenance that is rapid, fieldable, sensitive, cheap, and can be safely introduced into and recovered from the environment. This technology has broad potential as it can help solve object provenance challenges in a wide range of applications. Second, the global SARS-CoV-2 pandemic has highlighted in many countries the limitations of inadequate testing infrastructure and the pressing need for different testing modalities. Rapid, inexpensive, and sensitive testing is essential for disease surveillance and containment strategies to be effective. We optimized the isothermal nucleic acid detection method used for microbial spores to develop a SARS-CoV-2 point-of-care diagnostics that can detect down to 5 viral RNA molecules and can provide quantitative information in crude patient samples. This method can be quickly and easily adapted to future infectious disease outbreaks. Taken together, this thesis provides some groundwork for bringing isothermal nucleic acid detection methods closer to a future where fully field-deployable technologies may become a reality.