Rationally engineered nucleic acid-based diagnostic for infectious and genetic diseases in practical settings

Abstract

Accurate, timely, and cost-efficient nucleic acid-based testing is critical to the global effort to fight infectious and genetic diseases, which plays a vital role in selecting proper intervention measures and preventing the deterioration of epidemics. The rationally engineered DNA probes and primers with intended thermodynamic characteristics in nucleic acid circuitry are essential factors to achieve the desired function of the sensing platform thus providing solutions for bottlenecks existing in screening tests. This dissertation focus on building a series of nucleic acid sensing platforms to attribute to the development of large-scale screening for infectious pathogens and routine screening for genetic diseases and leveraging on the thermodynamic prediction of Gibbs free energy and melting temperature of DNA motifs based on Watson-Crick base pair nearest neighbors model. First, a sample-specific pooled testing assay using melting curve analysis was developed to solve the issues of large-scale screening for infectious individuals, and it can analyze five individual samples in one batch without the need for secondary testing. Subsequently, an extraction-free mass testing platform utilizing magnetic beads was developed to detect different types of clinical specimens in practical settings, which further improves the sensitivity and shortens the turnaround time during the sample preparation and sample pooling. The pools with six subsamples were identified in a single run without retesting. Third, a competitive probe-and-sink strategy based on toehold exchange reaction at an elevated temperature was studied to phase two single nucleotide polymorphism (SNP) sites in long DNA templates. This assay shows the ability to differentiate four haplotypes and their respective combination of diplotypes up to 20 nM of the target concentration. Finally, a universal reporting composition in melting curve analysis was applied in multiplex genotyping to reduce the complexity and cost. It achieves multiple detections of nucleic acid samples using one set of fluorophore and quencher-modified probes.

Date of Award	2023
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	I-ming HSING (Supervisor)