Nucleic acid-based systems for multiplexed and portable detection of pathogens
Embargo Date
2026-02-07
OA Version
Citation
Abstract
The rapid and accurate detection of biomarkers associated with disease is key to identification of pathogens, ensuring that patients receive timely treatment, and limiting the spread of infectious disease. Ribonucleic acid (RNA)- or deoxyribonucleic acid (DNA)-based molecular assays are powerful tools for disease detection since they can be programmed to interact with biological ligands or DNA/RNA molecules with their predictable base-pairing and thermodynamic properties.
This dissertation focuses on developing a series of nucleic acid-based systems for multiplexed and portable detection of pathogens. First, an assay that combines computer- designed aptamer-based switches (aptaswitches) with isothermal pre-amplification was developed to enable the detection of SARS-CoV-2 RNA and human control RNA down to 1 copy/μL and 0.24 copy/μL, respectively. To reduce time to result, handling steps, and the likelihood of cross-contamination, a one-pot diagnostic assay was implemented by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with an aptaswitch based on the Broccoli aptamer or Red Broccoli aptamer. Second, by taking advantage of the distinct spectral properties of aptamers with their companion fluorogens, three-channel single-reaction multiplexing reactions were developed for mosquito-borne viral diseases with orthogonal aptamer/fluorogen pairs providing strong fluorescence within 15 minutes. Furthermore, a two-channel, one-pot RT-LAMP/aptaswitch assay that enables simultaneous detection of SARS-CoV-2 RNA and an ACTB mRNA control was developed. Third, to enable the detection of small sequence differences and mutations, a forward-engineered aptaswitch that provides single-nucleotide-specificity was developed. This aptaswitch can simultaneously identify SARS-CoV-2 RNA and distinguish variants of interest, such as Alpha, Beta, Gamma, Delta, and Omicron. Fourth, a multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a assay was developed. Finally, a one-pot recombinase/RT-LAMP-based diagnostic assay was developed for respiratory disease detection, which enables the visible detection of SARS- CoV-2 RNA to as few as 10 copies in under 15 minutes at a constant temperature. These studies demonstrate that this assay has the potential to simultaneously detect multiple targets by adopting a recombinase-based DNA-strand exchange mechanism.