Microscale mRNA Amplification, Separation and Detection for Forensic and Clinical Point-of-Care Applications
Layne, Tiffany, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Landers, James, Chemistry, University of Virginia
Specific, sensitive identification of nucleic targets is an invaluable tool across numerous disciplines, including the forensic and clinical communities. The conventional assays and detection methods for these purposes are laborious, destructive, typically have single target detection, lack specificity and sensitivity, and are time-consuming for the scientist. The development of novel biochemical assays to alleviate these shortcomings can provide faster, multi-target reactions with minimal manual steps and are highly specific and sensitive for identification purposes. Biocompatible technology, such as microfluidics, can enhance an assay by providing rapid, multi-sample, cost-efficient automation for unique bioanalytical techniques. The work outlined in this dissertation discusses development of such centrifugal microdevices for novel amplification and separation methods and subsequent application to differentiation between forensically relevant body fluids, testing tolerance of transplanted organs, and detection of viral targets. Specifically, this dissertation presents two distinct approaches to forensic body fluid identification (bfID) using (1) an isothermal amplification method with colorimetric image analysis and (2) a polymerase chain reaction (PCR)-based primer panel for messenger RNA (mRNA) amplification followed by electrophoretic fluorescent detection. Additionally, this dissertation explores two microfluidic approaches identifying RNA targets in clinical characterization of SARS-CoV-2 infection status and transplanted organ tolerance.
Conventional methods for forensic bfID are ubiquitous, but reply upon time-consuming, laborious microscopic analysis or presumptive (bio)chemical, enzymatic, or immunological assays that are generally limited in specificity and lacking in sensitivity. The work presented in Chapter 2 details a novel method for forensic bfID via the optimization of the detection of five mRNA targets using loop-mediated amplification (LAMP) with a colorimetric indicator and image capture and analysis. This method allows for amplification of the mRNA target in <15 min providing single-fluid specificity and sensitivity in the picogram range for venous blood, semen, saliva, vaginal fluid, and menstrual blood. Chapter 3 describes the construction of a 3D-printed system for isothermal heating and automated image analysis. Along with the optimized amplification method described in Chapter 2, the combined system was evaluated via a single-blind mock study and compared to ‘gold standard’ conventional approaches. Chapter 4 details the adaptation of a panel of primers specific to five mRNA targets in various body fluids integral to forensic analysis to a portable, microfluidic platform. The time required for analysis was greatly diminished relative to traditional methods; while the conventional approach requires ≥3 hours of analytical time, the approach described herein is complete in less than 30 minutes, with on-disc reverse transcription and electrophoresis complete in 11 and <15 min, respectively. Finally, Chapter 5 shows successful detection of an RNA target for SARS-CoV-2 identification in clinical samples using an amplification method 2X faster on-disc than in-tube. This chapter also provides justification for a second clinical application to continue PCR optimization efforts, with the goal of a significantly reduced overall time and reagent cost, that assesses whether a transplanted liver is being tolerated or rejected. The results in this chapter demonstrate the utility of adapting biochemical assays to microfluidic platforms for rapid, point-of-care target for rapid pathogen detection and establishing patient tolerance of a transplanted organ.
PHD (Doctor of Philosophy)
Forensic Body Fluid Identification, Loop-mediated amplification, Organ Transplant Tolerance Markers, SARS-CoV-2, Messenger RNA
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