Qualitative and semiquantitative isothermal detection of nucleic acids for point-of-care testing applications
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Abstract
Nucleic acid amplification tests (NAATs) are widely implemented as infectious disease diagnostics due to their high sensitivity and specificity. Polymerase chain reaction (PCR) tests are the most commonly diagnostic NAAT. PCR requires the use of equipment for temperature cycling and instrumentation to monitor reaction progress and often require upstream sample preparation to isolate nucleic acids from a raw clinical samples. As such, PCR-based tests are most commonly used large and well-resourced healthcare systems where clinical samples are collected from patients and transported to a centralized testing laboratory.
Point-of-care diagnostic testing (POCT) is a complementary paradigm to centralized testing where testing infrastructure and consumables are disseminated throughout a healthcare system such that diagnostic testing can be performed on-site with the patient nearby. POCT methods enable test-and-treat strategies commonly implemented in STI screening programs and obviate issues associated with patient loss to follow up. When developing NAATs for POCT, it is often necessary to simplify assay protocols and reduce required overhead especially in low resourced settings where access to laboratory resources may be limited. Recently, isothermal NAA methods, which eliminate the need for costly thermocycling equipment, have garnered use in POCT applications often coupled with visual readout methods to eliminate instrumentation requirements.
In this work, I expand upon these existing isothermal NAA methods to develop POCT with both qualitative and semiquantitative detection capabilities. First, I developed a thermophilic helicase dependent amplification (tHDA) assay for the detection of the STI Trichomonas vaginalis (TV) and a competitive internal control. This assay is compatible with visual readout via a low cost lateral flow immunoassay (LFIA) device. I also developed a simple yet effective strategy for rapid nucleic acid extraction and enrichment from urine. When coupled, the sample preparation strategy and tHDA assay were able to detect TV at a concentration of 0.25 TV genomes per mL. I observed 96.6% sensitivity and 100% specificity when testing clinical urine samples. Second, I developed a novel test for semiquantitative detection of hepatitis B virus (HBV) DNA at two tunable thresholds with LFIA readout. Chronic HBV patients require serial viral load monitoring to inform initiation of antiretroviral therapy (ART) and ensure continued treatment efficacy for patients receiving ART. I demonstrated the tunability of the semiquantitative ligation and amplification assay by controllably setting two independent quantification thresholds to specific HBV viral load concentrations used in chronic HBV case management. This assay can be performed in under one hour and is compatible with isothermal tHDA.