Developing an environmentally friendly diagnostic system for the detection of respiratory diseases

Bozkurt, Hejan and Munir, Muhammad and Unterholzner, Leonie (2025) Developing an environmentally friendly diagnostic system for the detection of respiratory diseases. PhD thesis, Lancaster University.

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Abstract

Respiratory viruses are the most common infectious cause of morbidity and mortality in humans worldwide. Considering the events of the recent Coronavirus Disease 2019 (COVID-19) pandemic, there is an urgent need for rapid and accurate diagnostic methods to allow early disease detection and subsequent treatment. Although current gold standard testing methods, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are mainly accurate, they are limited by high costs, complex instrumentation, and slow turnaround times, particularly in low-resource settings. This thesis hypothesises that reverse transcription-recombinase polymerase amplification (RT-RPA), an isothermal amplification method, can offer a cost-effective, environmentally friendly alternative for detecting respiratory viruses with comparable diagnostic performance to RT-qPCR. In order to evaluate this hypothesis, RT-RPA was developed and tested to detect SARS-CoV-2 and other respiratory viruses. Assay performance was compared with RT-qPCR and reverse transcription-loop-mediated isothermal amplification (RT-LAMP) across 140 clinical samples. RT-RPA demonstrated high sensitivity and specificity, particularly with fluorescence-based detection, though lateral flow detection (LFD) offered better portability. TOPO TA cloning and in vitro transcription were used to generate and validate synthetic viral RNA for assay development. Further evaluations showed that RT-RPA achieved faster detection times and comparable or superior sensitivity for multiple viral targets, including cases where RT-PCR failed to detect low viral loads. Despite a small number of false positives, particularly for respiratory syncytial virus B (RSV-B), RT-RPA maintained its strong diagnostic accuracy. Overall, this work supports using RT-RPA as a robust alternative to RT-qPCR, especially for point-of-care testing (POTC) in low-resource settings, highlighting its potential for broad application in respiratory virus diagnostics.