Abstract
The emergence of the novel SARS-CoV-2 virus in December 2019 warranted the rapid development of diagnostic tests, vaccines, therapeutics, and public health measures to contain the spread of the virus. The rapid spread of the virus, coupled with the rise in new variants and co-/superinfections required the development of sensitive molecular diagnostic assays that enabled sample pooling, the detection of variants and co-/super-infecting viruses.Sensitive diagnostic tests are important to limit the proportion of false negatives. Despite the benefits offered, sample pooling and multiplexing are known to impact the sensitivity of molecular diagnostic assays. This thesis demonstrates the development of a loop mediated isothermal assay and reverse transcription- polymerase chain reaction-based (RT-PCR) assays to assess the impact of pooling, multiplexing and co-/superinfection on assay sensitivity, and develop methods to optimise the analytical sensitivity and specificity of such assays.
The work presented demonstrates that the inclusion of trehalose, Triton X-100, maintaining free Mg2+ and deoxynucleoside triphosphate concentrations, and optimal primer design, among others are essential in improving assay analytical sensitivity and specificity. The implementation of time cut-offs was also shown to improve assay analytical specificity. Furthermore, whilst pooling was expected to theoretically increase assay LoD, the results show that the use of a 1:5 sample pooling strategy did not impact the ability of the RT-PCR assay to detect samples positive for SARS-CoV-2. Differences in diagnostic algorithms were shown to lead to differences in assay LoD, which can have implications when validating new assays against comparators. Multiplexing was reported to increase the LoD of certain targets within an RT-PCR assay designed for the differentiation of SARS-CoV-2 variants but did not impact the LoD of an RT-PCR assay designed for the detection of common respiratory viruses (Influenza A, Influenza B and Respiratory Syncytial virus) – reiterating the importance of optimal assay design. However, multiplexing was shown to impact the signal intensities of target probes within both RT-PCR assays.
Taken together, this research demonstrates that multiplexing, co/super-infection, differing diagnostic algorithms and pooling all impact the limit of detection of an assay. However, understanding and optimising the factors that impact an assay of limit of detection can limit the impact on analytical sensitivity.
| Date of Award | 29 Mar 2023 |
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| Original language | English |
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| Supervisor | Hannah Leese (Supervisor) & Semali Perera (Supervisor) |