Evans, Catherine
(2025)
Development and evaluation of new methods to optimise the Actiphage RapidTM assay for detection of mycobacteria.
PhD thesis, University of Nottingham.
Abstract
Pathogenic mycobacteria including M. tuberculosis complex and M. avium subspecies paratuberculosis are pathogens of global importance posing a risk to human health and contributing to large economic losses in the agricultural industry. Due to nature, severity and implications of mycobacterial infections, there is a need for rapid and sensitive diagnostics. The characteristic, slow-growing nature of pathogenic mycobacteria introduces a barrier to diagnosing infections quickly when using culture-based methods. Histology lacks the sensitivity to detect low cell numbers, nor does it allow for species specific identification. ELISA assays are most used for detection of mycobacteria due to their inexpensiveness and quick turnaround from sampling to results. Although ELISA tests are specific, they have low sensitivity, especially in early stages of infection. To overcome these issues with standard mycobacterial methods of detection, the Actiphage® assay was developed to provide a reasonably quick, sensitive identification method by using a broad host- range Mycobacteriophage to lyse mycobacterial cells recovered from white blood cells and using endpoint PCR to identify species-specific signature DNA sequences extracted from the mycobacteria.
This project was sponsored by PBD Biotech Ltd. to develop, evaluate and optimise Actiphage RapidTM-qPCR based approaches for detection of mycobacteria from clinical blood and milk samples. Whilst qPCR kits are currently commercially available for the detection of M. avium subspecies paratuberculosis, many of these kits have been optimised for detection from faecal, tissue or organ samples. Therefore, whether these kits were compatible with DNA samples prepared using mycobacteriophage lysis and containing substances from blood known to inhibit PCR amplification remained to be explored. Evaluation of the use of commercial qPCR kits to detect mycobacterial DNA prepared from the Actiphage® assay was carried out. The results highlighted issues with using commercial kits including the need to implement a ∆Cq analysis method due to unspecific binding events of the Actiphage RapidTM assay negative control. Results also showed that trouble shooting was difficult with commercial qPCR kits, therefore, the company decided to develop and optimise an in-house qPCR assay to allow for more control over assay development. Whilst there was success in designing and using a novel qPCR kit for the detection of M. avium subspecies paratuberculosis and M. tuberculosis complex, results show that more optimisation is needed to efficiently incorporate an internal amplification control within the assay, something that is essential for a diagnostic kit. Nevertheless, experiments to detect M. avium subspecies paratuberculosis from blood of farmed goats by Actiphage RapidTM assay and qPCR using a commercial qPCR kit demonstrated that Actiphage RapidTM-qPCR assay approaches offer a more sensitive detection of M. avium subspecies paratuberculosis than ELISA based assays.
A second area of research focused on elucidating further applications of the Actiphage RapidTM assay. Areas of interest were using Actiphage RapidTM assay as a physical lysin to extract mycobacterial DNA for sequencing experiments, investigating restriction fragment length polymorphisms found in M. avium complex and using Actiphage RapidTM assay to aid in herd management. Exploiting single nucleotide polymorphisms within an insertion sequence of M. avium complex to develop a high-resolution melting analysis approach allowed for differentiation of M. avium subspecies paratuberculosis subtype, something that can be beneficial as an epidemiological tool. Finally, through testing of bovine bulk milk and individual cattle with Actiphage RapidTM assay, results were used to successfully isolate M. tuberculosis complex infected animals from a herd to control disease and work towards a disease-free status for the farm, validating that Actiphage RapidTM assay is a useful diagnostic tool for control of mycobacterial disease.
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