Evaluation of MDA and ONT sequencing for developing a point of care diagnostic for mycobacterium : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Science at Massey University, Manawatu, New Zealand. Embargoed until 30th October 2025

Abstract

Direct sequencing of pathogen DNA from clinical samples can significantly improve the speed of diagnosis, antimicrobial resistance prediction, and outbreak investigation. This approach is especially useful for slow-growing pathogens like Mycobacterium tuberculosis (MTB). However, since MTB may represent only 0.01% of the total DNA in a sputum sample, it is crucial to enrich MTB DNA by specific amplification and/or depletion of non-target DNA, including human and/or bacterial DNA. Here, we investigated the potential of selective multiple displacement amplification (MDA) using novel primers that might be used to characterise Mycobacterium DNA from young cultures or directly from sputum samples. In an earlier study, selective MDA primers were designed to preferentially bind to MTB DNA and not human DNA or the DNA from bacteria found to be commonly occurring in the upper respiratory tract of MTB patients. As suggested by analyses in this thesis, these primers will also amplify other closely related species of Mycobacterium.--Shortened abstract