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

Loading...
Thumbnail Image
Date
2023
DOI
Open Access Location
Journal Title
Journal ISSN
Volume Title
Publisher
Massey University
Rights
The Author
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. In the present study, MDA was coupled with Oxford Nanopore (ONT) MinION sequencing to help evaluate the potential MDA-nanopore sequencing as a point-of-care diagnostic. To assess the level of genome coverage of cultured M. bovis DNA with different selective MDA primers, both Illumina NovaSeq and Minion MK1C nanopore sequences were obtained. Selective primers that produced optimal coverage antimicrobial resistance genes in M. bovis were identified. It was found that mismatch priming played a significant role in the success of the MDA reactions. The implication of this for potential diagnostic applications has been discussed.
Description
Embargoed until 30th October 2025.
Keywords
Citation