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Title: The rapid detection of drug resistant mycobacteria
Author: Ahortor, Evans
ISNI:       0000 0004 8500 6988
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2019
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Mycobacterium abscessus is a multidrug resistant pathogen commonly isolated from patients with cystic fibrosis. Currently, there is no rapid diagnostic tool to detect the presence of M. abscessus. Rapid diagnosis followed by appropriate, prompt treatment remains the best curative approach to mitigate disease burden and halt transmission. A major bottle neck in developing a rapid diagnostic assay is DNA extraction. Mycobacterial cells are very difficult to lyse, the existing methods are time consuming resulting in long turnaround time to detect the pathogen. This study employed the use of microwave energy to rapidly release nucleic acids from microorganisms and test the ability to detect the released nucleic acids in a magnetic-bead-based sandwich hybridisation assay using specific DNA probes. Based on published genome sequences, probes targeting the rpoB and erm-41 genes of M. abscessus and M. smegmatis were designed. In a magnetic-bead-based sandwich hybridisation assay using these specific probes, M. abscessus and M. smegmatis were distinguished from non-specific isolates within 70 mins with a lower detection limit of 1 pg/μL. The disruptive effects of microwaves on biological structures has been attributed to the local generation of heat. The contribution, if any, of non-thermal factors is yet to be determined. To study the interaction of microwaves with cell membranes, the structure which represents the major barrier to DNA release, fluorescent microscopy was employed to examine the passage of different sized fluorescent dextran particles into bacterial and yeast cells following microwave exposure. The results show a transient membrane disruption, size dependent permeabilization of dextran particles into cells. In conclusion, a prototype hybridisation assay capable of detecting M. abscessus and M. smegmatis has been developed. The application of microwaves to cells induced membrane disruption allowing internalisation of varying sizes of fluorescent dextran particles and the release of intact DNA for detection in hybridisation assay.
Supervisor: Not available Sponsor: Cardiff University
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General)