Cardiff University | Prifysgol Caerdydd ORCA
Online Research @ Cardiff 
WelshClear Cookie - decide language by browser settings

The rapid detection of drug resistant mycobacteria

Ahortor, Evans 2019. The rapid detection of drug resistant mycobacteria. PhD Thesis, Cardiff University.
Item availability restricted.

[img]
Preview
PDF - Accepted Post-Print Version
Download (5MB) | Preview
[img] PDF (Cardiff University Electronic Publication Form) - Supplemental Material
Restricted to Repository staff only

Download (715kB)

Abstract

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.

Item Type: Thesis (PhD)
Date Type: Submission
Status: Unpublished
Schools: Pharmacy
Subjects: Q Science > Q Science (General)
Funders: Cardiff University
Date of First Compliant Deposit: 5 December 2019
Last Modified: 05 Dec 2019 10:16
URI: http://orca.cf.ac.uk/id/eprint/127104

Actions (repository staff only)

Edit Item Edit Item

Downloads

Downloads per month over past year

View more statistics