Date of Award


Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy


Department of Biological Sciences

First Advisor

Dr. S. Fanning

Second Advisor

Dr. B. Cryan


Throughout the 1990s, the incidence of methicillin-resistant Staphylococcus aureus has increased in many health care areas including acute and chronic care facilities, outpatient clinics and in the community. Once introduced into a health care environment, this nosocomial pathogen can spread rapidly and as MRSA are resistant to multiple antibiotics, treatment is often difficult. Therefore effective infection control measures are required to prevent cross-infection and further spread of endemic strains.

In this study, a sensitive and specific triplex-PCR assay was designed for MRSA detection, wherein three genes, the methicillin resistance gene (mecA). femA and the extracellular thermonuclease gene (rmc) were simultaneously amplified. The products of this triplex-PCR were analysed using a number of detection formats (including conventional agarose gel electrophoresis, “dot blot” analysis, colour amplified PCR detection system (CAPS) and fluorescent GeneScan technology).

Such PCR detection assays do not investigate the complex epidemiology of this pathogen. Rapid and accurate epidemiological typing methods are required to identify the source and track the spread of different MRSA clinical isolates. To this end, the genetic relationship(s) among two Cork University Hospital (CUH) MRSA outbreaks was successfully analysed using rep-PCR with a single RW3A primer, together with agarose gel electrophoresis and fluorescent GeneScan analysis.

The fluorescent triplex-PCR and RW3A fingerprinting strategies were multiplexed and this permitted the development of a combined detection and typing scheme termed COGEDET for MRSA strains.

In addition, a number of conserved RW3A-generated amplicons were characterised by cloning and sequencing to determine their genomic origins.

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