M.Sc. (Biochemistry) / Cholera, the highly epidemic diarrhoeal disease caused by Vibrio cholerae (V. cholerae) infection, continues to devastate many developing countries. Therefore, a rapid and sensitive method to identify pathogenic V. cholerae biotypes is imperative. Literature highlighted the sensitivity of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and high resolution melt curve analysis (HRM) for this purpose. It is necessary to develop these methods based on the virulence proteins/genes themselves in order to be future diagnostic tools capable of detecting distinct strains of V. cholerae and to determine their pathogenicity. Such virulence proteins/genes would require significant sequence variation among different strains of V. cholerae. The toxin-coregulated pilus protein A (TcpA) is a key player in V. cholerae infection and displays significant sequence variation at both the genetic and protein level. The hypothesis of this study is that the variation in the TcpA/tcpA protein and gene, respectively, can be used to differentiate between V. cholerae biotypes by using MALDI-TOF MS and/or HRM methods. The objectives were first to investigate established methods for the rapid isolation of surface proteins that would yield a high enough concentration of whole pili or TcpA subunits for future MS biotyping techniques. Two methods were evaluated: a) pili isolation by mechanical shearing b) crude extraction of the outer membrane proteins and associated surface proteins. Secondly, HRM-compatible oligonucleotides were designed for specific amplification of the variable regions within the tcpA gene in V. cholerae. The efficacy of these oligonucleotides was tested on V. cholerae reference strains and environmental isolates. Lastly, a current standard procedure for V. cholerae typing was evaluated. Pili were successfully isolated from bacterial cell colonies, but large quantities of starting material were required and much of the cell content was isolated with the pili. Alternative isolation and enrichment methods have been proposed and may prove promising for future MALDI-TOF MS biotyping. The current standard procedure for V. cholerae typing with multiplex PCR revealed some discrepancies between the multiplex PCR steps. Therefore, the current multiplex PCR procedure may result in inaccuracies during typing. The designed HRM-compatible oligonucleotide set designated Contig 1 successfully amplified a 101 bp region within the tcpA gene in Vibrios. This Contig 1 oligonucleotide set could possibly be used in future studies to differentiate between Vibrio species with HRM. Further studies would be needed for the development of MALDI-TOF/MS or HRM as future rapid and specific environmental monitoring systems based on the TcpA/tcpA protein or gene, respectively.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uj/uj:11646 |
| Date | 01 July 2014 |
| Creators | Kleynhans, Ronèl Elaine Susan |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Johannesburg |
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