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Gewählte Master / Diploma Thesis:

Elena Catalfamo (2011): Development of a Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) based method for the subtyping of a set of Listeria monocytogenes strains .
Master / Diploma Thesis - Institut für Lebensmittelwissenschaften, BOKU-Universität für Bodenkultur, pp 159. UB BOKU obvsg

Data Source: ZID Abstracts
Abstract:
Listeria monocytogenes is a food borne pathogen with the capacity to cause a rare but serious disease called listeriosis. L. monocytogenes serotypes 1/2a, 1/2b and 4b are responsible for the majority of human listeriosis cases. It is therefore critical to develop a valuable and rapid molecular subtyping method to allow fast outbreak detection and source tracking. Recently, Multiple- Locus Variable-Number Tandem- Repeat Analysis (MLVA) showed to be a valid method for the subtyping of L. monocytogenes although further validation of the protocol is necessary and only a limited number of publications is available. The aim of this work was to find the best combination of parameters for a multiplex PCR protocol in order to develop a gel based MLVA method for the subtyping of a set of L. monocytogenes reference strains. For this purpose, different parameters were tested and the gels were visually compared. Furthermore, selected amplicons were sequenced by capillary electrophoresis. The present study provides the initial step to set a MLVA protocol. The final aim is to develop a method based on capillary separation of multiplexed PCR products of VNTR loci labelled with multiple fluorescent dyes. The most appropriate primer combination was TR1+TR2+TR4+TR5 (primers according to Murphy et al., 2007). The best parameters for the multiplex PCR were set as follows: DyNAzyme™ Taq DNA polymerase (Finnzymes, Finland); 0.5 µl (conc.: 10 pmol) of each reverse and forward primer, 1 µl DNA template. The PCR programme was used according to Murphy et al. (2007) and the cycler showed no influence on the results. The gel electrophoresis yielded the best results using 1.5 % Biozym LE Agarose® (Biozym, Germany) and the PeqLab PerfectBlue™ Horizontal Maxigelsystem (PeqLab, Germany).

Beurteilende(r): Kneifel Wolfgang
1.Mitwirkender: Domig Konrad

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