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Optimization of a loop-mediated isothermal amplification method for testing Streptococcus pneumoniae infection
Friček, Matúš ; Jeřábek, Petr (advisor) ; Dračínská, Helena (referee)
Streptococcus pneumoniae is grampositive pathogen bacteria. According to World Health Organisation, diseases like pnemoniae or meningitidis are most commonly caused by S. pneumoniae infection. Gold standard for pathogen detection is currently polymerase chain reaction based on specific DNA amplification. During the last decades, many DNA amplification methods have been developed. One of them is loop mediated isothermal amplification (LAMP). Thanks to low instrumental requirements and simple interpretation, this method should be appropriate alternative for pathogen detection. Putting LAMP into clinical practise is complicated mainly because of common false positive results occurance in non DNA containing samples. Based on that, one of the aims of the thesis was to identificate and eliminate main sources of DNA contamination. According to the results, the main source of contamination seems to be the products from previous reactions. Nowdays, lytA and piaB genes are the most commonly used as targets for PCR detecion of S. pneumoniae. In available publications, there is not any mention about targetting LAMP method at piaB gene for detection of S. pneumoniae. That is why the aim of this bachelor thesis was to design optimal LAMP "primers" set for piaB gene detection. Within the optimization of the...
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The utilization of the uracil-DNA glycosylase in the elimination of carry-over contamination by products of the previous DNA amplifications
Zrnová, Adéla ; Martínková, Markéta (advisor) ; Prošková, Veronika (referee)
DNA amplification is an important tool in diagnosis of infectious diseases. The most commonly used method in laboratories is the polymerase chain reaction, which, does not meet the need for a fast and inexpensive method. It was therefore necessary to develop a method that would be sensitive, fast, specific and accessible, for example, in developing countries with limited access to instrumentation. It appears that LAMP could be this method. Unfortunately, the LAMP method encounters considerable false positivity, which must be prevented in order to be used in practice. The subject of the presented bachelor thesis is the introduction of a unique LAMP method to detect the presence of DNA Streptococcus pneumoniae and the study of the use of uracil- DNA glycosylase to prevent contamination of samples with DNA formed in previous amplification reactions. The LAMP method of DNA detection Streptococcus pneumoniae was successfully introduced, then the method was optimized even in the presence of deoxyuridine triphosphate. After determining the appropriate deoxyuridine triphosphate concentration for LAMP amplification, the resulting Streptococcus pneumoniae DNA amplification products were treated with uracil-DNA glycosylase. This procedure was shown to have the potential to prevent false positivity of other...
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