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Optimization of a loop-mediated isothermal amplification method for a detection of Haemophilus influenzae infection.
Zadáková, Kateřina ; Jeřábek, Petr (advisor) ; Svášková, Dagmar (referee)
Haemophilus influenzae is a gramnegative, rod-shaped and predominantly human pathogen. Infection caused by this microorganism is especially dangerous for children under five years of age, who are mainly at risk of pneumonia and meningitis.Althought vaccination is available against its most dangerous type b, it still causes problems, especially in developing countries. Due to the insufficient vaccination, strains that are more resistant to the antibiotics are more spreading in these countries. Equipment requirement of current detection options for H. influenzae infection(e.g. polymerase chain reaction) causes, that reliable detectionis a problem in poor countries. Loop-mediated isothermal amplification could be a cheap and reliable method. The aim of this bachelor thesis was the design of sets of primers and their applicationinthe LAMP reaction. The designed primers targetedthe ompP6 gene, which was alreadythe target of LAMP reaction in some studies and is also a frequent target in PCR detection.As a part of the optimization, different volumes of the reaction mixtures and multiple detection options were tested -colorimetric detectionusinganacid-base indicator and fluorescence detection using different dyes monitored in real time. Key words: Haemophilus influenzae, loop-mediated isothermal...
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Study of chicken antibodies against viral S protein of SARS-CoV-2
Křížová, Tereza ; Hodek, Petr (advisor) ; Svášková, Dagmar (referee)
In the beginning of the year 2020, the WHO declared a pandemic in relation with spreading of a new infection. The Covid-19 disease is mainly manifested by respiratory problems caused by the SARS-CoV-2 virus. It enters the host cell through the ACE2 receptor, to which it is bound by the RBD domain of the surface "spike" protein. A possible way of protection against viral infection is vaccination, e.g. with an mRNA vaccine, which induces the production of a "spike" protein to invoke an immune response and antibody production. Another possible alternative for protection is passive immunotherapy. In this thesis, antibodies from chicken eggs were chosen, which are extensively used in the prevention of various viral and bacterial diseases. They are characterized by high binding affinity to antigens, they are economically inexpensive and are considered a rapid, simple and safe method of passive protection. Antibodies IgY were prepared from egg yolks of chickens vaccinated with an mRNA vaccine encoding a "spike" protein. The reactivity of the prepared antibodies with the whole "spike" protein (expressed in insect cells) was tested by ELISA, demonstrating the ability of the antibodies to bind to the antigen with high affinity. Subsequently, their ability to interact with the RBD domain itself (expressed in...
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The effect of cancerogenic azo dye Sudan I on expression of biotransformation enzymes
Hejduková, Žaneta ; Svášková, Dagmar (advisor) ; Dračínská, Helena (referee)
Sudan I is a widely used azo dye which has the ability to cause carcinomas in organs and tissues of experimental animals. During reactions catalyzed by microsomal monooxygenase enzyme systems or cytoplasmic biotransformation enzymes, Sudan I is oxidized to reactive metabolites that covalently bind to nucleic acids and cause their damage. Sudan I can also be metabolized by reduction, e. g. by a DT-diaphorase enzyme (NQO1). Reduction of Sudan I is considered to be a detoxification reaction. In this work, the in vivo action of Sudan I is examined in terms of its ability to induce an expression of the biotransformation enzyme DT-diaphorase in tissues of rats treated with the azo dye. The aim of this work was to quantify the degree of NQO1 induction at mRNA level. After the isolation of total RNA from organs of rats treated with Sudan I, the RNA was converted to cDNA by reverse transcription using random hexamers as primers. Using specific probes, the abundance of mRNA for the enzyme NQO1 in the organs of treated rats was quantified by "real-time" PCR, relatively to the control gene with a constant expression (β-actin). Through comparing thus determined amounts of mRNA in individual organs of treated and untreated rats, it has been found that Sudan I had caused a significant increase in the expression...
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