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DNA microextraction from plant vegetable matrix
Cesnak, Filip ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of the thesis was the comparison of two DNA microextraction methods with the use of magnetic beads from food of plant origin. Samples had disparate and complex matrices and were either raw (broccoli) or processed (strawberry jam). The first method uses a magnetic separator for the manipulation of magnetic beads and was used as a standart for the comparison. The second method uses a paramagnetic needle, the advantage of which should be the possibility to isolate DNA of higher quality without a significant contamination by polyphenolic compounds or proteins. The former method was validated by statistic analysis of results obtained from both methods. DNA quality was judged by testing the amplificability of isolated DNA via PCR. The amplified products were visualised on an agarose gel with electrophoresis.
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Methods of molecular cytogenetics: evolution, analysis, comparison
Budková, Kateřina ; Šolc, Roman (advisor) ; Daňková, Pavlína (referee)
Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of chromosomes, especially during the cell division. Chromosomal abnormalities that may occur during this process are often associated with the emergence of various diseases. Cytogenetic methods are primarily used to detect these abnormalities. Traditional methods allow resolution at the level of whole chromosomes, the introduction of new molecular methods led to a significant refinement of these analyzes. Today's methods allow easy analysis of chromosomes to the level of individual sequences. Despite significant differences between classical and modern cytogenetic techniques, classical cytogenetic methods are not evil, but they are still widely used. Combining classical and molecular methods in order to obtain a comprehensive analysis of chromosomal rearrangements is no exception.
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Molecular genetic diagnosis of cystic fibrosis
Matějčková, Iva ; Krkavcová, Miroslava (advisor) ; Kotlas, Jaroslav (referee)
Cystic fibrosis is a very common and serious disease. It is an autosomal recessive disease with an incidence of 1 : 2500 - 1 : 4500. With this disease, CTFR gene mutations occur which are responsible for the failure of chloride channels. It is a multi-organ handicap, which primarily infects the respiratory and gastrointestinal system, but also affects the hepatobiliary system, reproductive system, and sweat glands. Cystic fibrosis can be diagnosed by a sweat test, molecular genetic diagnosis, or more rarely, using transepitel potential differences, which has not been introduced in the Czech Republic yet. Since 2009, the Czech Republic applies neonatal screening CF, which helps make a faster diagnosis to then be able to proceed to the rapid introduction of appropriate treatment. The introduction of an early treatment has significantly improved the prognosis of the patient. Currently CF patients can live on average from thirty to forty years. For the CFTR gene genetic testing is used postnatal, prenatal or preimplantation material. From these samples, DNA gets isolated and is further investigated using genetic methods, which may be based on the principle of PCR, reverse hybridization, restriction analysis, DNA sequencing or MLPA. Powered by TCPDF (www.tcpdf.org)
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Cytomegalovirus infection in transplant patients
Dvořák, Jan ; Tachezy, Ruth (advisor) ; Harant, Karel (referee)
Cytomegalovirus (HCMV) is a ubiquitous human -herpesvirus highly prevalent in the population. HCMV is transmitted by close contact between individuals. In infected person this virus causes mainly asymptomatic primary infection, after which the latency is established. In pregnant women HCMV infection can lead to abortions, defects of the fetus and congenital abnormalities of newborn babies. Even more serious complications are caused by this virus in the immunocompromised patients, especially those infected by HIV and in patients who undergo solid organ transplantation and hematopoietic stem cell transplantation. This work is a complex report about HCMV biology with emphasis on complications which HCMV causes in patients after solid organ transplantation and hematopoietic stem cell transplantation. This article also contains summary of the methods used for diagnostic of HCMV infection and monitoring and prevention of HCMV associated diseases. Keywords: Cytomegalovirus, hematopoietic stem cell transplantation, solid organ transplantation, detection, monitoring, polymerase chain reaction, cellular immunity, humoral immunity
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The application of magnetic particle for DNA isolation from selekted probiotic products for children
Vozárová, Petra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
In the food industry, it is important to correctly identify the species of bacteria and thier properties so that they can be used as a probiotic in dietary supplements. This is performed using DNA diagnostics. In the experimental part, the DNA from four probiotic dietary supplements for children was isolated. Magnetic particles P(HEMA-co-GMA) were tested for isolation. Isolated DNA was amplified by PCR and the presence of DNA of genus Lactobacillus, Bifidobacterium and Bacillus was demonstrated in the products according to the data declared by the manufacturer. The presence of species L.acidophilus, B.animalis in accordance with the data on the product has been demonstrated by PCR with species specific primers. Using PCR, the presence of L.casei, which was declared by the manufacturer, has not been proven in one product at given experimental conditions.
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Laboratory diagnostics of micrometastases in breast cancer patients
Mikulová, Veronika ; Zima, Tomáš (advisor) ; Svoboda, Marek (referee) ; Průša, Richard (referee)
Introduction: The presence of circulating tumor cells (CTC) in the peripheral blood has been associated with worse prognosis and early relapse in breast cancer patients. CTC determination in the peripheral blood has been considered as a liquid biopsy. The aim of this project was to analyze the presence of CTC followed by their molecular characterization with the potential use not only as a new biomarker for real-time monitoring of therapy efficacy but also as a suitable tool for patient's stratification and individualization of treatment for breast cancer. Methods: A total of 54 patients with diagnosed early breast cancer were enrolled into a prospective study. Ten millilitres of peripheral blood were sequentially collected to test for the presence and characterization of CTC during the follow-up of patients. CTC isolation and detection was performed by AdnaTest BreastCancer™ (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC- lysates. cDNA from isolated CTC has been further used for newly optimized qPCR assays for breast tumor and therapy resistance associated genes: TOP1, TOP2A, CSTD, ST6GAL, KRT19 and reference gene actin. qPCR results have been analyzed by Genex software (MultiD Analysis). Results: 195 blood samples have been...
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The analysis of DNA isolated from different types of probiotic products using real-time PCR and HRM analysis
Sedláková, Lucie ; Rittich, Bohuslav (referee) ; Trachtová, Štěpánka (advisor)
The aim of this diploma thesis was to introduce real-time PCR with high-resolution melting analysis for Bifidobacterium species. Currently a small number of publication, dealing with identification of Bifidobacterium species using high-resolution melting analysis, is available. According to publications dealing with identification of lactic acid bacteria were selected primers P1V1 and P2V1, LAC1 and LAC2, LsppUPF and LsppUPR, V3F and V3R, V6F and V6R. Using this primers bacterial DNA was amplified by real-time PCR with high-resolution melting analysis. After evaluation of the measured results efficiency of selected primers was verified on DNA izolated from complex sample of probiotic product. After further optimisation real-time PCR with high-resolution melting analysis could be suitable using selected primers for Bifidobacterium species.
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Plasmid DNAs interactions with lanthanoide compounds
Budko, Kateryna ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Recently much attention is given to lanthanides and their complexes as excellent catalysts for cleavage of nucleic acids. The thesis has been focused on the cleavage of plasmid and bacterial DNA by ions Nd3+ and Y3+ and by different carriers containing the lanthanide compounds. The creation of single-stranded nicks and double-stranded ones in the plasmid DNA molecules was studied by agarose gel electrophoresis. Verification of the cleavage of bacterial DNA was made by polymerase chain reaction using primers specific for the domain Bacteria and genus and species-specific primers. The results will be used in the development of the method that will allow perfect carriers`s coverage verification with the magnetic perovskit nucleus and other carriers with the lanthanide compounds.
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Use of PCR for species identification and searching of selected genes of lactobacilli
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic food products - food additives contain different species of probiotic bacteria. Accurate species identification with their characteristics is very important from the view of products quality. Methods of DNA diagnostics are used for these purposes. In this thesis DNA was isolated from 4 probiotic products. The presence of bacterial of genus Lactobacillus and species L. acidophilus, L. casei, L. plantarum, L. rhamnosus were detected in three products by PCR. This information was in accordance with the data provided by the manufacturer. Two sets of primers were used for identification of species. Using other primers sequences of genes such as bsh, lai and odc were detected in DNA isolated from the products. Differences were estimated among products concerning the detection of lai gene Lactobacillus acidophilus.
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Imunomagnetic separation of lactic acid bacteria using magnetic microparticles functionalised by antibodies
Vaňásek, Jakub ; Španová, Alena (referee) ; Trachtová, Štěpánka (advisor)
Immunomagnetic separation is based on binding of antibody with antigen, where antibody is bound to magnetic particle. In this thesis there were used particles of magnetic pearl cellulose with antiLactobacillus and antiBifidobacterium antibodies. Immunomagnetic separation method was optimalized and verified for its efficiency and specifity with bacterial and yeast cells. This cells were identified by polymerase chain reaction. Efficiency of immunomagnetic separation was verified on probiotic meat product, where Lactobacillus cells were isolated. With DNA from isolated Lactobacillus cells the high resolution melting was performed. The results show presence of several bacterial strains of Lactobacillus species.
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