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Characterization of selected yeast strains from food
Ostrihoňová, Katarína ; Vítová, Eva (referee) ; Vránová, Dana (advisor)
This bachelor´s thesis is focused on identification of yeasts of the cheeses by PCR-RFLP method and verifying the lipolytic activity of the yeast. In the theoretical part are processed basic information about yeast, their possible positive and negative effects on the quality of cheeses, the technology of production of cheeses, lipolysis and proteolysis in the cheese and of course of PCR-RFLP (The polymerase chain reaction-restriction fragment length polymorphism). Experimental section shows the isolation of DNA, identification of DNA by PCR made by amplification 5,8S-ITS sections of DNA using primers ITS1 and ITS4. The amplified DNA was purified by ethanol and then was subjected to restriction analysis with the enzymes HaeIII, HinfI, HhaI, TaqI. Then there is listed detection of the PCR product and the restriction fragments by gel electrophoresis. Lengths of the fragments obtained after electrophoresis will be used to identify yeast species isolated from cheeses. In the second part of the thesis in the experimental part we have dealt with evidence of lipolytic activity of the yeast by test on Spirit blue agar.
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Sample preparation for DNA analysis from foods of plant origin
Silná, Renata ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of high quality DNA is nessecary for many molecular biology applications. However, plant DNA contains high amonts of polysaccharides, polyphenols and various secondary metabolites, which decrease yield and quality of isolated DNA. The aim of this study was preparation of samples and different food matrices for DNA isolation DNA by magnetic particles. It was about 5 species of vegetable and 10 species of processed plant food. Homogenization of samples was performed in CTAB buffer. Isolation of plant DNA was performed by magnetic particles covered with carboxyl groups. All DNAs were isolated in conventional PCR qualities using primers for 700 bp amplicons, in the case of heat processed products for 220 bp ampilicons and for real time PCR. The efficiancy of separation of magnetic particles with DNA by magnetic separator and magnetic needle was compared. It was find out that DNA of higher purity was isolated using magnetic needle. The micromethod of isolation of plant DNA from homogenates with CTAB with magnetic particles is suitable for different processed food.
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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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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 Molecular Biology Techniques for Identification and Analysis of Probiotic Bacteria
Konečná, Jana ; Doškař, Jiří (referee) ; Kráčmar, Stanislav (referee) ; Obruča, Stanislav (advisor)
Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol-chloroform extraction and DNA precipitation in ethanol is time-consuming and requires the use of toxic phenol. Alternative method of DNA isolation is use of commercially available kits which, however, are expensive and their efficiency is low. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solid-phase DNA extraction. Magnetic microparticles P(HEMA – co – GMA) containing –NH2 group and nanoparticles PLL, whitch contains polylysine. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed on the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. After optimalization, the developed method was used for DNA isolation from real food supplements. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this thesis, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
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The monitoring of the lactic acid bacteria in the Moravian wines
Valicová, Markéta ; Španová, Alena (referee) ; Omelková, Jiřina (advisor)
The aim of this Master Degree Thesis was to monitor the total number of lactic acid bacteria occurring in grape must during wine production. The study was performed on the red wine grape variety Cabernet Moravia from organic vineyard and on the white wine grape variety Sauvignon from both organic and integrated vineyards. The isolation of pure cultures of lactic acid bacteria from mixed cultures and subsequently their identification by genus and species-specific PCR was also subject of the thesis. The experimental results show that the number of viable cells of lactic acid bacteria is influenced not only by the wine grape variety, whether it is a variety of red or white wine grape, but also by the way of wine growing. The method of wine growing also had an impact on the species representation of lactic acid bacteria in each variety.
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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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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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DNA analysis of nonpathogenic clostridia isolated from cheeses
Chroboková, Maria ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Polymerase chain reaction (PCR) is a molecular method which allows in vitro replication of nucleic acids. It allows the identification and quantification of microorganisms or to prove specific gene sequentions in different matrices of biological origin. Some nonpathogenic species of genus Clostridium cause damages of cheeses, so their identification and quantification is very important in cheesemaking. In this thesis, specific primers for genus Clostridium were tested. Bacterial DNA from culture collection strains and from strains isolated from damaged cheeses were used. Genus-specific region for Clostridium was amplified using specific primers. The PCR products (619 bp) were detected using electrophoresis in 1,8% agarose gel. Genus-specific character of primers was confirmed. DNA of Lactobacillus was used for negative control.
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