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Selective isolation of the genus Bifidobacterium bacteria from foods
Mizerovská, Lucie ; Šárka, Havlíková (referee) ; Rittich, Bohuslav (advisor)
Probiotic lactic acid bacteria (LAB) are very often used in food procesing industry, such as milk products, cheese and fermentsd salami production in nova days. In diploma thesis were tested symbiotic food supplements from different producers. Bacterial DNA was isolated from crude cell lysates of six food suplements by magnetic particles P(HEMA-co-GMA). PCR-ready DNAs were isolated. from all products The detection of Bifidobacterium bacteria identified by PCR was in agreement with those declared by the manufacturers. Magnetic particles with immobilized antibodies against Bifidobacterium were used in the next part of thesis. These particles were used for the isolation of target cells from two products with cell identification by genus specific PCR.
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DNA isolation from probiotic lactic acid bacteria in food additives
Tvrdíková, Jana ; Vojtíšková, Marie (referee) ; Španová, Alena (advisor)
In this work the functionalised magnetic particles were tested with streptavidin to selective DNA isolation. The method of selective DNA isolation was tested by using DNA probiotic strain Lactobacillus paracasei subsp. paracasei CCDM 211/06. A test was done on the biotinyl oligonucleotic particles, which was immobilised by containing streptavidin and it was used like a DNA probe for isolation complementary DNA chain by means of DNA/DNA hybridization. The primer R 5´ bio and the biotinyl denatured specific PCR product were tested for species Lb. paracasei as a DNA probe. These following experimental conditions were optimized for selective DNA isolation: temperature and time of hybridization, amount of DNA and the release of DNA from microspheres. Isolation of DNA was verified by PCR with specific generic primers. The specific generic PCR product was amplified in extent 250 bp, which was detected by using electrophoresis in agarose gel. This optimized method was successfully used in selective isolation of DNA Lactobacillus from a complementary sample of supplementary food (BIFI pangamin).
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The application of magnetic nano- and microparticles for the isolation of DNA from selected foods
Ráčková, Lucie ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
In thesis was verified micromethod for isolation of plant DNA from different vegetable (onion and broccoli) and plant food products in quality for application in polymerase chain reaction (PCR). The micromethod allows isolation DNA using magnetic particles from crude lysates of cells obtained by direct homogenization of plant tissues. Various methods of processing homogenates were compared. Homogenization was performed by lysis buffer containing cetyltrimethylammonium bromide (CTAB). The effect of the organic extraction agents was tested (chloroform-octanol and isopropanol). DNA was purified from homogenates by reversible adsorption on magnetic particles (four different types of magnetic particles were tested). The quality of isolated DNA was verified by UV spectrophotometry. The amplificabilty of DNA was tested by polymerase chain reaction (PCR). Specific primers for plant ribosomal DNA (rDNA) were used. PCR products of lenght 700 and 220 bp were detected by agarose gel electrophoresis. Differences in yield and quality of DNA were depended on the homogenate processing and magnetic particles used. The proposed procedure with two magnetic particles was tested for the isolation DNA from plan food products (spreads). DNA was amplified in PCR. Micromethod is suitable for DNA analysis of foods.
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The application of magnetic particles for DNA isolation from selected vegetable products
Akwari, Michala ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
Micromethod of DNA isolation using magnetic particles is one of the modern technological methods used in DNA isolation, and makes the process simpler, more effective and faster. The main aim of this study was to isolate the DNA from various plant (tomato) food products, using different types of magnetic particles. The results were compared and the quantity, purity and the possibility of amplication of the isolated DNA among samples were found to be different. The DNA isolation method using magnetic particles P(HEMA-co-GMA) or HPS B-M-NH2 was shown to be the most effective in achieving the above mentiond parametres. DNAs from the analysed samples of plant food products were isolated in sufficient quantity and quality to be used in the conventional PCR. Differences in the possibility of the amplification of the isolated DNA stored at -20 °C during more than a half year were not found.
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The use of magnetic microparticles for DNA isolation
Jelínek, Zdeněk ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
The effectiveness of magnetic microparticles in isolation of DNA from Lactobacillus rhamnosus CCM 1825T and DNA from chicken erythrocytes were studied in diploma thesis. Magnetic HEMA based microparticles coated by carboxylic groups and hyperbranched styrene-divinylbenzene particles (IMC AS ČR, Prague, Czech Republic) were used for DNA isolation. Magnetic microparticles Dynabeads® DNA DIRECT™ Universal (Dynal, Norway) based on polystyrene and MPG® Uncoated (PureBiotech, USA) based on magnetic glass were used as a control. The dependence of amount of eluted DNA on concentration of DNA in the base solution and the dependence of amount of eluted DNA on concentration of magnetic microparticles were studied. The affinity of magnetic microparticles to RNA for various concentrations of RNA solution was studied, too. The ability of tested particles to isolate DNA from real samples was validated using milk product Actimel. The quality of isolated DNA of Lactobacillus genus was proved using genus specific PCR.
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Magteic particles and their applications in biotechnology
Knápková, Monika ; Konečná, Jana (referee) ; Trachtová, Štěpánka (advisor)
The thesis is focused on the magnetic particles which are used in several biotechnological applications. The theoretical part deals with the specific properties of these nanoparticles and materials of which the nanoparticles can be made. There are also mentioned some of the biotechnological applications of magnetic particles. During the experimental part, selected types of magnetic particles were used to isolate nucleic acid. The quality of the isolated DNA with respect to purity was evaluated using the polymerase chain reaction and its modifications. High resolution analysis (HRM analysis) was also used to verify the quality of the isolated DNA and to resolution Lactobacillus casei and Lactobacillus rhamnosus. DNA isolation using magnetic carriers was successful. Commercially available MPG microcarriers and magnetic microparticles Fkol 77ox were the most suitable. In terms of purity magnetic nanoparticles F79/L3-PLL were the most suitable for the DNA isolation. The resolution of bacterial strains of Lactobacillus casei and Lactobacillus rhamnosus was not successful.
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Magnetic carriers and their practical use
Chlopková, Barbora ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The theoretical part summarizes the current knowledge for practical use of magnetic carriers in molecular diagnostics. It includes both already used methods and methods with potential for the future. In the experimental part was tested by use of magnetic media for isolation of DNA from a dairy product and a bacterial culture. It was confirmed that the magnetic carrier DNA was isolated in quantity and quality suitable for carrying out polymerase chain reaction.
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PROBIOTIC GENES OF SIGNIFICANT LACTIC ACID BACTERIA IN FOOD
Konečná, Jana ; Ševčovičová,, Andrea (referee) ; Doškař, Jiří (referee) ; Španová, Alena (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. 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 solidphase DNA extraction. 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 to the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. 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 contribution, 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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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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