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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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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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Recombinant probiotics
Surá, Tereza ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Theoretical part of this thesis focuses on present state and research of recombinant probiotics and their use in food industry and health care. It also concentrates on their beneficial effects on the health of individuals. The experimental part focuses on the identification of specific bacterial strain in a probiotics food supplements. The DNA was isolated from these products by use of magnetic microparticles and obtained DNA was subsequently analysed through polymerase chain reaction.
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Probiotics and their use in food industry
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic bacteria are defined as live microorganisms, which when consumed in the determining quantities, have healthy and beneficial effects. Most of probiotics belongs to the genera Lactobacillus and Bifidobacterium. These and other genera of microorganisms are successfully used in industry, including food industry at present. Probiotics are used primarily in dairy products and food additives in food idustry. Probiotic bacteria, like other organisms, can be to identifie by PCR method that allows amplifying specific regions of DNA. Polymerase chain reaction was performed after DNA isolation from bacterial cultures of three strains using phenol extraction method. PCR specific for the domain Bacteria and genus-specific PCR were used for the confirmation of the presence of bacteria of the genus Lactobacillus.
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Nucleic acid detection using magnetic microparticles
Pešková, Aneta ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
Modern and fast separation proces for nucleic acids isolation uses magnetic microparticles in routine laboratory practise. The comparision of DNA isolation efectivity by three types of magnetic microparticles P(HEMA-co-GMA) (A, B), PGMA and magnetic nanoparticles covered by poly-L-lysine (PLy) from probiotic products Pangamin was carried out. The quality and quantity of isolated DNA were verified by spektrofotometric measurments and gel electrophoresis. Possibility of amplifying of isolated DNA was verified by polymerase chain reaction (PCR) with specific primers for yeast and evaluated by agarose gel electrophoresis. The quantity of isolated DNA depended on the product and the type of magnetic particles. The highest amount of DNA was isolated by magnetic nanoparticles of poly-L-lysine (PLy) and magnetic microparticles P(HEMA-co-GMA) (A).
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The used of magnetic microparticles for isolation and prove of probiotic bacterial DNA in meat
Vašíček, Roman ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
This thesis deals with the isolation of probiotic DNA from meat products and its assesment by PCR methods. In this thesis is developed homogenization of samples of sausages with kopist, preparation of sausage cells lysates and isolation of DNA by using of magnetic microparticles. The DNA was isolated from sausage lysates by using magnetic microparticles. Isolated DNA was further amplified in genus and spesies-specific PCR methods. In tested products was proven presence of DNA of domain Bacteria, type Lactobacillus and Bifidobacterium. In one product was proven presence of species Lactobacillus acidophilus and Bifidobacterium animalis.
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Plasmid DNA interactions with lanthanoide compounds
Gőghová, Sabína ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
In the theoretical part of bachelor thesis, literature was summarised on the isolation and purification of plasmid DNA, the application of lanthanides as non enzymatic nucleases and practical importance of lanthanide compounds in medicine. In the experimental part of the thesis, plasmid DNA pUC19 was isolated by the method of alkaline lysis. Isolated plasmid DNA was cleaved by neodymium trichloride at 70 °C in different intervals. Gel electrophoresis was used for evaluation of experiments.
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Monitoring of Phytoestrogens Expression to the Milk and Milk Products
Watzková, Jarmila ; Rittich, Bohuslav (referee) ; Doležal, Petr (referee) ; Omelková, Jiřina (advisor)
Phytoestrogens are very intensively studied in recent years because they show different pharmacological effects on human health. For example, it was demonstrated that phytoestrogens reduce the risk of certain cancers (breast, prostate or colon), act as antioxidants in the body and affect the activity of certain enzymes. In addition, also it was demonstrated number of positive effects of soybean phytoestrogens on the health status of women in the menopause, such as reducing the risk of atherosclerotic cardiovascular disease or osteoporosis. Various technologically processed soy beans are important part of the feed rations mainly for high-lactating dairy cows, because they are a valuable source of the energy and high-quality protein, despite their negative effect on reproductive performance. Phytoestrogens consumed from the feed rations are hydrolyzed in the rumen of dairy cow and together with their metabolites are absorbed in the small intestine into the blood. They can be excreted from the body in urine or milk. From this perspective, cow's milk may become a source of phytoestrogens substances, especially equol in the human nutrition. On the other hand, some research results suggest possible potential risks associated with phytoestrogens intake in the diet on human health. At the some age (infants and young children) and group (vegetarians) categories may be dietary intake of phytoestrogens undiserable. According to most experts, a beneficial effect on human health prevail the potential risks. However, the risks associated with phytoestrogens intake in food can not be ignored and therefore phytoestrogens are still actual problem in the field of scientific research.
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Identification of selected probiotic bacteria in food additives
Bubeníková, Lucia ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
Bifidobacteria and lactobacilli are gram-positive bacteria which belong to the lactic acid bacteria group. They are ordinary constituents of gastrointestinal microflora, play a pivotal role in human nutrition and health, prevent pathogen colonization and maintain normal mucosal immunity. In present days they are used in production of functional food products and pharmaceutical additives. Polymerase chain reaction (PCR) is used to the detection and identification of bacteria which belong to the genus of Bifidobacterium and Lactobacillus. Specific primers for each genus are used in PCR reactions for in vitro amplification of definite part of DNA. In this work, total DNA was isolated from three additives (Pangamin Bifi plus, Biopron Junior and Probiodom) by the method of phenol extraction and amplified using genus-specific PCR primers giving amplicons of 523 bp (Bifidobacterium) and 250 bp (Lactobacillus). Occurrence of bacteria of both genera was proved in all of the three tested probiotic products.
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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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