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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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Analysis of DNA isolated from probiotic products using magnetic microparticles
Oliva, Jan ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
This thesis is interested in isolation and identification of probiotic bacteria in three different probiotic products using polymerase chain reaction (PCR). DNA in quality suitable for PCR was isolated from crude lysates using three different types of magnetic microparticles and phenol extraction. Identification genera and species of probiotic bacteria was proven using genus and species specific PCRs. Results were in accordance with data presented by manufacturers.
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Probiotic bacteria and authenticity of milk products
Storozhko, Viktoriya ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotics are living microorganisms that have positive effects on human health after consumption. The theoretical part of the bachelor thesis describes properties and health effects of probiotics and their use in food industry. The experimental part was focused on the isolation of PCR-ready DNA from two dairy probiotic products. The presence of target bacterial DNA was confirmed using PCR methods.
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Optimalisation of a new micromethod of DNA isolation from foods
Surá, Tereza ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The thesis were focused on the optimalization of micromethod for isolation of DNA in quality for polymerase chain reaction (PCR) using magnetic microparticles from plant food products. There were chosen a red beetroot (fresh, frozen, dried and sterilized) for the analysis and food products containing red beetroot. Different approaches of processing of homogenates were compared and optimized. The homogenates were prepared in lysis buffer with cetyltrimethylammonium bromide (CTAB) with different amounts of NaCl with or without addition of organic extraction agents chloroform-octanol and isopropanol. Microisolation of DNA was performed using magnetic particles P(GMA). The concentration of NaCl and polyethylene glycol (PEG) 6000 in separation mixtures was tested. The influence on quantity and purity of isolated DNA was compared and the optimum amounts of NaCl in CTAB buffer and optimal concentration of PEG 6000 in separation mixtures were compared. The optimized separation mixture for the DNA isolation from red beetroot was applied to food products containing red beetroot. Amplifiability of DNA was tested in conventional PCR using specific primers for plant DNA. PCR products of length 700 bp were detected by agarose gel electrophoresis.
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The application of magnetic particles for DNA isolation from thermally processed food products
Hronová, Aneta ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The thesis has been focused on testing of micromethod of DNA isolation using magnetic particles from thermic-managed food products in a quality suitable for polymerase chain reaction (PCR). Currant jams were selected for the analysis. These were homogenized using plastic copist and stomacher in lysis buffer with cetyltrimethylammonium bromide (CTAB). The effect of chloroform-octanol and isopropanol in the preparation of homogenates was tested. Homogenates were used for DNA isolation by magnetic particles. Rough fraction of DNA was purified by binding on the magnetic particles after centrifugation of the CTAB complexes with proteins, polyphenols and polysaccharides. Two types of magnetic particles were tested: microparticles of poly(hydroxyethylmethacrylate-co-glycidylmethacrylate) - P(HEMA-co-GMA) and nanoparticles of iron oxides covered by poly(L-lysine) - PLL. Isolated DNA was analyzed spectrophotometrically - it was assessed its concentration and contamination by polyphenols and proteins. After that, amplification of the DNA was tested in PCR. Primers specific for plant ribosomal DNA were used. PCR products of expected length 700 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from currant jams using magnetic particles was in PCR-ready quality.
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The use of magnetic microparticles for bacterial DNA isolation
Hrudíková, Radka ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.
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Study of reversible adsorption of nucleid acids on magnetic carriers
Šálek, Petr ; Ing.Daniel Horák, CSc. (referee) ; Rittich, Bohuslav (advisor)
Reversible adsorption of nucleid acids on magnetic carriers was studied in this diploma thesis. Magnetic P(HEMA-co-GMA) microspheres and magnetic glass particles were used. The aim of the study was to isolate DNA in suitable quality for polymerase chain reaction (PCR). Adsorption of DNA on magnetic carriers was achieved after DNA condensation by PEG and NaCl in separation mixture. PEGs of various molecular weight (600 and 6000 g/mol) and different concentrations of PEG in separation mixture (4, 8, 12, 16%) were used. Quantity of eluted DNA incerased with molecular weight and concentration of PEG in separation mixtures. Optimized experimental conditions were applied for the separation of DNA from chicken erythrocytes, purified DNA, DNA in crude lysates of bacterial cells of Lactobacillus paracasei ssp. paracasei CCDM 211/06 and from real samples (liquid dairy products, hard cheese). The presence of target DNA in eluates was tested using genus specific PCR (genus Lactobacillus) or species specific PCR (species Bifidobacterium longum) Aqueous two-phase system (liquid-liquid) was used for separation of DNA from real symplex, too. At first the condiotions aqueous two-phase systém creation were studied. It was created by 16% PEG of various molecular weight (600, 6000 g/mol) and by various concentration of ammonium sulphate. Reversible DNA adsorption on carboxyl group-containing magnetic nonporous P(HEMA-co-EDMA) microspheres for the isolation PCR-ready DNA from liquid dairy products containing PCR inhibitors was studied, too. The quality of isolated DNA was checked by PCR amplification.The presumption on the elimination of PCR inhibitors from DNA samples was confirmed.
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Probiotic bacteria and authenticity of milk products
Storozhko, Viktoriya ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotics are living microorganisms that have positive effects on human health after consumption. The theoretical part of the bachelor thesis describes properties and health effects of probiotics and their use in food industry. The experimental part was focused on the isolation of PCR-ready DNA from two dairy probiotic products. The presence of target bacterial DNA was confirmed using PCR methods.
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Optimalisation of a new micromethod of DNA isolation from foods
Surá, Tereza ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The thesis were focused on the optimalization of micromethod for isolation of DNA in quality for polymerase chain reaction (PCR) using magnetic microparticles from plant food products. There were chosen a red beetroot (fresh, frozen, dried and sterilized) for the analysis and food products containing red beetroot. Different approaches of processing of homogenates were compared and optimized. The homogenates were prepared in lysis buffer with cetyltrimethylammonium bromide (CTAB) with different amounts of NaCl with or without addition of organic extraction agents chloroform-octanol and isopropanol. Microisolation of DNA was performed using magnetic particles P(GMA). The concentration of NaCl and polyethylene glycol (PEG) 6000 in separation mixtures was tested. The influence on quantity and purity of isolated DNA was compared and the optimum amounts of NaCl in CTAB buffer and optimal concentration of PEG 6000 in separation mixtures were compared. The optimized separation mixture for the DNA isolation from red beetroot was applied to food products containing red beetroot. Amplifiability of DNA was tested in conventional PCR using specific primers for plant DNA. PCR products of length 700 bp were detected by agarose gel electrophoresis.
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The application of magnetic particles for DNA isolation from thermally processed food products
Hronová, Aneta ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The thesis has been focused on testing of micromethod of DNA isolation using magnetic particles from thermic-managed food products in a quality suitable for polymerase chain reaction (PCR). Currant jams were selected for the analysis. These were homogenized using plastic copist and stomacher in lysis buffer with cetyltrimethylammonium bromide (CTAB). The effect of chloroform-octanol and isopropanol in the preparation of homogenates was tested. Homogenates were used for DNA isolation by magnetic particles. Rough fraction of DNA was purified by binding on the magnetic particles after centrifugation of the CTAB complexes with proteins, polyphenols and polysaccharides. Two types of magnetic particles were tested: microparticles of poly(hydroxyethylmethacrylate-co-glycidylmethacrylate) - P(HEMA-co-GMA) and nanoparticles of iron oxides covered by poly(L-lysine) - PLL. Isolated DNA was analyzed spectrophotometrically - it was assessed its concentration and contamination by polyphenols and proteins. After that, amplification of the DNA was tested in PCR. Primers specific for plant ribosomal DNA were used. PCR products of expected length 700 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from currant jams using magnetic particles was in PCR-ready quality.
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