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Identification of lactic acid bacteria in fermented dairy products using amplification methods
Tycová, Martina ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Polymerase chain reaction (PCR) is molecular diagnostic method which allows the identification of lactic acid bacteria used in food industry. In this work species-specific PCR primers (targeted on highly conserved 16S rDNA region) were used for identification of bacteria of species Streptoccocus thermophilus in 10 randomly commercially accessible fermented milk products and for identification of species Streptococcus thermophilus in 25 lyophilisates collected in Culture Collection of Dairy Microorganisms Laktoflora (CCDM, Tábor, Czech Republic). The PCR products (968 bp) were detected using electrophoresis in 1,2 % agarose gel. Bacterial DNA was isolated from crude cell lysates by magnetic carriers P(HEMA co GMA) containing carboxyl groups. DNA was reversibly bind on their surface in the presence of high concentrations of poly(ethylene glycol) (PEG 6000) and sodium chloride. Phenol extraction of DNA was used as control. Streptococcus thermophilus strains were identificated using PCR in all analysed samples.
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Comparison of different types of magnetic carriers for DNA microisolation from foods
Koplík, Jerguš ; Lunerová,, Jana (referee) ; Kovařík, Aleš (advisor)
Micro-isolation of PCR-ready from fresh and dried legumes seeds and food products containing legumes (hummus) was tested. For optimization process magnetic microparticles PGMAox was used. Optimum weight plants and food material was 200 mg. For isolation of DNA from fresh legumes, mixture containing 500 L of CTAB extraction buffer, 1 L -mercaptoethanol and 500 L chloroform-octanol was used. For isolation of DNA from dried legumes seeds and food products volume of CTAB extraction buffer was increased to 1 000 L. To achieve higher purity of DNA some prepared homogenates was precipitate by isopropylalcohol. The optimized process of DNA isolation was used to prepare a homogenate from food products which DNA was isolated by different types of magnetic carriers. For comparison, non-porous magnetic carriers poly(glycidyl methacrylate) PGMAox, poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) P(HEMA-co-GMA)ox covered by carboxyl groups and porous fully crosslinked microparticles poly(styrene-co-divinylbenzene) HPS-B-M-22-NH2, magnetic porous glass MPG and nanoparticles of iron oxides covered by poly(L-lysine) PLL were used. In average the highest concentration and the best purity of DNA was isolated by magnetic carriers P(HEMA-co-GMA)ox a PGMAox.
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Applications of real-time PCR for characterization particles suitable for DNA isolation
Ondrejková, Martina ; Šálek, Petr (referee) ; Trachtová, Štěpánka (advisor)
The theoretical part of the diploma thesis was focused on core-shell type magnetic carriers, used mainly in medical, molecular-biological and biochemical applications. Encapsulation of the core is essential for these applications due to the decrease od non-specific protein adsorbtion, increase of biocompatibility and the possible functionalization of magnetic carriers. In the experimental part, the DNA (E. coli) was amplified by real-time PCR in the presence of poly(hydroxymethacrylate-co-glycidylmethacrylate) (P(HEMA-co-GMA)) magnetic carriers with/without carboxyl groups. The inhibitory effect of different concentrations of magnetic carriers in the PCR mixture was evaluated from the calibration curve parameter values obtained by regression analysis. The presence of a specific PCR product was verified by agarose gel electrophoresis. Most of magnetic carriers without carboxyl groups extinguished the fluorescence in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, without inhibition of DNA amplification - the carriers were biocompatible. Magnetic carriers with carboxyl groups extinguished the fluorescence in the lower concentration range (0,4 – 4,0 g.l-1 in the PCR mixture). Their inhibition of amplification was in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, from the concentration 0,8 g.l-1 in the PCR mixture, the inhibition did not occur and the carriers were biocompatible. The results do not depend on the characteristic properties of the magnetic carriers but on the presence of the carboxyl groups on the surface of the carrier and the degree of coverage of the magnetic core by the polymer. Real-time PCR has become an effective tool for studying magnetic core encapsulation and the influence of functional groups on the surface of the polymeric layer.
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Isolation of bacterial DNA from foods using magnetic carriers
Bubeníková, Lucia ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was the selective isolation of bacterial DNA with help of magnetic carriers covered by streptavidine (PGMA-NH2-STV, MPG® Streptavidin). Conditions of functionalisation of carriers using two biotinylated probes were optimized: the amount of carrier, the amount of probe, binding of biotinyled probe to streptavidine. Purified DNA Lactobacillus was used for hybridization. DNA binding to the probe (DNA/DNA hybridization) and nospecific adsorption of DNA to the carrier were tested. Target DNA eluted from the carrier was identified using PCR with primers R16-1 and LbLMA1-rev and with primers P_eub and F_eub. The amount of probe bound to the carrier was estimated using UV spectrophotometry. It was estimated that biotinyled probe can be used for functionalisation in concentration 5 pmol/µl added to the carrier in the ration carrier : probe 1:1. It was shown that nonspecific DNA adsorption to the MPG® Streptavidin is significantly lower than to the carrier PGMA-NH2-STV.Using DNA/DNA hybridization and the MPG® Streptavidin, DNA from pure culture Lactobacillus was isolated. Procedure was applicated for DNA isolation from milk products.
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Reversible immobilisation of DNA on newly designed magnetic carriers
Kubisz, Petr ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of work was an optimization of separation deoxyribonucleic acid (DNA) with the use of nucleic acid reversible adsorption to the surface of magnetic particles coated by functional groups. Six carriers were verificated for DNA isolation: P (HEMA-co-GMA) ox, F-kol B 30 ox, F-kol 77 ox, F-kol B100 ox, F-kol 135 ox, coated with carboxyl groups and Perovskit 439 (coated by silicone). Bacterial DNA was isolated by phenol extraction procedure, first. DNA was reversibly bond to magnetis carrier in the presence of high concentration of NaCl ( 5 M) and poly (ethylene glycol) (PEG 6000). The final PEG and NaCl concentrations of 16.0 % (w/v) and 2.0 M, respectively, were used.DNA was eluted into TE buffer. The quality of extracted DNA was checked by PCR amplification. It was found out that although different quantities of DNA were isolated, the quality of isolated DNA was always compatible with PCR. Nanoparticles Perovskit 439 had the best separative characteristics in comparison to the other magnetic carriers because highest amounts of DNA was isolated. However, next optimisation of DNA separation procedure is required for the use of studied microspheres in real samples.
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Comparison of different types of magnetic carriers for DNA microisolation from foods
Koplík, Jerguš ; Lunerová,, Jana (referee) ; Kovařík, Aleš (advisor)
Micro-isolation of PCR-ready from fresh and dried legumes seeds and food products containing legumes (hummus) was tested. For optimization process magnetic microparticles PGMAox was used. Optimum weight plants and food material was 200 mg. For isolation of DNA from fresh legumes, mixture containing 500 L of CTAB extraction buffer, 1 L -mercaptoethanol and 500 L chloroform-octanol was used. For isolation of DNA from dried legumes seeds and food products volume of CTAB extraction buffer was increased to 1 000 L. To achieve higher purity of DNA some prepared homogenates was precipitate by isopropylalcohol. The optimized process of DNA isolation was used to prepare a homogenate from food products which DNA was isolated by different types of magnetic carriers. For comparison, non-porous magnetic carriers poly(glycidyl methacrylate) PGMAox, poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) P(HEMA-co-GMA)ox covered by carboxyl groups and porous fully crosslinked microparticles poly(styrene-co-divinylbenzene) HPS-B-M-22-NH2, magnetic porous glass MPG and nanoparticles of iron oxides covered by poly(L-lysine) PLL were used. In average the highest concentration and the best purity of DNA was isolated by magnetic carriers P(HEMA-co-GMA)ox a PGMAox.
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Applications of real-time PCR for characterization particles suitable for DNA isolation
Ondrejková, Martina ; Šálek, Petr (referee) ; Trachtová, Štěpánka (advisor)
The theoretical part of the diploma thesis was focused on core-shell type magnetic carriers, used mainly in medical, molecular-biological and biochemical applications. Encapsulation of the core is essential for these applications due to the decrease od non-specific protein adsorbtion, increase of biocompatibility and the possible functionalization of magnetic carriers. In the experimental part, the DNA (E. coli) was amplified by real-time PCR in the presence of poly(hydroxymethacrylate-co-glycidylmethacrylate) (P(HEMA-co-GMA)) magnetic carriers with/without carboxyl groups. The inhibitory effect of different concentrations of magnetic carriers in the PCR mixture was evaluated from the calibration curve parameter values obtained by regression analysis. The presence of a specific PCR product was verified by agarose gel electrophoresis. Most of magnetic carriers without carboxyl groups extinguished the fluorescence in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, without inhibition of DNA amplification - the carriers were biocompatible. Magnetic carriers with carboxyl groups extinguished the fluorescence in the lower concentration range (0,4 – 4,0 g.l-1 in the PCR mixture). Their inhibition of amplification was in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, from the concentration 0,8 g.l-1 in the PCR mixture, the inhibition did not occur and the carriers were biocompatible. The results do not depend on the characteristic properties of the magnetic carriers but on the presence of the carboxyl groups on the surface of the carrier and the degree of coverage of the magnetic core by the polymer. Real-time PCR has become an effective tool for studying magnetic core encapsulation and the influence of functional groups on the surface of the polymeric layer.
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Isolation of bacterial DNA from foods using magnetic carriers
Bubeníková, Lucia ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was the selective isolation of bacterial DNA with help of magnetic carriers covered by streptavidine (PGMA-NH2-STV, MPG® Streptavidin). Conditions of functionalisation of carriers using two biotinylated probes were optimized: the amount of carrier, the amount of probe, binding of biotinyled probe to streptavidine. Purified DNA Lactobacillus was used for hybridization. DNA binding to the probe (DNA/DNA hybridization) and nospecific adsorption of DNA to the carrier were tested. Target DNA eluted from the carrier was identified using PCR with primers R16-1 and LbLMA1-rev and with primers P_eub and F_eub. The amount of probe bound to the carrier was estimated using UV spectrophotometry. It was estimated that biotinyled probe can be used for functionalisation in concentration 5 pmol/µl added to the carrier in the ration carrier : probe 1:1. It was shown that nonspecific DNA adsorption to the MPG® Streptavidin is significantly lower than to the carrier PGMA-NH2-STV.Using DNA/DNA hybridization and the MPG® Streptavidin, DNA from pure culture Lactobacillus was isolated. Procedure was applicated for DNA isolation from milk products.
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Reversible immobilisation of DNA on newly designed magnetic carriers
Kubisz, Petr ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of work was an optimization of separation deoxyribonucleic acid (DNA) with the use of nucleic acid reversible adsorption to the surface of magnetic particles coated by functional groups. Six carriers were verificated for DNA isolation: P (HEMA-co-GMA) ox, F-kol B 30 ox, F-kol 77 ox, F-kol B100 ox, F-kol 135 ox, coated with carboxyl groups and Perovskit 439 (coated by silicone). Bacterial DNA was isolated by phenol extraction procedure, first. DNA was reversibly bond to magnetis carrier in the presence of high concentration of NaCl ( 5 M) and poly (ethylene glycol) (PEG 6000). The final PEG and NaCl concentrations of 16.0 % (w/v) and 2.0 M, respectively, were used.DNA was eluted into TE buffer. The quality of extracted DNA was checked by PCR amplification. It was found out that although different quantities of DNA were isolated, the quality of isolated DNA was always compatible with PCR. Nanoparticles Perovskit 439 had the best separative characteristics in comparison to the other magnetic carriers because highest amounts of DNA was isolated. However, next optimisation of DNA separation procedure is required for the use of studied microspheres in real samples.
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Identification of lactic acid bacteria in fermented dairy products using amplification methods
Tycová, Martina ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Polymerase chain reaction (PCR) is molecular diagnostic method which allows the identification of lactic acid bacteria used in food industry. In this work species-specific PCR primers (targeted on highly conserved 16S rDNA region) were used for identification of bacteria of species Streptoccocus thermophilus in 10 randomly commercially accessible fermented milk products and for identification of species Streptococcus thermophilus in 25 lyophilisates collected in Culture Collection of Dairy Microorganisms Laktoflora (CCDM, Tábor, Czech Republic). The PCR products (968 bp) were detected using electrophoresis in 1,2 % agarose gel. Bacterial DNA was isolated from crude cell lysates by magnetic carriers P(HEMA co GMA) containing carboxyl groups. DNA was reversibly bind on their surface in the presence of high concentrations of poly(ethylene glycol) (PEG 6000) and sodium chloride. Phenol extraction of DNA was used as control. Streptococcus thermophilus strains were identificated using PCR in all analysed samples.
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