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DNA extraction from plant tissues for polymerase chain reaction analysis
Trojánek, Zdeněk ; RNDr.Roman Matyášek, CSc. (referee) ; Rittich, Bohuslav (advisor)
Extraction of nucleic acids is an important step for all molecular biological studies. The process of isolation of plant DNA is complicated due to the presence of polyphenols, polysaccharides and other metabolites. They can be co-isolated with DNA and act as PCR inhibitors. The aim of this study was to compare CTAB extraction procedure, Qiagen DNA easy kit, direct homogenization, carboxyl-functionalised magnetic non-porous HEMA based microspheres and combination of the above mentioned methods for DNA isolation from different plants. The DNA was evaluated regarding concentration, purity and amplification in PCR. All methods provided DNA that could be used in downstream PCR applications. However, there were differences regarding yield, purity, labour intensiveness and cost. Combination of direct homogenization and magnetic microspheres coated by carboxyl groups was isolated DNA from various plants and plant foods in a quality suitable for convectional PCR, real time PCR and restriction analysis. This method is fast, simple and does not require work with harmful substances.
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Selective isolation of of the genus Lactobacillus bacteria from foods
Novotná, Eva ; Šárka, Havlíková (referee) ; Rittich, Bohuslav (advisor)
Probiotic lactic acid bacteria of genus Lactobacillus play an important role in the digestive tract of human. They are used in food processing and they are the part of food supplements. Lactic acid bacteria of the genus Lactobacillus can be identificated by polymerase chain reaction (PCR). Bacterial DNA was isolated from cell lysates of 4 synbiotic food suplements by magnetic particles P(HEMA-co-GMA). Isolated DNA was amplified by genus-specific and species-specific primers. Magnetic particles with immobilized antibodies against Lactobacillus bacteria were used in the next part of thesis. These particles were used for isolation target cells from products with their identification by genus specific PCR.
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Identification of bacteria of Lactobacillus acidophilus species in probiotic products
Sznapková, Veronika ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Probiotic lactic acid bacteria (LAB) are an important part of fermented dairy products, pharmaceuticals and food supplements. At present, rapid and accurate identification of bacteria is carried out using molecular biological methods based on DNA amplification. The aim of the thesis was to identify by non-cultivation bacteria of genus Lactobacillus and bacteria of species Lactobacillus acidophilus in complex matrices at total of seven different food supplements. Total DNA was isolated from crude cell lysates using magnetic carrier P(HEMA-co-GMA). Amplificability of DNA was verified by PCR using primers specific for the domain Bacteria. In next step isolated DNA was amplified using primers specific for the genus Lactobacillus and species Lactobacillus acidophilus to demonstrate the presence of this bacterial genus and species declared by the producers. The results of bacteria identification obtained by PCR were compared with declared specification given by the producers.
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Identification of probiotic Bifidobacterium strains in dairy products
Zovčáková, Monika ; Horák, Daniel (referee) ; Španová, Alena (advisor)
Lactobacilli are dominant bacteria of the vaginal flora. Lactobacillus-containing probiotics products are used for the treatement and profylaxis of bacterial urogenital infections. This work is focused on DNA identification and species identification of probiotic bacteria in 5 different vaginal tablets using molecular-genetic methods. Total DNA isolated from complex matrix of vaginal tablets was used for amplification in polymerase chain reaction. DNA was isolated from crude cell lysates by magnetic particles P(HEMA-co-GMA) and by method of phenol extraction. Identification of species of probiotic bacteria was verified using genus-specific and species-specific PCRs. Results of bacterial identification obtained by PCR were compared with declared specification given by producers. Bacteria of genus Lactobacillus were proved in all tablets whereus species identification was in accordance with the stated composition in 1 tablet only.
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Molecular identification of selected species of lactic acid bacteria and bifidobacteria in food additives
Riegelová, Kristýna ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic lactic acid bacteria and bifidobacteria are natural part of microflora of gastrointestinal tract. In the present, day they are grossly exploited in food processing industry. The aim of the work was molecular identification of bacteria of genus Lactobacillus and Bifidobacterium in complex matrices of two food additives. Total DNA was isolated from crude cell lysates by magnetic particles P(HEMA-co-GMA). Isolated DNA was amplified in genus-specific and species-specific PCRs. Amplicons were detected by agarose gel electrophoresis. Results were compared with declared specification given by producers in three different batches.
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Isolation of PCR-ready DNA from dairy products for baby nutrition
Mantlová, Jana ; Eva, Kvasničková (referee) ; Španová, Alena (advisor)
The work was focused on isolation of PCR-ready DNA and the identification of probiotic lactic acid bacteria that were isolated from five milk product for infant nutrition. DNA was isolated from crude cell-lysates of the products by magnetic P(HEMA-co-GMA) microspheres. DNAs isolated from crude cell lysates of control strains using phenol extraction method were used as positive controls. Using PCRs DNA of genera Bifidobacterium and species B. animalis, B. bifidum, B. breve, B. infantis, B. longum and Streptococcus thermophilus species were identified in products. The results obtained are consistent with the data declared by the manufacturers.
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Identification of lactic acid bacteria in food products using amplification methods
Jurečková, Nela ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
The aim of the study was isolation of genomic DNA from 7 milk products (yogurts/ yogurt milk) in quality for use it in PCR. Magnetic microparticles (P(HEMA-co¬-GMA)) were used for the DNA isolation. DNA was reversibly adsorbed to the microspheres in the presence of 8% poly(ethylene glycol) PEG 6000 and 5 M sodium chloride. The adsorbed DNA was released from the microspheres in a low ionic strength TE buffer and use as DNA matrix in genus specific PCR Lactobacillus. The presence of DNA of genus Lactobacillus was detected in all analysed products.
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Identification of lactic acid bacteria in hard cheeses using amplification methods
Herzogová, Jitka ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Diploma thesis was focused on identification of lactic acid bacteria of species Lactococcus lactis and subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris using species and subspecies specific polymerace chain reaction (PCR). PCR method was used for identification of bacteria of species Lactococcus lactis in 10 samples of hard cheeses. The method of sample preparation was evaluated for hard cheeses with the aim to receive sufficient amount of cells for the preparation of crude cell lysates. Whole DNA in quality suitable for PCR was separated using magnetic microspheres P(HEMA-co-GMA) in the presence of polyethylenglycol (PEG 6000) and sodium chloride. DNA isolated by phenol extraction was used as control of DNA isolation. PCR was used to the analysis of 7 strains of Lactococcus lactis from Collection of dairy microorganisms Laktofora (CCDM). Altogether 5 or 2 strains were identified into subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris, respectively.
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DNA extraction from plant tissues for polymerase chain reaction analysis
Trojánek, Zdeněk ; RNDr.Roman Matyášek, CSc. (referee) ; Rittich, Bohuslav (advisor)
Extraction of nucleic acids is an important step for all molecular biological studies. The process of isolation of plant DNA is complicated due to the presence of polyphenols, polysaccharides and other metabolites. They can be co-isolated with DNA and act as PCR inhibitors. The aim of this study was to compare CTAB extraction procedure, Qiagen DNA easy kit, direct homogenization, carboxyl-functionalised magnetic non-porous HEMA based microspheres and combination of the above mentioned methods for DNA isolation from different plants. The DNA was evaluated regarding concentration, purity and amplification in PCR. All methods provided DNA that could be used in downstream PCR applications. However, there were differences regarding yield, purity, labour intensiveness and cost. Combination of direct homogenization and magnetic microspheres coated by carboxyl groups was isolated DNA from various plants and plant foods in a quality suitable for convectional PCR, real time PCR and restriction analysis. This method is fast, simple and does not require work with harmful substances.
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