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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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Testing of food authenticity using DNA analysis
Kocianová, Vanda ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Authenticity verification of food products is an important step before their release to the market. This testing ensures that the product contains what the label states and also confirms the medical harmlessness. One of methods possible to use for authenticity verification is polymerase chain reaction (PCR). The experimental part was focused on the DNA isolation using magnetic particles on PCR – ready quality from food product – ketchup. DNA isolated from seeds and stalk of tomato were used as a controls. Amplificalibity of DNA isolated with magnetic particles using magnetic separator and needle was compared. The method requires further optimization.
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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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DNA isolation using newly designed magnetic carriers
Machan, Radoslav ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
Theoretical part of the master thesis was aimed on giving an overview of basic characteristics of magnetic particles, their morphology, basic methods of their synthesis, interaction with DNA and recent applications in biotechnology and biomedicine. The experimental part of thesis was aimed on application of new designed magnetic particles for isolation of both lactobacilli DNA and calf thymus DNA. Two types of magnetic beads were used: hyperbranched poly(glycidyl methacrylate-co-[2-(methacryloyloxy) ethoxy]acetic acid-co-ethylene dimethylacrylate) microbeads covered with amino groups (P(GMA-MOEAA-EDMA)-NH2) and magnetic non-porous poly(2-hydroxyethyl methacrylate) microbeads covered with carboxyl groups (P(HEMA-co-GMA)-COOH). For both types of microbeads two different protocols for preparation of separation mixtures with two different concentrations od poly(ethyleneglycol) 6000 (PEG 6000) as condensation agent were tested. Differences among both types of magnetic microbeads and DNAs used were found. It was shown that both types of microbeads are suitable for DNA isolation in the presence of 8% PEG 6000.
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The application of magnetic particles for DNA isolation from cereal products
Starenkova, Anastasiia ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The thesis has been focused on micro method for isolation of PCR- ready DNA iusing magnetic particles from cereal products. Cereal biscuits and cereal products for babies were selected for the analysis. These were homogenized using plastic copist in lysis buffer with cetyltrimethylammonium bromide (CTAB). The homogenates were purified using chloroform- octanol mixture. The effect of isopropanol in the preparation of homogenates was tested, too. Homogenates were used for DNA isolation by magnetic particles. Two ways to isolate magnetic particles with bounded DNA (magnetic separator and magnetic needle have been tested. Isolated DNA was analyzed spectrophotometrically its concentration and purity were assessed. . After that, amplification of the DNA was tested in PCR. Two sets of primers specific for plant ribosomal DNA were used for their amplification. PCR products of expected length 700 bp and 220 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from seeds and cereal products using magnetic particles was in PCR-ready quality.
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DNA microextraction from plant vegetable matrix
Cesnak, Filip ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of the thesis was the comparison of two DNA microextraction methods with the use of magnetic beads from food of plant origin. Samples had disparate and complex matrices and were either raw (broccoli) or processed (strawberry jam). The first method uses a magnetic separator for the manipulation of magnetic beads and was used as a standart for the comparison. The second method uses a paramagnetic needle, the advantage of which should be the possibility to isolate DNA of higher quality without a significant contamination by polyphenolic compounds or proteins. The former method was validated by statistic analysis of results obtained from both methods. DNA quality was judged by testing the amplificability of isolated DNA via PCR. The amplified products were visualised on an agarose gel with electrophoresis.
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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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Intestinal metabolism of bilirubin
Jirásková, Alena ; Branny, Pavel (advisor) ; Španová, Alena (referee) ; Pátek, Miroslav (referee)
CONCLUSIONS In this study we focused on the process of bilirubin reducfion catalyzed by an anaerobic intestinal bacterium C' peýingen.s. We aimed to undertake analysis of bile pigments metabolized by C. perfringens and their respective reduction products and to identify gene(s) encoding protein(s) involved in metabolism of bilirubin. Analysis of bile pigments metabolized by C. perfringens and their respective reduction products 1) The C. perfringens strain BRI isolated from neonatal stools reduces a variety of different bile pigments indicating that this broad substrate speciÍicity could be an effective tool for disposal of electrons produced in catabolic pÍocesseswithin thesebacteria. 2) The examined strain reduces UCB only to the level of urobilinogen. Other bacterial sfoains and species, absent in neonates, are presumed to be essential for catabolism to the level ofstercobilinogen. Identification of gene(s)involved in bilirubin metabolism 1) The C. perfringens strain BRl is resistant to the transformation of plasmid DNA mediatedby electroporation and thereforeit is not a candidate suitable for transposonmutagenesis. A transformab|e C. peýingens P90.2,2. strain was found to reduce bilirubin. Rapid and simple method suitable for electroporation of this strain was developed providing transformation...
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