|
|
Optimization of calcium chloride concentration for removal of polysaccharide contamination during plant DNA isolation
Frnčová, Ekaterina ; Šlosárová, Katarína (referee) ; Fialová, Lenka (advisor)
The greatest difficulty in isolating DNA is the presence of contaminants that cause side effects. Polysaccharides are the most common contaminants in fruits. They can distort the results in spectrophotometric determination of purity or act as inhibitors in PCR analysis together with other substances (for example, proteins or phenolic substances). The aim of this work was to investigate the influence of different concentrations of calcium chloride on the process of DNA isolation. In the experimental part, DNA from the apple was isolated using different concentrations of calcium chloride. The isolation was carried out four times, and each time the sample was adjusted in different ways. It was found that the isolation method used works only with a sample that has been lyophilized. Isolation of DNA from fresh fruit provided very low yields. Probably, this was due to the large water content in the sample, and the proportion of the solid component was smaller. Subsequently, PCR analysis and electrophoresis were performed to determine the amplifiability of the isolated DNA. Two sets of primers with different specificity were used for this analysis. Amplifiability was confirmed only when using primers specific to apple DNA when using 100 mM solution of CaCl2. Other samples have been amplifiable using both types of primers. Probably, samples isolated using a 100 mM solution of CaCl2 had a larger amount of inhibitors that do not affect all PCR reactions equally, which may also indicate a small effectiveness of this amount of CaCl2.
|
|
|
Testing and optimization of protocols for removal of contaminants during DNA isolation from hibiscus blossoms
Brabcová, Martina ; Kubalová, Michaela (referee) ; Fialová, Lenka (advisor)
The Roselle (Hibiscus sabdariffa) is a widely used plant in food industry, especially in tea production. The hibiscus flower is rich in polyphenolic substances, which are a major problem in DNA isolation. This work deals with the modification of the CTAB isolation protocol to ensure concentration and purity of DNA sufficient for its amplification. DNA isolated according to the CTAB protocol did not have sufficient concentration and purity for amplification due to high contamination with polyphenolic substances. A procedure with double incubation of plant tissue in CTAB buffer containing 3% polyvinylpyrrolidone (PVP) was proposed to remove it. These isolates were successfully amplified. The effect of PVP concentration (1-5%) in CTAB buffer on the concentration and purity of isolated DNA was also investigated. PCR results showed that double incubation with CTAB buffer had a greater effect on the removal of polyphenolic compounds than changing the PVP concentration. Subsequently, the modified isolation protocol was also successfully used to isolate amplifiable DNA from commercial tea blends.
|
|
|
Evaluation of the presence of probiotic bacteria in a food supplement using molecular biological methods
Dvornyi, Nikolai ; Brázda, Václav (referee) ; Smetana, Jan (advisor)
Probiotics are beneficial microorganisms that, when administered properly, can provide health benefits to the host, such as aiding digestion and boosting the immune system. Their effectiveness and benefits depend on the specific strain of microorganism. These strains are often from the genera Lactobacillus, Bifidobacterium, and represent natural hosts of the gastrointestinal tract. This bachelor thesis focuses on the identification of probiotic bacteria in a commercially available dietary supplement Linex® Forte using molecular biological methods, mainly polymerase chain reaction (PCR). The aim was to verify the presence of the claimed probiotic cultures of Lactobacillus acidophilus, Bifidobacterium animalis, and to compare the results with the manufacturer's claims. Therefore, two DNA isolation methods were used: phenolchloroform extraction and the commercial purification kit OMNI International. The isolated DNA was then analyzed by domain, genus and species-specific PCR. The results show that the methods used are suitable for the identification of probiotic bacteria and confirm their presence in the selected product.
|
|
|
Utilization of PCR technique for identification of probiotic bacteria in daily hygiene product
Horobets, Yuliia ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
Probiotic bacteria have traditionally been used in the food industry, but their applications have now expanded to the prevention and treatment of various diseases. Increasing evidence supports the efficacy of bacteria of the genus Lactobacillus in the oral cavity has led to the application of probiotic strains of this genus in mouthwashes and other personal care products. In this bachelor thesis, DNA was isolated by two methods, then quantified spectrophotometrically and amplified via conventional PCR. The results of the polymerase chain reaction were detected by gel electrophoresis. The presence of probiotic bacteria species Lactobacillus delbrueckii, Lactobacillus plantarum and Lactobacillus pentosus was confirmed.
|
|
|
Verification of the presence of probiotic bacteria in a cosmetic product using the polymerase chain reaction (PCR) technique
Raevskaya, Vera ; Kroupová, Zuzana (referee) ; Smetana, Jan (advisor)
This bachelor thesis focuses on the verification of the presence of probiotic bacteria in a cosmetic product using the polymerase chain reaction (PCR) technique. The theoretical part provides an overview of probiotics, their application in cosmetics and their effect on skin condition. Furthermore, the most commonly used molecular biological methods for the identification of probiotic organisms are described. In the practical part, the work focuses on the isolation of bacterial DNA from cosmetic products and its subsequent amplification by PCR. The amplified products were analyzed by agarose gel electrophoresis and detected by UV irradiation. The results showed the presence of bacteria of the genera Lactobacillus and Bifidobacterium, but the presence of bacteria of the genus Lactococcus, which were declared by the manufacturer together with the above genera, was not confirmed.
|
|
|
Selective isolation of the genus Bifidobacterium bacteria from foods
Mizerovská, Lucie ; Šárka, Havlíková (referee) ; Rittich, Bohuslav (advisor)
Probiotic lactic acid bacteria (LAB) are very often used in food procesing industry, such as milk products, cheese and fermentsd salami production in nova days. In diploma thesis were tested symbiotic food supplements from different producers. Bacterial DNA was isolated from crude cell lysates of six food suplements by magnetic particles P(HEMA-co-GMA). PCR-ready DNAs were isolated. from all products The detection of Bifidobacterium bacteria identified by PCR was in agreement with those declared by the manufacturers. Magnetic particles with immobilized antibodies against Bifidobacterium were used in the next part of thesis. These particles were used for the isolation of target cells from two products with cell identification by genus specific PCR.
|
|
|
Study of genome of Metschnikowia yeasts by molecular methods
Schneiderwindová, Nicole ; Skoumalová, Petra (referee) ; Němcová, Andrea (advisor)
Yeasts of the genus Metschnikowia belonging to the family Metschnikowiacea are yeasts characterized by vegetative propagation through multilateral budding. These are yeasts widely distributed in nature. More than 35 species occurring have been defined in the wild. They most often occur on flowers, fruits, but also on insects or human skin. They have a wide range of uses due to their antifungal effects in agriculture and the cosmetics industry. This bachelor thesis deals with the study of usage of molecular methods to characterize selected species of yeasts of the genus Metschnikowia. It focuses on a detailed description of the yeast cell structure, karyotype and methods of reproduction in the theoretical part of the work. In the practical part on optimization and description of molecular methods including pulse gel electrophoresis methods used to separate the yeast genome and their subsequent observation of changes in individual parts of genome. First, the yeast was cultured under special conditions that are characteristic of Metschnikowia yeasts, then yeast DNA was isolated using methods suitable for DNA isolation, which was further examined by the PFGE molecular method. The DNA isolation procedure was first optimized for individual yeast strains, as it was necessary to verify the required ratio of low melting agarose to isolated DNA. That was because of it was important for the resulting gel blocks to be suitable for measurement by PFGE analysis. By optimizing the method was possible to create ideal blocks of isolated yeast DNA, which were subsequently subjected to PFGE analysis. Several measurements of PFGE analysis were performed at different time intervals in order to separate small and large yeast chromosomes. The CHEF standard of the yeast Hansenula wingei and the standard of the yeast Schizosaccharomyces pombe were used for the measurements. According to the measurement results, it can be determined that the yeast DNA isolation procedure and subsequent analysis by pulsed gel electrophoresis were successful, as the number of chromosomes of all used yeast species of the genus Metschnikowia was determined.
|
|
|
Plasmide DNA isolation from bacteria and transfection to HEK293 cell line
Měsíčková, Klára ; Fohlerová, Zdenka (referee) ; Svoboda, Ondřej (advisor)
The isolation of plasmid DNA is an important and often used method in microbiology. The isolation itself is preceded by preparation of bacterial competent cells and by amplification of the plasmids. In this stage, plasmids CHR2, ASAP1, ASAP-3, ASAP-5 and Kir2.1. are first amplified in E.Coli bacteria of the DH5 strain and then isolated through the method of phenol-chloroform extraction. Gel electrophoresis and transfection to cellular line HEK293 are used for determining the correctness of the isolation.
|
|
|
Authenticity of natural plant component in cosmetics products
Kubalová, Michaela ; Fialová, Lenka (referee) ; Němcová, Andrea (advisor)
The purpose of this thesis was to study the authenticity of selected natural ingredients in cosmetic products. These were specifically cosmetic products that contained citruses, mint or lavender. Commercially available isolation kits were used for DNA isolation. The presence of plant origin DNA was verified by PCR method using primers specific for the ITS2 region of plants. The presence of limonene, a significant allergen contained in said plants, was determined in the samples by PCR method using primers for limonene synthase. At the same time, its presence was verified by HPLC method. In addition, two primers were tested for lavender and monitored for their efficacy, with no significant difference in the usage.
|
|
|
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.
|
guest ::
