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The influence of polysaccharide contamination on molecular-biology analysis
Žylková, Kateřina ; Strečanská, Paulína (referee) ; Fialová, Lenka (advisor)
The presence of polysaccharides in DNA isolation and its subsequent analysis often leads to undesirable effects. Polysaccharides together with other metabolites (phenolics, proteins) can act as inhibitors of PCR. In this work, the effect of polysaccharide contamination on the analyzed DNA was investigated. In the experimental part, DNA samples were isolated from two exotic fruits (mango, banana), from which the concentration of polysaccharides was then determined. The analysis showed that by adding CaCl2, the polysaccharide content of the samples was significantly lower. After checking the amplification of the DNA samples with added CaCl2, it was found that CaCl2 itself inhibited PCR and therefore had to be removed from the samples. After purification, the amplification of the DNA was reverified and it was found that the DNA with CaCl2 after purification gave the best results. These results were further verified by agarose gel electrophoresis, which confirmed that a reduction in the polysaccharide content of the samples helped DNA amplification. It was also observed that it depends on the type of polysaccharides present in the source plant material. Banana DNA showed better amplification results than mango DNA. This is due to the different chemical composition of these fruits. Banana, unlike mango, does not contain polysaccharides that would significantly contaminate the isolated DNA.
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Use of advanced molecular biology techniques for detailed characterization of probiotic bacteria from a dietary supplement
Folwarczná, Tereza ; Trachtová, Štěpánka (referee) ; Smetana, Jan (advisor)
The aim of the diploma thesis was the identification of probiotic bacteria from a probiotic food supplement in the form of syrup. DNA was isolated from the complex matrix of the product in sufficient purity and quality suitable for the real-time PCR method followed by PCR-HRM. Specific primers for the genera Lactobacillus, Bifidobacterium and Streptococcus and at the species level for the species Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis and Streptococcus thermophilus were used for amplification by real-time PCR and PCR-HRM. After optimizing the conditions for specific primers, all declared bacteria were detected in the product.
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The utilization of the uracil-DNA glycosylase in the elimination of carry-over contamination by products of the previous DNA amplifications
Zrnová, Adéla ; Martínková, Markéta (advisor) ; Prošková, Veronika (referee)
DNA amplification is an important tool in diagnosis of infectious diseases. The most commonly used method in laboratories is the polymerase chain reaction, which, does not meet the need for a fast and inexpensive method. It was therefore necessary to develop a method that would be sensitive, fast, specific and accessible, for example, in developing countries with limited access to instrumentation. It appears that LAMP could be this method. Unfortunately, the LAMP method encounters considerable false positivity, which must be prevented in order to be used in practice. The subject of the presented bachelor thesis is the introduction of a unique LAMP method to detect the presence of DNA Streptococcus pneumoniae and the study of the use of uracil- DNA glycosylase to prevent contamination of samples with DNA formed in previous amplification reactions. The LAMP method of DNA detection Streptococcus pneumoniae was successfully introduced, then the method was optimized even in the presence of deoxyuridine triphosphate. After determining the appropriate deoxyuridine triphosphate concentration for LAMP amplification, the resulting Streptococcus pneumoniae DNA amplification products were treated with uracil-DNA glycosylase. This procedure was shown to have the potential to prevent false positivity of other...
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Comparison of different molecular-biological approaches for detecting the presence of DNA of the pathogenic bacterium Haemophilus influenzae
Smrčka, Tomáš ; Martínková, Markéta (advisor) ; Dračínská, Helena (referee)
Haemophilus influenzae is one of the main initiators of meningitis and pneumonia in children. Implementation of fast, cheap and instrumentally accessible method for detection of this pathogen would enable an early and targeted treatment of patients. The development of amplification methods in the last decades enables, apart from commonly used PCR method, application of alternative approaches, such as the LAMP. The focus of this Bachelor thesis was the study (research) of the alternative method LAMP as a tool for detection of Haemophilus influenzae. The LAMP method was successfully implemented for Haemophilus influenzae, however, it has contended with the false positive results of negative control in case of longer incubation times. Therefore, the optimized LAMP method was designed in presence of deoxyuridine triphosphate and uracil-DNA glycosylase. Its aim was to change the structure of LAMP products via the incorporation of uracils to amplified regions of DNA and subsequent removal of uracils with influence of uracil-DNA glycosylase, and therefore prevent their replication during potential contamination of reaction mixtures and consequently reduce the risk of false positive results of negative controls to minimum. The concentration of deoxyuridine triphosphate in reaction mixtures was optimized...
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Use of high resolution melting analysis for the study of lactic acid bacteria
Knápková, Monika ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
Currently, there is a growing interest in the use of probiotic products, and there are many of them in the market. With the growing interest, greater emphasis is placed on the identification of declared probiotic microorganisms. Precise identification of microbial composition is often a difficult task and it requires more advanced methods especially in the field of molecular diagnostics. The diploma thesis was focused on the verification of the presence od declared probiotic microorganisms in probiotic food supplements GS Laktobacily Forte 21, Biopron 9 Premium and Linex® Forte. DNA was isolated from the complex matrices by phenol extraction, commercial kit and magnetic carriers F79/L3-PLL in the quality suitable for PCR. Subsequently, the isolated DNA was amplified by real-time polymerase chain reaction using genus- and species-specific primers. The specific PCR product was subjected to agarose gel electrophoresis, whereas species identification was not always in compliance with the data declared by producers. The next part of the thesis was focused on polymerase chain reaction with high-resolution melting analysis to distinguish bacterial strains belonging to the Lactobacillus group and to identify probiotic microorganisms present in the complex matrices of the probiotic food supplements. Eight primer sets were tested (V1F HRM a V1R-HRM, CHAU-V3F a CHAU-V3R, CHAU-V6F a CHAU-V6R, LAC2 a LAC4, LAC1 a LAC2, P1V1 a P2V1, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT). Three primer pairs (V1F HRM a V1R-HRM, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT) were evaluated as the most suitable for distinguishing Lactobacillus bacterial strains.
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Distribution of Heterobasidion and Armillaria root rots in Vallombrosa fir forest, Italy
Dálya, László Benedek
This work intends to describe the present condition of Vallombrosa forest (Tuscany, Italy) from the phytopathological point of view. The chronic disease caused by Heterobasidion and Armillaria root rots is a key factor affecting the vitality of silver fir plantations of the region. Detailed knowledge about their distribution could help to control the pathogens. Systematic sampling and survey of damages on trees were undertaken at 52 points. Identification of different species from soil and fungal samples was accomplished by DNA-based methods (TSCP, nested PCR, RFLPs analysis). The high presence of both parasitic fungi was detected under a wide range of ecological conditions. Data analysis indicates the strong spreading potential of the pathogens even into new habitats, especially in connection with water stress of their hosts.
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Analysis of the composition of selected probiotic products by PCR-HRM
Tomanová, Barbora ; Španová, Alena (referee) ; Trachtová, Štěpánka (advisor)
This work was focused on the detection of probiotic bacteria in four different probiotic products (probiotic cream, probiotic tampons, oral probiotics and soy beverages with probiotics). The viability of the bacteria contained in the products was verified. Complex matrices of the products were used to isolate DNA in a quality suitable for the PCR method, followed by identification of the declared bacterial genus and species. Amplification was achieved with conventional PCR and real-time PCR, genus- and species-specific primers were used. Bacteria, of the genus Lactobacillus and Bacillus and bacterial species Lactobacillus pentosus, Lactobacillus rhamnosus, Lactobacillus fermentum and Lactobacillus gasseri, were proven to be within the products. Subsequently, the DNA from mixed bacterial species in the probiotic tampon were distinguished using PCR-HRM. Five sets of primers were used to test this. Two sets of primers (primers P1V1, P2V1 and V1F-HRM, V1R-HRM) were evaluated as the most suitable for resolution.
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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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Probiotics in food products
Silná, Renata ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Probiotics are living microorganisms with a positive effect on the consumer when they are added to food in adequate amount. The best known probiotic are lactic acid bacteria and yeast Saccaromyces cerevisiae var. boulardii. The theoretical part of the thesis is focused on using probiotics microorganisms in food. In the experimental part of the thesis were prepared crude lysates from three food products and the presence of bacterial DNA was proved by PCR method.
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