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Use of Molecular Biology Techniques for Identification and Analysis of Probiotic Bacteria
Konečná, Jana ; Doškař, Jiří (referee) ; Kráčmar, Stanislav (referee) ; Obruča, Stanislav (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. Alternative method of DNA isolation is use of commercially available kits which, however, are expensive and their efficiency is low. 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 solid-phase DNA extraction. Magnetic microparticles P(HEMA – co – GMA) containing –NH2 group and nanoparticles PLL, whitch contains polylysine. 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 on the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. After optimalization, the developed method was used for DNA isolation from real food supplements. 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 thesis, 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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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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Sample preparation for DNA analysis from foods of plant origin
Silná, Renata ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of high quality DNA is nessecary for many molecular biology applications. However, plant DNA contains high amonts of polysaccharides, polyphenols and various secondary metabolites, which decrease yield and quality of isolated DNA. The aim of this study was preparation of samples and different food matrices for DNA isolation DNA by magnetic particles. It was about 5 species of vegetable and 10 species of processed plant food. Homogenization of samples was performed in CTAB buffer. Isolation of plant DNA was performed by magnetic particles covered with carboxyl groups. All DNAs were isolated in conventional PCR qualities using primers for 700 bp amplicons, in the case of heat processed products for 220 bp ampilicons and for real time PCR. The efficiancy of separation of magnetic particles with DNA by magnetic separator and magnetic needle was compared. It was find out that DNA of higher purity was isolated using magnetic needle. The micromethod of isolation of plant DNA from homogenates with CTAB with magnetic particles is suitable for different processed food.
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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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Methods of molecular cytogenetics: evolution, analysis, comparison
Budková, Kateřina ; Šolc, Roman (advisor) ; Daňková, Pavlína (referee)
Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of chromosomes, especially during the cell division. Chromosomal abnormalities that may occur during this process are often associated with the emergence of various diseases. Cytogenetic methods are primarily used to detect these abnormalities. Traditional methods allow resolution at the level of whole chromosomes, the introduction of new molecular methods led to a significant refinement of these analyzes. Today's methods allow easy analysis of chromosomes to the level of individual sequences. Despite significant differences between classical and modern cytogenetic techniques, classical cytogenetic methods are not evil, but they are still widely used. Combining classical and molecular methods in order to obtain a comprehensive analysis of chromosomal rearrangements is no exception.
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Molecular genetic diagnosis of cystic fibrosis
Matějčková, Iva ; Krkavcová, Miroslava (advisor) ; Kotlas, Jaroslav (referee)
Cystic fibrosis is a very common and serious disease. It is an autosomal recessive disease with an incidence of 1 : 2500 - 1 : 4500. With this disease, CTFR gene mutations occur which are responsible for the failure of chloride channels. It is a multi-organ handicap, which primarily infects the respiratory and gastrointestinal system, but also affects the hepatobiliary system, reproductive system, and sweat glands. Cystic fibrosis can be diagnosed by a sweat test, molecular genetic diagnosis, or more rarely, using transepitel potential differences, which has not been introduced in the Czech Republic yet. Since 2009, the Czech Republic applies neonatal screening CF, which helps make a faster diagnosis to then be able to proceed to the rapid introduction of appropriate treatment. The introduction of an early treatment has significantly improved the prognosis of the patient. Currently CF patients can live on average from thirty to forty years. For the CFTR gene genetic testing is used postnatal, prenatal or preimplantation material. From these samples, DNA gets isolated and is further investigated using genetic methods, which may be based on the principle of PCR, reverse hybridization, restriction analysis, DNA sequencing or MLPA. Powered by TCPDF (www.tcpdf.org)
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Cytomegalovirus infection in transplant patients
Dvořák, Jan ; Tachezy, Ruth (advisor) ; Harant, Karel (referee)
Cytomegalovirus (HCMV) is a ubiquitous human -herpesvirus highly prevalent in the population. HCMV is transmitted by close contact between individuals. In infected person this virus causes mainly asymptomatic primary infection, after which the latency is established. In pregnant women HCMV infection can lead to abortions, defects of the fetus and congenital abnormalities of newborn babies. Even more serious complications are caused by this virus in the immunocompromised patients, especially those infected by HIV and in patients who undergo solid organ transplantation and hematopoietic stem cell transplantation. This work is a complex report about HCMV biology with emphasis on complications which HCMV causes in patients after solid organ transplantation and hematopoietic stem cell transplantation. This article also contains summary of the methods used for diagnostic of HCMV infection and monitoring and prevention of HCMV associated diseases. Keywords: Cytomegalovirus, hematopoietic stem cell transplantation, solid organ transplantation, detection, monitoring, polymerase chain reaction, cellular immunity, humoral immunity
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The application of magnetic particle for DNA isolation from selekted probiotic products for children
Vozárová, Petra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
In the food industry, it is important to correctly identify the species of bacteria and thier properties so that they can be used as a probiotic in dietary supplements. This is performed using DNA diagnostics. In the experimental part, the DNA from four probiotic dietary supplements for children was isolated. Magnetic particles P(HEMA-co-GMA) were tested for isolation. Isolated DNA was amplified by PCR and the presence of DNA of genus Lactobacillus, Bifidobacterium and Bacillus was demonstrated in the products according to the data declared by the manufacturer. The presence of species L.acidophilus, B.animalis in accordance with the data on the product has been demonstrated by PCR with species specific primers. Using PCR, the presence of L.casei, which was declared by the manufacturer, has not been proven in one product at given experimental conditions.
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Laboratory diagnostics of micrometastases in breast cancer patients
Mikulová, Veronika ; Zima, Tomáš (advisor) ; Svoboda, Marek (referee) ; Průša, Richard (referee)
Introduction: The presence of circulating tumor cells (CTC) in the peripheral blood has been associated with worse prognosis and early relapse in breast cancer patients. CTC determination in the peripheral blood has been considered as a liquid biopsy. The aim of this project was to analyze the presence of CTC followed by their molecular characterization with the potential use not only as a new biomarker for real-time monitoring of therapy efficacy but also as a suitable tool for patient's stratification and individualization of treatment for breast cancer. Methods: A total of 54 patients with diagnosed early breast cancer were enrolled into a prospective study. Ten millilitres of peripheral blood were sequentially collected to test for the presence and characterization of CTC during the follow-up of patients. CTC isolation and detection was performed by AdnaTest BreastCancer™ (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC- lysates. cDNA from isolated CTC has been further used for newly optimized qPCR assays for breast tumor and therapy resistance associated genes: TOP1, TOP2A, CSTD, ST6GAL, KRT19 and reference gene actin. qPCR results have been analyzed by Genex software (MultiD Analysis). Results: 195 blood samples have been...
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