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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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Identification of probiotic bacteria in complex food matrices and dietary supplements
Cesnak, Filip ; Konečná, Jana (referee) ; Trachtová, Štěpánka (advisor)
Theoretical part of the thesis deals with the general characteristics of probiotic bacteria, their effect on human health and with technological demands imposed on them. Furthermore the thesis deals with actual methods of bacterial identification, especially on the base of polymerase chain reaction. Presence of the declared probiotic bacteria was analysed with the polymerase chain reaction (PCR) and the DNA was isolated from the sample of food additive with the use of magnetic particles.
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Valuation of works in the land consolidation
Naňka, Martin ; Konečná, Jana (referee) ; Tichá, Alena (advisor)
The aim is to comprehensively describe the problems of land consolidation plans and prepare budgets of joint facilities performed in a frame of the landscaping. For individual plans of common facilities, cards of budgetary indicators will be processed. Finally, we will evaluate the resulting processed budgets and budget indicators.
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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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Magteic particles and their applications in biotechnology
Knápková, Monika ; Konečná, Jana (referee) ; Trachtová, Štěpánka (advisor)
The thesis is focused on the magnetic particles which are used in several biotechnological applications. The theoretical part deals with the specific properties of these nanoparticles and materials of which the nanoparticles can be made. There are also mentioned some of the biotechnological applications of magnetic particles. During the experimental part, selected types of magnetic particles were used to isolate nucleic acid. The quality of the isolated DNA with respect to purity was evaluated using the polymerase chain reaction and its modifications. High resolution analysis (HRM analysis) was also used to verify the quality of the isolated DNA and to resolution Lactobacillus casei and Lactobacillus rhamnosus. DNA isolation using magnetic carriers was successful. Commercially available MPG microcarriers and magnetic microparticles Fkol 77ox were the most suitable. In terms of purity magnetic nanoparticles F79/L3-PLL were the most suitable for the DNA isolation. The resolution of bacterial strains of Lactobacillus casei and Lactobacillus rhamnosus was not successful.
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Nurses' duty of conifidentiality and the consequences of its breach
Benešová, Pavla ; Prošková, Eva (advisor) ; Konečná, Jana (referee)
The aim of my thesis was to find out what is the legal awareness of nurses in the issue of confidentiality. Furthermore, it was examined whether nurses know what are the consequences for unauthorized access to the medical records. The theoretical part of this thesis describes the mandatory confidentiality issue and its consequences. And it defines the concepts of personal and sensitive personal data. In addition the conditions under which it is required to break confidentiality and the consequences of breach of legal obligation are mentioned. Describe the legal responsibility of nurses. In the practical part of the work I use qualitative methods of data collection, interview, for me to respond to major and minor research questions. I use interviews conducted with nurses, for doing analysis of the case and to describe the measures taken by the employer at the Municipal Hospital in Litoměřice.
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