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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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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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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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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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