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DNA isolation and identification of nonpathogenic species of clostridia isolated from cheeses
Sedláček, Zbyněk ; Eva, Kvasničková (referee) ; Rittich, Bohuslav (advisor)
In the food industry are requested speedy and accurate methods for identification of bacteria in microbiological testing of products. Molecular diagnostic methods are based on isolation of DNA from bacterial cells which is amplified in polymerase chain reaction (PCR). The result is fragment DNA about specific size, characteristic for genus or species of bacteria. The aim of the work was isolation of PCR-ready DNA. DNA has been isolated from 8 strains of genus Clostridium. Procedure of cell lysis was optimized in order to find the optimal concentration of EDTA and proteinase K in lysing buffer. DNA was isolated by phenol extraction and using magnetic microspheres. Concentrations 10 mM of EDTA and 10 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by phenol extraction. Concentrations 10 mM of EDTA and 15 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by magnetic microspheres. Isolated DNA was checked by gel electrophoresis, quantificated by spectrophotometry and tested in PCR. Individuals species were distinguished in denaturing gradient gel electrophoresis (DGGE).
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PCR identification of selected bacteria in cheeses
Gregušová, Barbora ; Burdychová, Radka (referee) ; Španová, Alena (advisor)
This work is focused on identification and species specification of a collection of 44 clostridial strains using molecular-genetic methods. DNA of bacteria isolated from late-blowing defected cheeses was used. The purified DNA was diluted to 10 ng/µl and its ability to be amplified was verified by PCR with universal primers. By genus-specific PCR was proved that DNA of all samples belongs to Clostridium genus. Based on species-specific PCR reactions, it was determined that 7 strains belong to C. butyricum and 12 strains belong to C. tyrobutyricum. 15 strains were positively detected in both species-specific PCRs and therefore identified as mixed cultures. Another 10 strains were not classified into tested species. Strains with the gene encoding the enzyme hydrogenase hydA were searched using PCR. Specific PCR products for this gene were detected in 30 strains of the analysed collection. Especially intense were the amplicons by all strains belonging to C. butyricum species.
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Hydrogen production by the bacteria of the genus Clostridium
Filová, Dagmar ; Kvasničková, Eva (referee) ; Rittich, Bohuslav (advisor)
Bachelor thesis deals with hydrogen production using bacteria of Clostridium genus - specifically C. butyricum and C. tyrobutyricum. Theoretical part analyzes means of hydrogen production divided into biological and non - biological methods. Thesis is further aimed at practical use of these microorganisms for hydrogen production purposes and introduces cheese whey as one of utilizable growth medias. In experimental part, there was used polymerase chain reaction for identification of bacteria genus Clostridium and species Clostridium butyricum and Clostridium tyrobutyricum. PCR products were reamplified using the same primers for specifity confirmation purposes. Presence of gene producing hydrogenase A enzyme was proved by PCR, too.
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DNA isolation and identification of nonpathogenic species of clostridia isolated from cheeses
Sedláček, Zbyněk ; Eva, Kvasničková (referee) ; Rittich, Bohuslav (advisor)
In the food industry are requested speedy and accurate methods for identification of bacteria in microbiological testing of products. Molecular diagnostic methods are based on isolation of DNA from bacterial cells which is amplified in polymerase chain reaction (PCR). The result is fragment DNA about specific size, characteristic for genus or species of bacteria. The aim of the work was isolation of PCR-ready DNA. DNA has been isolated from 8 strains of genus Clostridium. Procedure of cell lysis was optimized in order to find the optimal concentration of EDTA and proteinase K in lysing buffer. DNA was isolated by phenol extraction and using magnetic microspheres. Concentrations 10 mM of EDTA and 10 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by phenol extraction. Concentrations 10 mM of EDTA and 15 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by magnetic microspheres. Isolated DNA was checked by gel electrophoresis, quantificated by spectrophotometry and tested in PCR. Individuals species were distinguished in denaturing gradient gel electrophoresis (DGGE).
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|
|
PCR identification of selected bacteria in cheeses
Gregušová, Barbora ; Burdychová, Radka (referee) ; Španová, Alena (advisor)
This work is focused on identification and species specification of a collection of 44 clostridial strains using molecular-genetic methods. DNA of bacteria isolated from late-blowing defected cheeses was used. The purified DNA was diluted to 10 ng/µl and its ability to be amplified was verified by PCR with universal primers. By genus-specific PCR was proved that DNA of all samples belongs to Clostridium genus. Based on species-specific PCR reactions, it was determined that 7 strains belong to C. butyricum and 12 strains belong to C. tyrobutyricum. 15 strains were positively detected in both species-specific PCRs and therefore identified as mixed cultures. Another 10 strains were not classified into tested species. Strains with the gene encoding the enzyme hydrogenase hydA were searched using PCR. Specific PCR products for this gene were detected in 30 strains of the analysed collection. Especially intense were the amplicons by all strains belonging to C. butyricum species.
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|
|
Hydrogen production by the bacteria of the genus Clostridium
Filová, Dagmar ; Kvasničková, Eva (referee) ; Rittich, Bohuslav (advisor)
Bachelor thesis deals with hydrogen production using bacteria of Clostridium genus - specifically C. butyricum and C. tyrobutyricum. Theoretical part analyzes means of hydrogen production divided into biological and non - biological methods. Thesis is further aimed at practical use of these microorganisms for hydrogen production purposes and introduces cheese whey as one of utilizable growth medias. In experimental part, there was used polymerase chain reaction for identification of bacteria genus Clostridium and species Clostridium butyricum and Clostridium tyrobutyricum. PCR products were reamplified using the same primers for specifity confirmation purposes. Presence of gene producing hydrogenase A enzyme was proved by PCR, too.
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