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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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Isolation of DNA from probitic products using solid carriers
Bonczek, Ondřej ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Microbial DNA was isolated from lysed cells of Lactobacillus genus in probiotic products. Reversible adsorption DNA on the surface of carboxyl coated nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) magnetic particles and silicagel coated manganase Perovskite nanoparticles. DNA was adsorbed on the surface of the particles in the presence of 16 % poly(ethylenglycol) (PEG 6000) and 2 M sodium chloride (NaCl) concentrations. The adsorbed DNA was released from particles by low ionic strength TE buffer (pH= 8.0). The quality of isolated DNA was checked by spectrofotometric measurement and PCR amplification. DNA samples isolated using magnetic particles and phenol extraction method (control method) were PCR-ready. The DNA isolated from lysed cells of probiotic products was quantificated in real-time qPCR.
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Use of PCR for species identification and searching of selected genes of lactobacilli
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic food products - food additives contain different species of probiotic bacteria. Accurate species identification with their characteristics is very important from the view of products quality. Methods of DNA diagnostics are used for these purposes. In this thesis DNA was isolated from 4 probiotic products. The presence of bacterial of genus Lactobacillus and species L. acidophilus, L. casei, L. plantarum, L. rhamnosus were detected in three products by PCR. This information was in accordance with the data provided by the manufacturer. Two sets of primers were used for identification of species. Using other primers sequences of genes such as bsh, lai and odc were detected in DNA isolated from the products. Differences were estimated among products concerning the detection of lai gene Lactobacillus acidophilus.
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Analysis of DNA isolated from probiotic products using magnetic microparticles
Oliva, Jan ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
This thesis is interested in isolation and identification of probiotic bacteria in three different probiotic products using polymerase chain reaction (PCR). DNA in quality suitable for PCR was isolated from crude lysates using three different types of magnetic microparticles and phenol extraction. Identification genera and species of probiotic bacteria was proven using genus and species specific PCRs. Results were in accordance with data presented by manufacturers.
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|
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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.
|
|
|
Use of PCR for species identification and searching of selected genes of lactobacilli
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic food products - food additives contain different species of probiotic bacteria. Accurate species identification with their characteristics is very important from the view of products quality. Methods of DNA diagnostics are used for these purposes. In this thesis DNA was isolated from 4 probiotic products. The presence of bacterial of genus Lactobacillus and species L. acidophilus, L. casei, L. plantarum, L. rhamnosus were detected in three products by PCR. This information was in accordance with the data provided by the manufacturer. Two sets of primers were used for identification of species. Using other primers sequences of genes such as bsh, lai and odc were detected in DNA isolated from the products. Differences were estimated among products concerning the detection of lai gene Lactobacillus acidophilus.
|
|
|
Analysis of DNA isolated from probiotic products using magnetic microparticles
Oliva, Jan ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
This thesis is interested in isolation and identification of probiotic bacteria in three different probiotic products using polymerase chain reaction (PCR). DNA in quality suitable for PCR was isolated from crude lysates using three different types of magnetic microparticles and phenol extraction. Identification genera and species of probiotic bacteria was proven using genus and species specific PCRs. Results were in accordance with data presented by manufacturers.
|
|
|
Isolation of DNA from probitic products using solid carriers
Bonczek, Ondřej ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Microbial DNA was isolated from lysed cells of Lactobacillus genus in probiotic products. Reversible adsorption DNA on the surface of carboxyl coated nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) magnetic particles and silicagel coated manganase Perovskite nanoparticles. DNA was adsorbed on the surface of the particles in the presence of 16 % poly(ethylenglycol) (PEG 6000) and 2 M sodium chloride (NaCl) concentrations. The adsorbed DNA was released from particles by low ionic strength TE buffer (pH= 8.0). The quality of isolated DNA was checked by spectrofotometric measurement and PCR amplification. DNA samples isolated using magnetic particles and phenol extraction method (control method) were PCR-ready. The DNA isolated from lysed cells of probiotic products was quantificated in real-time qPCR.
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