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Optimization of DNA isolation form yogurt cultures and their detection by RT-PCR
Šurková, Alice ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
The thesis has optimized DNA isolation from pure yoghurt cultures and yoghurt products. The isolated DNA was than subjected to RT-PCR analysis. In the first part of the thesis, DNA isolation from pure yoghurt cultures using a commercial kit was evaluated as more effective than isolation by phenol extraction and magnetic microparticles. To assess the quality and quantity of DNA obtained the spectrophotometric determination of concentration and purity and qPCR were used. DNA of a total of ten pure yoghurt cultures in a quality suitable for PCR was obtained using the commercial kit. In the second part of the thesis, bacterial DNA was isolated from yoghurt products using the same commercial kit with a previous sample washing by lysation solution. DNA of six yoghurt products was isolated this way. Furthermore, two packages of homemade yoghurt were mad of each product, of which DNA was isolated in the same way. DNA obtained from yoghurts was subjected to RT-PCR using six pairs of primers (V3_F a V3_R, V6_F a V6_R, V1_F a V1_R, GroHRM_F a GroHRM_R, UPF a UPR, P1V1 a P2V1) and using the pure cultures DNA as a positive controls. The results confirmed the presence of cultures declared in each yoghurt and their ability to multiply after inoculation into a new medium (milk).
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Optimization of DNA isolation form yogurt cultures and their detection by RT-PCR
Šurková, Alice ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
The thesis has optimized DNA isolation from pure yoghurt cultures and yoghurt products. The isolated DNA was than subjected to RT-PCR analysis. In the first part of the thesis, DNA isolation from pure yoghurt cultures using a commercial kit was evaluated as more effective than isolation by phenol extraction and magnetic microparticles. To assess the quality and quantity of DNA obtained the spectrophotometric determination of concentration and purity and qPCR were used. DNA of a total of ten pure yoghurt cultures in a quality suitable for PCR was obtained using the commercial kit. In the second part of the thesis, bacterial DNA was isolated from yoghurt products using the same commercial kit with a previous sample washing by lysation solution. DNA of six yoghurt products was isolated this way. Furthermore, two packages of homemade yoghurt were mad of each product, of which DNA was isolated in the same way. DNA obtained from yoghurts was subjected to RT-PCR using six pairs of primers (V3_F a V3_R, V6_F a V6_R, V1_F a V1_R, GroHRM_F a GroHRM_R, UPF a UPR, P1V1 a P2V1) and using the pure cultures DNA as a positive controls. The results confirmed the presence of cultures declared in each yoghurt and their ability to multiply after inoculation into a new medium (milk).
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