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Testing of primers for real-time PCR-HRM analysis of fruit products containing one fruit species
Boháčová, Barbora ; Dzurendová, Simona (referee) ; Fialová, Lenka (advisor)
Determining the authenticity of fruit products, which are often counterfeited by substituting part of a more expensive fruit with a cheaper but botanically similar fruit, is a current topic in the food industry. A prime example is the dilution of apricot products with peach puree. This study focuses on testing specific primers AGS18 and PdCass to reveal the true proportion of apricot content in model products mixed with peach puree using PCR analysis followed by HRM analysis. Testing of these primers for authenticity determination revealed limited utility of AGS18 primers due to the formation of small amount or no specific products during reactions with fruit DNA. PdCass primers required PCR condition optimization, but subsequently, specific DNA sequences from fruit leaves could be amplified in sufficient quantities. PCR analysis of DNA from model products with PdCass primers provided specific products in samples with apricot content of 70% or less. HRM analysis of samples and calculation of GCP did not distinguish purees with 0%, 10%, and 30% apricot content. However, purees with 50% and 70% apricot content exhibited significantly different melting curves compared to other samples.
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Development of methods for genetic analysis of plant foods
Fialová, Lenka ; Brázda, Václav (referee) ; Doškař, Jiří (referee) ; Márová, Ivana (advisor)
Multiplex real-time PCR-HRM is an approach which has gained some attention in recent years. It has already found applications in clinical diagnostics and food authenticity and safety control. Compared to its corresponding singleplex PCR assays, an optimized multiplex PCR assay provides the same information in a fraction of time. First part of this work dealt with isolation of DNA from both fresh fruits and processed commercial products. Six different DNA isolation protocols were tested with fresh fruits – three silica column-based kits, two magnetic carrier-based kits and one conventional protocol. One method was chosen as the most suitable and was applied to DNA isolation from commercial products. These experiments also involved optimisation of the chosen method. The second part of this work was focused on the development of a triplex real-time PCR assay for simultaneous detection of blueberry, strawberry and raspberry, and its application on DNA isolated from commercial products. During DNA isolation, calcium chloride was shown to be a promising agent for removal of pectin from samples. In several samples, presence of raspberry DNA was confirmed by singleplex PCR. We found out that for accurate results of food analysis by this assay, further optimization of its parameters would be needed.
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