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Development of real-time PCR assay for detection of total bacteria in beverage emulsions
The rapid growth and expansion of the soft drinks market and the necessity to meet and maintain the consumers’ expectations of having high quality products safe for consumption, have both drawn the attention to the need for rapid and sensitive methods for the detection of potential microbial contami...
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| Format: | Thesis |
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AUC Knowledge Fountain
2015
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| Summary: | The rapid growth and expansion of the soft drinks market and the necessity to meet and maintain the consumers’ expectations of having high quality products safe for consumption, have both drawn the attention to the need for rapid and sensitive methods for the detection of potential microbial contaminations. This has made the current conventional culture-based methods inconvenient due to the relatively long periods of time they need to yield results, in addition to their relatively low sensitivity. In contrast, real-time PCR is a rapid and sensitive molecular detection technique, capable of providing quick detection and quantification methods of specific DNA sequences even if the quantity of the starting material is small. In this study, a real-time PCR assay for the determination of total bacteria in one of the microbiologically sensitive constituents of soft drinks, called beverage emulsions, was successfully developed. This included the development of a DNA extraction protocol and the selection of a set of universal primers targeting a conserved region in the 16S rDNA of bacteria. The quantification strategy was based on a standard curve and a calculation method for the conversion of the determined DNA concentrations to bacterial cells numbers. This enabled the sensitive determination of total bacteria in beverage emulsions in the range between 10 fg/µL and 100 ng/µL, corresponding to 2 and 2 x 107 cells of Escherichia coli, respectively, in 6 to 8 hours instead of 7 days required by the pour plate method. Further optimization of the developed assay may allow the determination of viable bacterial cells, which will extend the scope of the developed assay applications in this industry, in the future. |
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