Abstract

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.

Department

Chemistry Department

Degree Name

MS in Chemistry

Graduation Date

2-1-2015

Submission Date

July 2015

First Advisor

Azzazy, Hassan M. E.

Committee Member 1

Mostafa, Ahmed

Committee Member 2

Abdel-Ghaffar, Abdel-Rahman B.

Extent

99 p.

Document Type

Master's Thesis

Library of Congress Subject Heading 1

Polymerase chain reaction.

Library of Congress Subject Heading 2

Bacteria.

Rights

The author retains all rights with regard to copyright. The author certifies that written permission from the owner(s) of third-party copyrighted matter included in the thesis, dissertation, paper, or record of study has been obtained. The author further certifies that IRB approval has been obtained for this thesis, or that IRB approval is not necessary for this thesis. Insofar as this thesis, dissertation, paper, or record of study is an educational record as defined in the Family Educational Rights and Privacy Act (FERPA) (20 USC 1232g), the author has granted consent to disclosure of it to anyone who requests a copy.

Institutional Review Board (IRB) Approval

Not necessary for this item

Comments

I owe my deepest gratitude to my advisor, Dr. Hassan Azzazy. I’d like to thank him for the opportunities he gave me, the doors he opened for me, the important roles he played in many turning points in my life, and for helping me making critical decisions and taking big steps in my academic and professional careers. I’d like to acknowledge the support the management of Cairo Concentrate Plant - Atlantic Industries has given to me. I’d like to specially thank my direct manager, Amr Mahgoub, for the interest and flexibility he has shown during the course of my enrollment in the chemistry graduate program. I’d like to share the credit of my work with my dear friend and colleague at the Novel Diagnostics and Therapeutics Research Group (founded and directed by Dr. Azzazy) at AUC, Mai Mansour. She has always been there, and this work would never have been possible without her encouragement, guidance, and valuable advices. A special thanks to the rest of my “teammates” at the Novel Diagnostics and Therapeutics Research Group for their help and encouragement. It’s an honor and a great pleasure being part of this team. Finally, I’d like to thank my dear family for the continuous support they give me in every possible way.

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