Abstract

Tuberculosis (TB) is still one of the most significant causes of morbidity and mortality worldwide. According to WHO, TB causes 2 million deaths and more than 9 million new cases annually; the overwhelming majority of TB cases occur in developing countries where accurate diagnosis of TB remains a challenge. This work aims to develop a rapid nano-gold assay for specific detection of mycobacterium tuberculosis complex (MTBC). In the first version of the assay, DNA was extracted from clinical isolates grown on LJ media. 16s rDNA regions were amplified by PCR then the genus and species of MTB were confirmed by semi-nested PCR. Spherical gold nanoparticles (AuNPs; 13 nm) were synthesized by citrate reduction method of HAuCl4 and characterized by spectrophotometry and SEM. In the first assay, the 16srDNA amplicons were denatured (95 oC, 30 s) then allowed to anneal (48 oC, 30 s) with genus- and species-specific oligotargeters in a hybridization buffer contaning NaCl (40 nM). This was followed by the addition of unmodified AuNPs (14 nM). In case of a positive specimen, the AuNPs aggregated and the solution color changed from red to blue. The solution retained red color in case of negative specimen. This assay was further optimized to specificially differentiate MTBC from other mycobacterial strains. In the second version of the assay, MTBC was directly detected in the extracted genomic DNA. Species-specific oligotargeter was added to genomic DNA and denatured for 3 min at 95 oC followed by annealing at 48 oC for 1 min. AuNPs were added and solution color changed from red to blue in case of MTBC-positive specimens. The assay detection limit was 1 ng for PCR product and 40 ng for genomic DNA. The assay showed 100% sensitivity and specificity (n = 27) as compared with automated liquid culture system (MGIT) and semi-nested PCR. Following DNA extraction according to standard procedures, the assay turnaround time is about 1 hour. In conclusion, we have developed a nano-gold assay prototype for direct detection of MTBC as a low cost alternative to current amplification-based detection platforms. The developed assay is simple, sensitive, rapid, and shows a great potential in the clinical diagnosis of TB especially in developing countries with low resource settings.

Department

Biotechnology Program

Degree Name

MS in Biotechnology

Graduation Date

2-1-2011

Submission Date

February 2012

First Advisor

Azzazy, Hassan

Extent

NA

Document Type

Master's Thesis

Library of Congress Subject Heading 1

Tuberculosis.

Library of Congress Subject Heading 2

Mycobacteria.

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 would like first to express my deepest and sincere gratitude to my advisor Prof. Hassan M. E. Azzazy (Professor at Chemistry department, School of Science and Engineering, The American University in Cairo) for his continuous support, help, and motivation through my Biotechnology Master Thesis study and research years. I profoundly appreciate his valuable and instructive comments during the process of writing. This thesis would not have been possible without his supervision and guidance throughout the whole practical and research work done. It is an honor for me to have him as an advisor. My sincere thanks also go to my Biotechnology (AUC), Chemistry (AUC), and Microbiology (MUST) departments' colleagues who kept inspiring me through my academic years. Besides, I would like to express my sincere gratitude to Tamer Samir for his patience and assistance. Last but not the least, I would like to thank my family who where always there for me. Filly, I would like to acknowledge Misr University for Science and Technology (MUST), Faculty of Pharmacy, Microbiology department and the U.S. val Medical Research Unit No.3 (MRU-3) for supporting us with the mycobacteria strains' D and their collaborative support.

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