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Construction of a Molecular Assay for Detection of Drug Resistant Tuberculosis Using Improved Linear-After-The-Exponential PCR

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Construction of a Molecular Assay for Detection of Drug Resistant Tuberculosis Using Improved Linear-After-The-Exponential PCR Construction of A Molecular Assay for Detection of Drug Resistant Tuberculosis Using Improved Linear-After-The-Exponential PCR Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biology Lawrence J. Wangh, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology by Ruth-Love Damoah February 2017 Copyright Ruth-Love Damoah © 2017 Acknowledgements I would like to thank God for leading me to this great opportunity and for giving me the strength to do this work. I am eternally grateful to Professor Lawrence Wangh for giving me this project and for guiding me through it. I am thankful that he also provided both the financial and moral support to make this possible and for all the great thoughts and encouragement. I am also grateful to John Rice for working with me to complete this project successfully. I am grateful for all the time spent analyzing data and contemplating solutions. I am also grateful to Aquiles J. Sanchez for taking time to explain difficult concepts from the very beginning, and also for constant support. I am grateful also to Kenneth Pierce, Adam Osborne and the rest of the Wangh lab for their constant support and for being very accommodating. I am truly grateful for all the help I received throughout this project Brandeis biology department and from friends and family. It is because of you all that I made it this far. Thank you all for teaching me that impossible just means no one has found a way to do it just yet. I truly appreciate you all. God bless you. iii Abstract Construction of A Molecular Assay for Detection of Drug Resistant Tuberculosis Using Improved Linear-After-The-Exponential PCR A thesis presented to the Department of Biology Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Ruth-Love Damoah Tuberculosis continues to plague mankind about three centuries after the first epidemic in modern history in the 17th-18th century. In spite of the advances made in understanding the disease and in diagnostics and treatment options since then, eradicating TB is proving difficult. Cases of antibiotic resistance are complicating both the diagnostic and treatment processes of the disease. A cheap, reliable, sensitive assay using Linear-After-The-Exponential PCR (LATE-PCR was invented in the Wangh laboratory) has been developed to meet the Point-Of-Care (POC) diagnosis needed to effectively combat TB. The nature of the assay and the method of amplification make it applicable to all sample types using less costly technology. Using this Assay, as few as five, rpoB gene copies from Mycobacterium tuberculosis can be selectively amplified in samples containing as many as 58,000 copies of human genomic DNA; PCR efficiency was 105.58%. In addition, the Assay can correctly distinguish thirty-five out of thirty-eight different mutations that are known to cause resistance to rifampicin and all thirty-eight strains are can be distinguished from wild-type drug sensitive rpoB gene sequences using Lights-Off Only Probe technology (also invented in the Wangh Laboratory). Although no mutant strain was scored as wild type, not all mutations could be distinguished from each iv other. Thus, the resolving power of Lights-Off Only Probes will have to be enhanced if it is clinically important to uniquely distinguish all types of mutations. v Table of Contents List of Tables ..................................................................................................................................... ix List of Figures ..................................................................................................................................... x Overview ........................................................................................................................................... 1 In the Beginning ................................................................................................................................. 4 About Mycobacterium tuberculosis: ......................................................................................................... 4 History .................................................................................................................................................. 4 Ending the First Epidemic ..................................................................................................................... 5 The Recent Epidemic ................................................................................................................................. 6 The Horror Today ................................................................................................................................. 6 Back with a Vengeance ........................................................................................................................ 8 The Mycobacterium tuberculosis Complex (MTBC) ............................................................................ 10 Basic Morphology ................................................................................................................................... 10 The Capsule ........................................................................................................................................ 10 Complex cell wall ................................................................................................................................ 11 Long Generation Time ........................................................................................................................ 11 Genetic component and adaptability................................................................................................. 12 Coevolution ..................................................................................................................................... 14 The Theory of the Coevolution of TB and Host ....................................................................................... 14 Cell Wall Specialization....................................................................................................................... 15 Host Related Adaptations .................................................................................................................. 16 Major Means of Evolution .................................................................................................................. 16 Why TB Has Not Been Eradicated ........................................................................................................... 17 Tuberculosis as a Disease ................................................................................................................. 18 Disease Cycle........................................................................................................................................... 18 Immunobiology ............................................................................................................................... 20 Cellular Response .................................................................................................................................... 20 Involved Immune Cells and Killing Mechanisms ..................................................................................... 20 Survival Strategy ..................................................................................................................................... 21 Pathogenicity and Virulence ................................................................................................................... 22 Treatment of TB ............................................................................................................................... 25 Available and Approved Drugs ............................................................................................................... 25 General Guidelines .................................................................................................................................. 26 Drug Susceptibility and Resistance ......................................................................................................... 27 New Drugs .............................................................................................................................................. 27 Beyond Drug Therapy ............................................................................................................................. 28 TB Diagnosis .................................................................................................................................... 29 History ..................................................................................................................................................... 29 Recent Introduction of Molecular Diagnostics ....................................................................................... 29 What Has Not Been Achieved ................................................................................................................. 30 Aim of This Research ........................................................................................................................ 33 Gene Targets ........................................................................................................................................... 34 RNA polymerase Subunit Beta (rpoB) and Region of Difference 9 (rd9)............................................ 34 Sample
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