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Copyright and Use of This Thesis This Thesis Must Be Used in Accordance with the Provisions of the Copyright Act 1968 COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Copyright Service. sydney.edu.au/copyright Development and application of molecular screening test to detect genetic polymorphisms within the G6PD gene in malaria infected individuals Elias Hanania University of Sydney Student ID 500578836 A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Science (Honours) December 2020 Supervised by: Associate Professor Rogan Lee and Professor Wieland Meyer Parasitology Laboratory NSW Pathology, ICPMR, Westmead Hopsital Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology The Westmead Institute for Medical Research THE UNIVERSITY OF SYDNEY Honours Thesis 3 Acknowledgements First and foremost, I would like to express my sincere gratitude and appreciation for my supervisors, Professor Wieland Meyer, and Associate Professor Rogan Lee, for their invaluable support and expertise throughout my honours year; without them, completion would not have been possible. I would also like to express my appreciation for the remarkable molecular mycology research lab at the Westmead Institute for Medical Research, including Alex Kan, Christianne Prosser, Krystyna Maszwsla, and Dr. Laszlo Irinyi, for their extensive assistance, encouragement, and support. I want to thank my colleagues for their advice, company, and humour, making my honours journey pleasurable. Lastly, I want to thank my friends and family for their steadfast love and encouragement, which kept me motivated throughout the year. Thank you all. 4 Table of Contents Compliance Statement 2 Acknowledgements 3 List of Figures 7 List of Tables 9 List of Abbreviations 11 Abstract 14 Chapter 1: Introduction 17 1.1 Malaria overview 18 1.1.2 Causative agents of malaria and their taxonomy 18 1.1.2 Epidemiology of malaria 20 1.1.3 Plasmodium vivax life cycle 22 1.1.4 Clinical disease 23 1.2 Treatment of malaria 24 1.2.1 Primaquine and Tafenoquine 25 1.3 What is Glucose-6-Phosphate Dehydrogenase? 27 1.3.1 Glucose-6-Phosphate Dehydrogenase deficiency 27 1.3.2 Global presence of G6PD mutations 28 1.4 Testing for G6PD deficiency 30 1.4.1 Current methodology of testing for G6PD deficiency 30 1.4.2 Current typing-based methods for detection of G6PD mutations 32 1.4.3 Proposed methodology for G6PD mutation typing: PCR 33 1.5 Project significance and problem statements 34 1.6 Hypothesis 35 1.7 Aims 35 Chapter 2: Materials and Methods 36 2.1 Materials 37 2.2 Methods 37 2.2.1 DNA Extraction 37 2.2.1.1 Extraction of genomic DNA from whole blood 37 2.2.1.2 Extraction of genomic DNA from dried blood spots 38 2.2.2 Extracted DNA quality control 38 2.2.3 Preparation of primer solution 39 2.2.4 Protocol development 39 2.2.4.1 PCR protocol 39 2.2.5 Gel Electrophoresis 41 2.2.6 DNA extraction and purification from gel 41 5 2.2.7 Primer dilution for sequencing 42 2.2.8 PCR sequencing and variant analysis 42 Chapter 3: Molecular Assay Optimisation 44 3.1 Primer design selection 45 3.2 Optimisation of the PCR amplification conditions 45 3.2.1 G6PD locus 1 45 3.2.2 G6PD locus 2 48 3.2.3 G6PD locus 3 53 3.2.5 G6PD locus 5 57 3.3 Summary of the final optimised amplification conditions 62 Chapter 4: Application of the designed molecular assay to samples from Southern Thailand 64 4.1 Plasmodium vivax isolates from Southern Thailand 65 4.2 Results of the application of the newly designed PCR protocol to samples from Southern Thailand 65 4.3 Summary 69 Chapter 5: Application of the new molecular assay to samples from Australian travellers 71 5.2 Blood samples from Australian travellers infected with Plasmodium falciparum 72 5.2 Results obtained from the application of the new PCR protocol to samples from Australian travellers 72 5.3 Summary 76 Chapter 6: Discussion 77 6.1 Study importance 78 6.2 Aims 78 6.3 Results 79 6.4 DNA extraction 79 6.5 PCR optimisation 80 6.6 Sequence analysis 81 6.7 Pilot molecular surveillance results of G6PD genotypes from Southern Thailand and Australian travellers 82 6.8 Direction for future research 84 6.9 Concluding remarks 85 References 87 Appendices 102 6 Appendix I. Reagent list and chemical manufacturers 103 Appendix II. List of equipment and materials used and their product manufacturers 104 Appendix III. List of commercial kits and manufacturers 105 Appendix IV. List of bioinformatics programs and databases used 106 Appendix V. Reference G6PD GenBank sequence with attached designed primers for all 5 loci and Asian reported G6PD mutations 107 Appendix VI. Patients from Southern Thailand information summary 121 Appendix VII. Reference G6PD GenBank sequence aligned to sequences of Southern Thailand and Australian travellers samples and mutation sequences covering all reported Asian G6PD mutations 122 Appendix VIII. Patients from Australian travellers information summary 131 List of Figures Chapter 1 Introduction Figure 1. Global endemicity of P. vivax. 21 Figure 2. The life cycle of P. vivax. 22 Figure 3. Molecular structure of chloroquine 25 Chapter 2 Materials and Methods Figure 4. Primer dilution calculation 42 Figure 5. DNA electrophoresis image of the amplification products of the G6PD locus 1. 46 Chapter 3 Molecular Assay Optimisation Figure 6. DNA electrophoresis image of the amplification products of the G6PD locus 1. 46 Figure 7. DNA electrophoresis image of the amplification products of the G6PD locus 1. 47 Figure 8. DNA electrophoresis image of the amplification products of the G6PD locus 1. 48 Figure 9. DNA electrophoresis image of the amplification products of the G6PD locus 2. 49 Figure 10. DNA electrophoresis image of the amplification products of the G6PD locus 2.1. 50 Figure 11. DNA electrophoresis image of the amplification products of the G6PD locus 2.1. 51 Figure 12. DNA electrophoresis image of the amplification products of the G6PD locus 2.2. 52 Figure 13. DNA electrophoresis image of the amplification products of the G6PD locus 2.2. 53 Figure 14. DNA electrophoresis image of the amplification products of the G6PD locus 3. 54 Figure 15. DNA electrophoresis image of the amplification products of the G6PD locus 3. 55 Figure 16. DNA electrophoresis image of the amplification products of the G6PD locus 4. 56 Figure 17. DNA electrophoresis image of the amplification products of the G6PD locus 4. 57 Figure 18. DNA electrophoresis image of the amplification products of the G6PD locus 5. 58 Figure 19. DNA electrophoresis image of the amplification products of the G6PD locus 5. 59 Figure 20. DNA electrophoresis image of the amplification products of the G6PD locus 5. 59 8 Figure 21. DNA electrophoresis image of the amplification products of the G6PD locus 5.1. 60 Figure 22. DNA electrophoresis image of the amplification products of the G6PD locus 5.1. 61 Chapter 4 Application of the designed molecular assay to samples from Southern Thailand Figure 23. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 1. 66 Figure 24. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 2. 66 Figure 25. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 3. 67 Figure 26. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 4. 67 Figure 27. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 5. 68 Figure 28. Alignment of sequenced DNA samples from Southern Thailand to G6PD gene (NG_009015) and the consensus sequences of all possible mutations of interest in Asia 69 Chapter 5 Application of the designed molecular assay to samples from Australian travellers Figure 29. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 1. 73 Figure 30. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 2. 73 Figure 31. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 3. 74 Figure 32. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 4. 74 Figure 33. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 5. 75 Figure 34. Alignment of sequenced DNA samples from Australian travellers to the G6PD gene (NG_009015) and the consensus sequences of all possible mutations of interest. 76 List of Tables Chapter 1 Introduction Table 1. Subgenera of the Plasmodium species. 19 Table 2. Classification of human protozoa of the genus Plasmodium.
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