Isothermal Nucleic Acid Assays for the Detection of HIV Drug Resistance and Sickle Cell Disease in Low-Resource Settings
| dc.contributor.advisor | Richards-Kortum, Rebecca R | |
| dc.creator | Natoli, Mary Elizabeth | |
| dc.date.accessioned | 2020-04-27T19:00:00Z | |
| dc.date.available | 2021-05-01T05:01:12Z | |
| dc.date.created | 2020-05 | |
| dc.date.issued | 2020-04-24 | |
| dc.date.submitted | May 2020 | |
| dc.date.updated | 2020-04-27T19:00:00Z | |
| dc.description.abstract | HIV treatment has become more widely available and effective in the past several decades, with major global health initiatives targeted at increasing diagnosis, treatment, and viral suppression. However, as treatment for HIV has become more widely available and effective, HIV drug resistance has emerged as a major challenge, with a disproportionate effect in low- and middle-income countries (LMICs). A lack of resources in these settings has made it difficult to implement effective strategies to differentiate ARV therapy from true instances of drug resistance. 90% of HIV drug resistance is caused by four specific single-nucleotide polymorphisms (SNPs). Sickle cell disease (SCD) is another disease of global importance that results from a point mutation in the β-globin gene which causes red blood cells to sickle. This in turn leads to painful vaso-occlusion and a host of other clinical consequences. The goal of this thesis work was to develop low-cost nucleic acid tests that can improve early detection of these two conditions in low-resource settings. The first part of this thesis work focused on the development of a method to discriminate M184V, the most common HIV-1 drug resistance mutation, from wild type DNA, and to detect the products in a paper-based enzyme-linked immunosorbent assay (ELISA) format. First, a section of HIV-1 reverse transcriptase is isothermally amplified using a recombinase polymerase amplification (RPA) assay. Next, an oligonucleotide ligation assay (OLA) is used to selectively label the mutant and wild type amplified sequences. Finally, a lateral flow ELISA differentiates between OLA-labeled products with or without M184V. Our method shows 100% sensitivity when tested with samples that contained 200 copies of mutant DNA and 800 copies of wild type DNA prior to amplification. When integrated with sample preparation, this method may detect HIV-1 drug resistance at a low cost and at a rural hospital laboratory. The second part of this thesis work focused on point-of-care detection of sickle cell disease (SCD). SCD is a common, life-threatening disorder caused by a point mutation in the β-globin gene. Early diagnosis through newborn screening is known to reduce mortality; however, the high cost and complexity of conventional diagnostic methods limit the scope and sustainability of newborn screening for SCD in resource-limited areas worldwide. Although several point-of-care tests are currently in development, antibody-based tests cannot be used in patients who have recently received a blood transfusion. Here we describe the development of a rapid, low-cost nucleic acid test that detects the point mutation that causes the formation of sickle hemoglobin (HbS) in one round of isothermal amplification and in an enclosed tube. When tested with a set of clinical samples, our assay demonstrated 100% sensitivity for both the βA globin and βS globin alleles, and 94.7% and 97.1% specificity for the βA globin allele and βS globin allele, respectively (n=91). Finally, sample-to-answer genotyping of genomic DNA is demonstrated from capillary blood in <30 minutes at a cost of <5 USD. This work demonstrates the potential utility of a point-of-care, sample-to-answer nucleic acid test for SCD that will be easily adapted to other disease-causing point mutations in genomic DNA. Overall, this thesis covers the progress made toward several technologies to detect point mutations using isothermal amplification, and contributes to the growing field of scientific knowledge on point mutation detection in resource-limited settings. | |
| dc.embargo.terms | 2021-05-01 | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.citation | Natoli, Mary Elizabeth. "Isothermal Nucleic Acid Assays for the Detection of HIV Drug Resistance and Sickle Cell Disease in Low-Resource Settings." (2020) Diss., Rice University. <a href="https://hdl.handle.net/1911/108379">https://hdl.handle.net/1911/108379</a>. | |
| dc.identifier.uri | https://hdl.handle.net/1911/108379 | |
| dc.language.iso | eng | |
| dc.rights | Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder. | |
| dc.subject | isothermal amplification | |
| dc.subject | nucleic acid amplification | |
| dc.subject | nucleic acid amplification tests | |
| dc.subject | recombinase polymerase amplification | |
| dc.subject | sickle cell disease | |
| dc.subject | HIV drug resistance | |
| dc.subject | point mutation | |
| dc.subject | single nucleotide polymorphism | |
| dc.title | Isothermal Nucleic Acid Assays for the Detection of HIV Drug Resistance and Sickle Cell Disease in Low-Resource Settings | |
| dc.type | Thesis | |
| dc.type.material | Text | |
| thesis.degree.department | Bioengineering | |
| thesis.degree.discipline | Engineering | |
| thesis.degree.grantor | Rice University | |
| thesis.degree.level | Doctoral | |
| thesis.degree.name | Doctor of Philosophy |
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