Researcher ORCID Identifier

0000-0001-5321-5222

Date Degree Awarded

Spring 5-13-2023

Degree Type

Open Access Dissertation

Degree Name

PHD in Applied Life Sciences

First Thesis/Dissertation Advisor

Travis Schlappi, Ph.D.

Terms of Use & License Information

Terms of Use for work posted in Scholarship@Claremont.

Abstract

Nucleic acid amplification tests (NAATs) are among the diagnostic tests with the highest sensitivity and specificity. However, they are more complex to develop than other diagnostic tests such as biochemical tests and lateral flow immunoassay tests. Polymerase chain reaction (PCR) is the gold standard for NAATs. PCR requires thermal cycling to achieve clonal amplification of the target pathogen DNA for diagnosis. Thermal cycling poses a challenge in the development of PCR diagnostics for point-of-care (POC) settings. Loop-mediated isothermal amplification (LAMP) offers an isothermal method for NAATs diagnostics. The advancement of the microfluidics field significantly enhances the development of LAMP diagnostics devices for POC testing. Another challenge with NAATs, is the limitation in the development of multiplex NAATs. Multiplexing however, occupies an important role in the efforts to address the antimicrobial resistance global crisis. Multiplexing will help to provide more thorough and complete diagnostics of infections, and enable doctors to prescribe the most effective antibiotics to the patients. This will help slow the emergence of antibiotic resistant pathogens. We are currently in a period of discovery void, with regards to antibiotics discovery. At this rate, more pathogens are becoming resistant to the antibiotics that we have, faster than we are developing new classes of antibiotics. According to the World Health Organization (WHO) interagency coordination group on AMR report to the secretary general of the United Nations, by 2050, there will be 10 million annual deaths globally, as a result of AMR-related events. There will also be $55 billion productivity losses globally due to AMR. In addition, there will be a total of $ 1 trillion in healthcare costs, and 28 million people will be living in poverty, as a result of the economic impact of uncontrolled AMR. Another area where multiplex diagnostics play a crucial role is infection control in the era of epidemics and pandemics. The increasing prevailing frequency of global pandemics stresses the need for the development of highly accurate and decentralized POC diagnostics. Over the last ten years, there have been more than 30 epidemics and pandemics around the world, including SARS-CoV-2, Monkey pox, India black fungus, Dengue fever, Measles, Zika, Avian influenza, Influenza A and Ebola. With advancing technology and international commerce and relations, we are now more connected than ever. This means that if there are no developments to make molecular tests more accessible at the POC, the future waves of epidemics and pandemics will have faster spread, further reach and more devastating impacts on the lives of the 8 billion people on our planet. We have developed a diagnostic method for executing droplet microfluidics LAMP via a microparticle primer payload mechanism and have demonstrated it with urinary tract infection (UTI) pathogens. With inspiration from overhang PCR and RNA-Seq, we engineered LAMP primers with 5’ polythymidine (PolyT) oligonucleotide (PolyT is placed in the middle of the Forward inner primers and Backward inner primers). The PolyT sequence is recognized by a biotinylated capture oligonucleotide engineered with a polyadenylated (PolyA) polynucleotide on the 3’ end. The streptavidin-coated microparticles functionalized with the PolyA oligonucleotide and PolyT primers, capture their specific target DNA and deliver the cargo into emulsion droplets of LAMP reagents for amplification. This platform provides the ability to multiplex by coding specific pathogen target DNA with different fluorescent signatures of the microparticles.

Rights Information

© 2023 Jonas A Otoo

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