DEVELOPMENT OF A RAPID DIAGNOSTIC ASSAY FOR THE COLORIMETRIC DETECTION OF BACTERIAL AND VIRAL PATHOGENS USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION COUPLED WITH A PEPTIDE NUCLEIC ACID AND GOLD NANOPARTICLE REPORTER SYSTEM
Date of Award
Master of Science
Clinical Molecular Genetics
Dr. Matthew Jennings
Detection of pathogenic agents remains pivotal to the control and prevention of infectious disease. While diagnostic methods continue to break barriers, the need for accurate, sensitive, and rapid methods persists. Current methods for pathogen detection, including culture, immunochemical, or molecular-based techniques, are subject to significant limitations that restrict their clinical utility. Point-of-care (POC) tests have received attention for the diagnostic screening of infectious disease, with benefits of simplicity, affordability, and convenience. This study describes the development of a novel loop-mediated isothermal amplification (LAMP) reaction followed by a peptide nucleic acid (PNA) probe and gold nanoparticle (AuNP) detection system that results in a colorimetric assay. By employing modified LAMP primers, the target-dependent, co-amplification of a novel, sequence is generated, which serves as the target for PNA/AuNP sequence-specific detection. This investigation verified the generation of a pathogen-dependent, novel sequence via pH-sensitive colorimetric detection, turbidimetric detection, restriction digestion, and with a molecular beacon. The generation of the pathogen-dependent sequence permits the complimentary binding of the PNA that is responsible for determination of the aggregative state of the AuNPs, resulting in a visual colorimetric detection system. Viral (human immunodeficiency virus type 1) and bacterial (Staphylococcus aureus) models were used to establish this LAMP technique, augmented with PNA/AuNP detection, for the potential for universal identification of any microbial nucleic acid biomarker. This investigation is the first to report sequence-specific colorimetric detection of LAMP products with a PNA/AuNP detection method, while being amenable to POC testing for infectious disease.
Brown, William, "DEVELOPMENT OF A RAPID DIAGNOSTIC ASSAY FOR THE COLORIMETRIC DETECTION OF BACTERIAL AND VIRAL PATHOGENS USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION COUPLED WITH A PEPTIDE NUCLEIC ACID AND GOLD NANOPARTICLE REPORTER SYSTEM" (2018). All NMU Master's Theses. 546.