Abstract
Multi-Drug Resistant Tuberculosis is an emerging threat to the global control of tuberculosis. MDR-TB is the product of single-nucleotide polymorphisms on the genome sequence of drugsusceptible ·rs, which render it resistant to at least rifampin (RIF) and isoniazid (INH), the two first-line drugs typically used to treat TB. Resistance to RIF is a reliable indicator of multi-drug resistance. Disease hotspots have been determined to be primarily in South Asia, specifically in China, India and the Russian Federation. The disease is a growing cause for concern in SubSaharan Africa, home to 60% of all HIV affected individuals worldwide, as HIV infection is a primary risk factor ofMDR-TB and it has a 90% mortality rate among HIV patients. Currently, the most rapid methods of MDR-TB detection use symmetric polymerase chain reaction to amplify relevant gene sequences to detect mutations by use of fluorescent molecular beacons. These assays, while successful at detecting MDR-TB, are not as accurate or informative. In addition, there is currently no method for rapid and reliable testing for MDR-TB in the field. This thesis provides a quick, reliable asymmetric PCR assay for the detection ofMDR-TB using Linear-After-The-Exponential-PCR (LATE-PCR). This assay is sensitive enough detect single nucleotide changes that give rise to drug resistance in as low as 10 copies ofMDR-TB, even in the presence of 10,000,000 human genomes, and is designed to be used either in laboratory settings or in the field using the Bio-Seeq, a portable PCR machine designed by Smiths Detection.