Loop-mediated isothermal amplification with TAMRA- modified dUTPs: Development and strand displacement-based detection methods of M. tuberculosis genes associated with antibiotic resistance

The global burden of antimicrobial resistance requires rapid, accessible diagnostic tools capable of detecting drug-resistant pathogens at point-of-care settings. This thesis presents the development of an innovative molecular diagnostic approach combining fluorescent labeling of genetic targets with isothermal amplification technology to address the diagnostic challenges posed by drug-resistant tuberculosis. A loop-mediated isothermal ampliYication (LAMP)-based detection system was established to target eight Mycobacterium tuberculosis genes, encompassing both species identification markers and genes with mutations conferring resistance to both first- and second-line antibiotics. Additionally, clinical relevance was established through successful amplification of tuberculosis DNA extracted from simulated sputum samples, validating this amplification technique for real world application. The research focused on incorporating TAMRA-modified nucleotides directly during amplification, eliminating the need for secondary labeling procedures typically required in conventional diagnostic workflows. Characterization of the developed method revealed that while TAMRA-dUTP integration caused measurable delays in amplification efficiency, amplification reactions maintained sufficient incorporation functionality for robust target detection. Michaelis-Menten kinetic measurements provided quantitative evidence that Bst 3.0 DNA polymerase retains catalytic competency despite structural modifications introduced by TAMRA incorporation. Furthermore, multiple reaction enhancers were tested for strategic optimization of TAMRA-dUTP incorporation during LAMP, and results showed that T4 gene 32 protein resolved the balance between fluorescent incorporation and amplification efficiency. Two target detection strategies were implemented and validated with this work: solution-based fluorescent double-stranded DNA probes for amplification verification and a microarray platform for multiplex mutation-specific identification. For single target detection, the microarray system successfully discriminated resistance-conferring single nucleotide polymorphisms from wild-type sequences, demonstrating the proof-of-concept for resistance profiling. While multiplex measurements across multiple gene targets showed cross-hybridization and signal variability, the fundamental approach proved viable for simultaneous pathogen identification and resistance characterization. This work establishes a foundation for a detection system that could be incorporated into a portable, user-friendly diagnostic device, suitable for resource-constrained environments where tuberculosis remains problematic. The methodological framework developed here offers a pathway toward decentralized resistance characterization, potentially transforming treatment decision-making by providing clinicians with useful diagnostic information within hours rather than weeks.