The drug resistance in tuberculosis (TB) is a rising concern for the diagnosis and treatment of the disease. Being able to detect the presence of drug resistance accurately and rapidly in the patient strain is essential for improving individual treatment outcomes and reducing further transmission of resistant strains, which are more costly and difficult to treat than drug-susceptible strains. However, the current methods come in short in point-of-care (POC) settings, due to problems such as long processing time, high complexity, and necessity for specialized personnel/equipment. Oligonucleotide ligation assay (OLA) provides a high sensitivity and specificity against TB drug resistance, and here, I have developed a novel lateral flow test (LFT) device that incorporates OLA into it, which have shown comparable specificity and sensitivity against traditional protocol of OLA in lab setting followed by LFT. Moreover, the simplicity of the design enables further incorporation of other techniques such as isothermal DNA amplification, for a compact, one-step TB drug resistance diagnostic device for low-resource environment. 

Serratia marcescens is an opportunistic pathogen that can infect multiple human organs and is responsible for many healthcare-associated infections. It has a mortality risk of up to 58% and early diagnosis is crucial for timely treatment. S. marcescens secretes a unique restriction endonuclease, which has been recognized as a virulent factor and thus can be used as a diagnostic biomarker. To detect this restriction enzyme biomarker, I have designed and investigated a model system using novel restriction endonuclease mediated DNA strand displacement (resDSD), adapted from the enzyme-free DNA strand displacement (DSD) reaction. In a typical DSD circuit, a DNA input “invading” strand invades a duplex DNA substrate, replacing the previous incumbent strand through branch migration to reveal a fluorescence molecule. In contrast, my resDSD circuit employs a restriction endonuclease enzyme input. In my design, the toehold region is concealed and blocked by a strand that the restriction enzyme can cleave. Once cleaved, the toehold region is exposed, allowing an invading strand to hybridize and initiate the DSD cascade. This study represents the first demonstration of the resDSD system. To validate the concept, I used commercially-available restriction endonuclease BamHi instead of S marcescens’ endonuclease. I will also modify E. coli 5-alpha competent strain (c2987h) to secrete BamHi in place of S. marcescens. By investigating this innovative resDSD approach, I aim to establish a reliable method for detecting bacterium such as S. marcescens based on its secretion of the restriction endonuclease. Such a diagnostic tool could contribute to early detection and prompt treatment of infection caused by this opportunistic pathogen or similar pathogens in healthcare settings.