Globally, tuberculosis, a disease caused by Mycobacterium tuberculosis (Mtb) complex species, stands as a leading cause of death from a single infectious agent. Even though antituberculosis drugs are available, treatment is challenging due to the point-mutations in the bacterial genome that lead to antibiotic resistance. For example, the katG and rpoB genes may contain point mutations that cause resistance to first-line antibiotics isoniazid and rifampicin, respectively. Current diagnostics lack a quick, sensitive, inexpensive test to determine resistance to both drugs, which would be indicative of multi-drug tuberculosis. This research aims to develop an assay to discern multi-drug resistant forms of tuberculosis using split deoxyribozyme sensors. For the sensor design, a catalytic core of an RNA-cleaving deoxyribozyme is split into two parts, with each part elongated with a target-recognizing fragment (“arm”). In the presence of a fully complementary target sequence, but not the one containing point mutations, the catalytic core of the deoxyribozyme can be re-formed from the two parts, which allows cleavage of a fluorophore-quencher reporter. The target-binding arms of the sensor were optimized in terms of signal-to-background ratio and selectivity of target recognition using synthetic targets corresponding to the fragments of the katG and rpoB genes with point-mutation sites. The optimized sensors sequences were used to interrogate the targets obtained by amplifying the correspondent fragments of the Mtb total DNA using Linear-After-The-Exponential (LATE) PCR, which allows synthesis of a single-stranded amplicon. The signal triggered by cognate targets can be read using a portable fluorometer, which eliminates the need to use a sophisticated real-time PCR instrument for the assay. The success of the split deoxyribozyme assay can establish an affordable and user friendly molecular diagnostic assay where a sample can be amplified and analyzed in a single tube.

Analysis of multi-drug resistant mycobacterium tuberculosis using LATE PCR and split deoxyribozyme sensors

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.