Multicomponent deoxyribozyme-based fluorescent assays for species-specific detection of nontuberculous mycobacteria

Laila Ghasseminia, Gavin Dunton, Ryan P. Connelly, Yulia Gerasimova

University of Central Florida

Mycobacteria are a genus of acid-fast bacilli that can be grouped into species that cause tuberculosis, known as Mycobacterium tuberculosis complex (MTC), and species that do not cause tuberculosis, known as non-tuberculous mycobacteria (NTM). NTM are opportunistic pathogens, which cause pulmonary infections. Currently, NTM infections are on the rise.¹ Individuals with preexisting conditions like lung disease, and autoimmune disorders are at the highest risk for pulmonary NTM infections.² The current gold standard of NTM identification includes culture-based methods and sputum smear microscopy (SSM), which either can take up to months (culture), or do not always provide definitive results (SSM). Timely identification of the causative agent of an NTM infection is critical for proper prescription of antibiotics, treatment plan development, as well as from surveillance standpoint.

In this work, a molecular assay intended to differentiate between different clinically relevant mycobacterial species was developed. This assay is based on multicomponent deoxyribozyme (Dz) probes that display catalytic activity commensurate with the presence of a specific nucleic acid sequence in the sample. Upon the target-dependent formation of the catalytic core, cleavage of a universal reporter substrate occurs, and increased fluorescence is observed. The fluorescence is quantified with a portable dual-channel fluorometer. This assay is executed in two tiers. Tier 1 uses Dz strands designed to differentiate mycobacteria from non-mycobacterial species, as well as determine if the mycobacterial species detected belongs to Mycobacterium tuberculosis complex (MTC). Tier 2 uses Dz strands designed to differentiate between three clinically relevant NTM species: M. abscessus, M. avium and M. kansasii. The assay has shown to be effective with PCR and transcription-mediated amplification products, and does not display diminished efficacy when the components are lyophilized, demonstrating its usefulness in a clinical setting.

(1) Dahl, V. N. et al. International Journal of Infectious Diseases, 2022, 125, 120-131.

(2) American Thoracic Society. https://www.thoracic.org/patients/patient-resources/resources/ntm.pdf.