Species Typing of Nontuberculous Mycobacteria by Use of Deoxyribozyme Sensors

Abstract

BACKGROUND Nontuberculous mycobacteria (NTM) species are a rising threat, especially to patients living with pulmonary comorbidities. Current point-of-care diagnostics fail to adequately identify and differentiate NTM species from Mycobacterium tuberculosis (Mtb). Definitive culture- and molecular-based testing can take weeks to months and requires sending samples out to specialized diagnostic laboratories. METHODS In this proof-of-concept study, we developed an assay based on PCR amplification of 16S ribosomal RNA (rRNA) rrs genes by using universal mycobacterial primers and interrogation of the amplified fragments with a panel of binary deoxyribozyme (BiDz) sensors to enable species-level identification of NTM (BiDz-NTMST). Each BiDz sensor consists of 2 subunits of an RNA-cleaving deoxyribozyme, which form an active deoxyribozyme catalytic core only in the presence of the complimentary target sequence. The target-activated BiDz catalyzes cleavage of a reporter substrate, thus triggering either fluorescent or colorimetric (visually observed) signal depending on the substrate used. The panel included BiDz sensors for differentiation of 6 clinically relevant NTM species (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium kansasii, and Mycobacterium gordonae) and Mtb. RESULTS Using the fluorescent BiDz-NTMST assay, we successfully identified the species of 38 clinical isolates. In addition, a subset of strains was tested with visual BiDz sensors, providing proof-of-concept for species typing of NTM by the naked eye. CONCLUSIONS The BiDz-NTMST assay is a novel platform for rapid identification of NTM species. This method is highly specific and significantly faster than current tools and is easily adaptable for onsite diagnostic laboratories in hospitals or clinical laboratories.

Cascade of deoxyribozymes for the colorimetric analysis of drug resistance in Mycobacterium tuberculosis

“…Previously, we have designed the sDz/PDz systems reporting flavivirus-specific targets (Reed et al, 2019), and species-typing of mycobacteria (Wood et al, 2019). However, no differentiation of point mutations was shown.…”

“…For fluoroquinoloneresistance (FQ R ), a gyrA gene fragment with a site of an A>G substitution in the case of D94G mutant (Pantel et al, 2016) was targeted. Finally, we designed the sDz/PDz cascade targeting a fragment of MTC 16S rRNA, as described earlier (Wood et al, 2019). The 16S rRNA-specific probe along with a set of the point-mutation differentiating probes would enable both MTC detection and DST.…”

“…PCR). To address the issue, we have developed a post-amplification signal amplification cascade with visual output (Gerasimova et al, 2013;Reed et al, 2019;Wood et al, 2019). This work combines split probes and a visual signal amplification cascade with isothermal target amplification via the nucleic acid sequence-based amplification (NASBA) technique (Compton, 1991;Kievits et al, 1991;Deiman et al, 2002), and for the first time applies the technology for differentiation of genotypes of drug-resistant and drug-susceptible MTC species.…”

Evolution of Hybridization Probes to DNA Machines and Robots

“…The assay was performed at room temperature and did not require annealing . Similar strategy for analyte-binding arm design was shown to be efficient for other types of multicomponent probes, including deoxyribozymes and G-quadruplex-forming binary probes. , …”

“…48 Similar strategy for analyte-binding arm design was shown to be efficient for other types of multicomponent probes, including deoxyribozymes and G-quadruplex-forming binary probes. 49,50 4. LIMIT OF DETECTION A typical limit of detection (LOD) for a conventional fluorescent probe is 0.1−1 nM, with some exceptional probes detecting down to 10 pM analyte.…”