Multiplex detection of extensively drug resistant tuberculosis using binary deoxyribozyme sensors

Abstract

Current diagnostic tools for Mycobacterium tuberculosis (Mtb) have many disadvantages including low sensitivity, slow turnaround times, or high cost. Accurate, easy to use, and inexpensive point of care molecular diagnostic tests are urgently needed for the analysis of multidrug resistant (MDR) and extensively drug resistant (XDR) Mtb strains that emerge globally as a public health threat. In this study, we established proof-of-concept for a novel diagnostic platform (TB-DzT) for Mtb detection and the identification of drug resistant mutants using binary deoxyribozyme sensors (BiDz). TB-DzT combines a multiplex PCR with single nucleotide polymorphism (SNP) detection using highly selective BiDz sensors targeting loci associated with species typing and resistance to rifampin, isoniazid and fluoroquinolone antibiotics. Using the TB-DzT assay, we demonstrated accurate detection of Mtb and 5 mutations associated with resistance to three anti-TB drugs in clinical isolates. The assay also enables detection of a minority population of drug resistant Mtb, a clinically relevant scenario referred to as heteroresistance. Additionally, we show that TB-DzT can detect the presence of unknown mutations at target loci using combinatorial BiDz sensors. This diagnostic platform provides the foundation for the development of cost-effective, accurate and sensitive alternatives for molecular diagnostics of MDR- and XDR-TB.

Species Typing of Nontuberculous Mycobacteria by Use of Deoxyribozyme Sensors

“…We have previously reported the limit of detection (LOD) of a fluorescent BiDz assay targeting Mtb as 10-15 genome equivalents (50-75 fg) (21). In this work, we determined the LOD for the IPDz-substrate based colorimetric assay by PCR amplification from 2-fold serial dilution of Mtb chromosomal DNA (0.125-4ng DNA) and subsequent detection with vBiDz sensors as described in Materials and Methods.…”

“…BiDz sensors offer unique advantages over traditional molecular methods. The catalytic feature of the BiDz sensors enables amplification of signal in the presence of a specific analyte resulting in lower limits of detection compared to traditional probe-based molecular detection methods (16,21). Most importantly, careful design of BiDz sensors can enable differentiation of DNA or RNA analytes that differ by a single nucleotide (16,(21)(22)(23).…”

Development of a technological platfom for creating innovative anti-tb drugs effective against multi-drug-resistant strains

“…Исследования в области разработки быстрых, точных и простых в эксплуатации систем диагностики лекарственно устойчивых форм туберкулёза ведутся во многих странах мира [15,16]. В рамках представляемого проекта предполагается разработать и внедрить в практику новую технологическую платформу для экспрессной молекулярной диагностики МЛУ и ШЛУ-ТБ в клинических образцах (мокроте, моче) при использовании системы бинарных дезоксирибозимных сенсоров [17]. Эта технология позволит одновременно обнаруживать ДНК/РНК МЛУ-ТБ непосредственно в клинических образцах и идентифицировать точечные мутации, обусловливающие формирование множественной и широкой лекарственной устойчивости.…”

Towards Nanomaterials for Cancer Theranostics: A System of DNA-Modified Magnetic Nanoparticles for Detection and Suppression of RNA Marker in Cancer Cells

“…Notably, the signal appears only in the presence of the DNA/RNA analyte species which are needed for assembling the catalytic BiDZ core. A BiDZ sensor was applied for molecular diagnostics of diseases [21][22][23]. The MaBiDZ system, which includes DNA-functionalized magnetic nanoparticles, was developed for the detection of a specific biomarker inside cancer cells.…”