title: 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.
authors.affiliation: Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA.
authors.forename: Hillary N
authors.initials: HN
authors.lastname: Bengtson
authors.affiliation: Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Homolka
authors.affiliation: Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany; German Center for Infection Research, Borstel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Niemann
authors.affiliation: Universidade Federal do Rio Grande, Rio Grande, RS, Brazil.
authors.forename: Ana Júlia
authors.initials: AJ
authors.lastname: Reis
authors.affiliation: Universidade Federal do Rio Grande, Rio Grande, RS, Brazil.
authors.forename: Pedro Eduardo
authors.initials: PE
authors.lastname: da Silva
authors.affiliation: Department of Chemistry, College of Sciences, University of Central Florida, Orlando, FL, USA.
authors.forename: Yulia V
authors.initials: YV
authors.lastname: Gerasimova
authors.affiliation: Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA; Department of Chemistry, College of Sciences, University of Central Florida, Orlando, FL, USA.
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Kolpashchikov
authors.affiliation: Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA. Electronic address: kyle.rohde@ucf.edu.
authors.forename: Kyle H
authors.initials: KH
authors.lastname: Rohde
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D021881:DNA, Catalytic / Q000737:chemistry / Q000302:isolation & purification*; D054908:Extensively Drug-Resistant Tuberculosis / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification / Q000472:pathogenicity; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology
chemical_list: D021881:DNA, Catalytic; D007538:Isoniazid; D012293:Rifampin
keywords: Binary deoxyribozyme; Diagnostics; Drug resistance; Multiplex PCR; Nucleic acid sensors; Tuberculosis