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.

Keywords: Binary deoxyribozyme; Diagnostics; Drug resistance; Multiplex PCR; Nucleic acid sensors; Tuberculosis.

Fig. 1. Schematic diagram of binary deoxyribozyme sensors (Dz)

A. Principle of BiDz sensor: Dz_a and Dz_b DNA strands hybridize to adjacent positions of the DNA/RNA analyte and reform a deoxyribozyme catalytic core, which cleaves the fluorescent substrate (F-sub). F-sub contains a 5′ fluorescein (FAM) label and 3′ black hole quencher which are in close proximity to ensure low fluorescence of the intact substrate. When the F-sub is cleaved, the fluorophore is separated from the quencher allowing for the increase of fluorescence. B. A panel of BiDz sensors was designed for the detection of SNPs in multiplex PCR amplicons. In the presence of the complementary (specific) analyte, Dz_a and Dz_b bind to the analyte and F-sub forming the sensor with catalytic activity, which cleaves F-sub and produce a fluorescent signal. Alternatively, in the presence of a mismatched (nonspecific) analyte the catalytic core of the sensor cannot be formed and the fluorescence of F-sub remains quenched.

Fig. 2. Validation of BiDz sensors for the detection of Mtb and drug-resistance conferring SNPs

A. BiDz sensors designed for specific detection of WT sequences. B. BiDz sensors designed for specific detection of mutations (MT) responsible for drug resistance. This data represents the signal to background (S/B) ratios obtained after 45 min of incubation in the presence of synthetic DNA analytes. The rpoB 531 mutant sensor shown corresponds to MT1 (Table S1). A student’s t-test was completed for each sensor. All comparisons (detection of WT vs MT analyte) resulted in p-values < 0.05.

Fig. 3. Use of the TB-DzT assay for the detection of drug resistant Mtb strains

Each panel (A–F) represents the drug resistance profiles produced by the TB-DzT assay in the presence of CDC1551 (WT) Mtb DNA and five strains containing drug resistant SNPs (MT1-5). The inset box indicates the phenotype of each strain which was confirmed by sequencing.

Fig. 4. Detection of heteroresistance via TB-DzT assay

This data represents the average S/B ratios and standard deviations for 5 independent experiments. Each sensor was tested in the presence of WT (dark grey bars), WT+10% MT4 (light grey bars), or 100% MT4 (white bars) multiplex PCR analytes. A student’s t-test was completed to establish statistical significance in the presence of specific analytes. All SNP specific sensors yield statistically significant S/B ratios in the presence of specific analytes compared to nonspecific analytes (p<0.05).

Fig. 5. Combinatorial sensor assay for the detection of multiple drug resistant SNPs

This data demonstrates the detection of synthetic DNA analytes using BiDz sensors in 30 min. In these experiments Dz_a and Dz_b strands were at 15 nM. All sensors were tested in the presence of 1nM synthetic analytes wt (dark grey), mt1 (medium grey), mt2 (light grey), mt3 (white).