Associate Professor University of Washington, United States
Introduction:: Tuberculosis (TB) is a respiratory infection caused by Mycobacterium tuberculosis which disproportionately affects low- and middle-income countries. Over 1.6 million people died from TB infection in 20211. The emergence of drug-resistant TB (DR-TB) strains is a growing public health threat which complicates the antibiotic treatment regime and exacerbates the disease’s spread. Current diagnostic methods for TB and DR-TB are challenging in low-resource settings because of their long turnaround times, reliance on expensive laboratory equipment, risk in sample collection, and difficulty with sample collection for pediatric and HIV-positive patients.
To address these challenges, we are a developing a method to specifically detect DR-TB in urine samples. Urine is easy to collect, poses minimal transmission risk, and contains short (< 100 base pair) fragments of cell-free DNA (cfDNA). Conventional purification and amplification methods, however, do not efficiently extract and detect cfDNA fragments. Additionally, these methods do not have the specificity necessary to differentiate fragments containing mutations associated with drug-resistance.
Materials and Methods:: Urine cfDNA was extracted using streptavidin-coated magnetic beads conjugated to a biotinylated single-stranded DNA probe. To achieve cfDNA fragment amplification with single-nucleotide specificity, a ligation-dependent probe amplification scheme was developed. Briefly, the hybridization of target DNA to upstream (5’) and downstream (3’) probes containing universal primer binding sites enabled sequence-dependent enzymatic ligation with Taq DNA ligase. The ligated probe was then detected using PCR or loop-mediated isothermal amplification (LAMP).
Results, Conclusions, and Discussions:: Using our bead-based extraction protocol, we have achieved near 100% recovery of target cfDNA from 10 mL urine with a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL)2. Additionally, we have incorporated the ligation-dependent amplification scheme into this protocol and demonstrated two key components: 1) the ligated sequence is efficiently amplified by PCR while conjugated to streptavidin-coated magnetic beads via 5’ biotinylation and 2) the bead-conjugated 5’ probe and 3’ probe can be ligated in a target concentration dependent manner. Future work will focus on optimizing the specificity and efficiency of the ligation reaction as ligation specificity will be critical to DR-TB sequence differentiation. Additionally, multiplexing via the introduction of additional probe pairs could enable the simultaneous detection of multiple mutation sites. We envision a < 2hr workflow involving three steps: 1) bead-based extraction, 2) sequence-specific ligation, 3) isothermal amplification detection. By obviating labor-intensive bacterial culture or expensive sequencing, our approach has the potential to greatly simplify DR-TB detection, particularly in low-resource settings.
Oreskovic, A., & Lutz, B. R. (2021). Ultrasensitive hybridization capture: Reliable detection of < 1 copy/mL short cell-free DNA from large-volume urine samples. PLOS ONE, 16(2), e0247851. https://doi.org/10.1371/journal.pone.0247851
