Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples

• DOI: 10.1371/journal.pone.0247851
• Publication Date: 2021-02-26T18:30:00Z
• Authors: Amy Oreskovic; Barry R. Lutz
• Keywords: Hybridization probe

Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there no consensus on its optimal pre-analytical methodology, including the extraction step. Due to short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA not efficiently recovered by conventional silica-based methods. To maximize sensitivity assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized magnetic beads improve from large-volume samples. Our recovers near 100% (95% CI: 82.6-117.6%) target-specific 10 mL urine, independent fragment (25-150 bp), has limit detection ≤5 copies double-stranded (0.5 copies/mL). Pairing ultrashort qPCR design, can amplify as 25 bp. enables amplification in single well, tolerates variation sample composition, effectively removes non-target DNA. protocol improves upon both existing methods previous hybridization-based preparation protocols. Two key innovations contribute strong performance our method: two-probe system enabling recovery strands dual biotinylated probes, which ensure consistent, high facilitating probe density bead surface, improving thermostability probe-bead linkage, eliminating interference endogenous biotin. We originally designed for tuberculosis diagnosis cfDNA, expect it will be versatile across targets, may useful other types applications beyond cfDNA. make accessible new users, present detailed straightforward guidelines designing probes.