A rapid ionic liquid-based DNA extraction method for molecular diagnostics of urinary tract infections.

Rapid and reliable DNA extraction from urine is a critical bottleneck in advancing molecular diagnostics for urinary tract infections (UTIs) in both centralized and decentralized settings. Here, we present an ionic liquid-based DNA extraction method (IL-DEx) that enables recovery of bacterial DNA from urine samples in under 30 min using minimal equipment and no hazardous chemicals. IL-DEx was benchmarked against a widely used commercial kit (QIAamp DNA Mini Kit, QIAGEN) using reference strains, clinical isolates, and spiked urine samples. For gram-negative bacteria, IL-DEx achieved comparable DNA yields (47%-102% relative efficiency), while recoveries from gram-positive bacteria were lower (0.7%-8%) but sufficient for downstream detection. Quantitative PCR (qPCR) revealed linear DNA recovery across five to six orders of magnitude (10⁸-10² CFU/mL, R² > 0.99), with detection limits of ~10²-10³ CFU/mL for gram-negatives and ~10³-10⁴ CFU/mL for gram-positives using 1 mL of urine. Clinical evaluation with 13 patient urine samples (ten culture-positive, three culture-negative) demonstrated that IL-DEx reliably enabled pathogen detection by qPCR and full-length 16S rRNA gene sequencing (Oxford Nanopore). Performance was comparable to three other extraction methods tested head-to-head, including the QIAamp DNA Mini Kit (QIAGEN), the MagaZorb DNA Mini-Prep Kit (Promega), and a phenol-chloroform extraction method. These findings establish IL-DEx as the first ionic liquid-based approach evaluated for DNA recovery from clinical urine samples, providing a fast, simple, and low-cost method suitable for integration into molecular workflows for UTI diagnostics across diverse laboratory and clinical settings.IMPORTANCEUrinary tract infections (UTIs) are among the most common infections worldwide and a major driver of antibiotic use. Rapid and accurate diagnosis is critical to guide therapy, reduce inappropriate antibiotic prescriptions, and improve patient outcomes. While molecular diagnostics can drastically reduce time to identify uropathogens, their implementation remains constrained by upstream DNA extraction-a step that is often laborious, cost-intensive, or incompatible with rapid diagnostic workflows. We developed a fast, simple, and low-cost DNA extraction method (IL-DEx) that uses an ionic liquid and magnetic beads to recover bacterial DNA directly from urine. IL-DEx eliminates hazardous reagents and complex equipment while delivering performance comparable to established extraction kits. By streamlining this critical pre-analytical step, IL-DEx enables faster molecular diagnostics and broadens access to modern UTI testing. Its simplicity and robustness position it as a valuable tool for improving diagnostic speed, antimicrobial stewardship, and patient care across healthcare settings.