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 minutes 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 (10–10^2; CFU/ml, R^2; >0.99), with detection limits of ~10^2;–10^3; CFU/ml for gram-negatives and ~10^3;–10^4; CFU/ml for gram-positives using 1 ml 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.