Threshold-guided multiplex PCR-LFA: a step toward UTI pathogen detection.

Urinary tract infections (UTIs) represent a major global healthcare burden, as both their detection and management remain challenging. Although culture-based methods remain the gold standard for diagnosis, they require 24-48 hours for pathogen identification, limiting rapid clinical decision-making. Nucleic acid amplification tests (NAATs) offer improved sensitivity and reduced turnaround time; however, their translation into a device format remains limited due to operational complexity. In this context, paper-based lateral flow assays (LFAs) offer a promising alternative. In this work, we develop a multiplex polymerase chain reaction (PCR) integrated with a paper-based LFA for the detection of UTI-causing pathogens. For the first time, the assay integrates threshold-guided PCR amplification in accordance with established clinical guidelines, enabling the selective detection of pathogen-specific signals, clinically significant bacterial loads (≥105 CFU per mL), and rapid turnaround time (less than an hour). Representative Gram-negative (Escherichia coli) and Gram-positive (Enterococcus faecalis) uropathogens were selected for this purpose, and molecular assays were designed directly from the isolates, eliminating the need for DNA extraction and thereby reducing the assay time. The multiplex assay was performed on a single LFA strip within an hour. In addition, ImageJ-based analysis was performed to quantify the T/C signal intensities and determine the cut-off values for distinguishing pathogenic conditions. The proposed approach is a demonstration of molecular diagnostics on paper-based platforms for UTI detection, which can potentially be used as an adjunctive decision-support tool to complement the standard culture method, highlighting the potential of threshold-driven PCR-LFA systems to enhance interpretability and enable adaptable disease diagnostics.