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

Dakshita S Sharma1, Sumit G Gandhi2, Manish Ranjan3

  • 1Department of Chemical Engineering, Indian Institute of Technology Jammu (IIT), Jammu and Kashmir, India. dharitri.rath@iitjammu.ac.in.

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Summary

This study introduces a rapid, paper-based diagnostic test for urinary tract infections (UTIs). The novel assay combines polymerase chain reaction (PCR) with lateral flow assays (LFAs) for quick and accurate pathogen detection.

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Area of Science:

  • Molecular Diagnostics
  • Point-of-Care Testing
  • Biotechnology

Background:

  • Urinary tract infections (UTIs) pose a significant global health challenge, with current diagnostic methods lacking speed and efficiency.
  • Traditional culture-based UTI diagnosis takes 24-48 hours, delaying critical clinical decisions.
  • Nucleic acid amplification tests (NAATs) offer faster results but face challenges in device integration.

Purpose of the Study:

  • To develop a novel multiplex polymerase chain reaction (PCR) integrated with a paper-based lateral flow assay (LFA) for rapid UTI pathogen detection.
  • To achieve selective detection of clinically significant bacterial loads (≥10^5 CFU/mL) within an hour.
  • To demonstrate the potential of threshold-driven PCR-LFA systems for adaptable disease diagnostics.

Main Methods:

  • Development of a multiplex PCR assay targeting common UTI pathogens (e.g., *Escherichia coli*, *Enterococcus faecalis*) directly from isolates, eliminating DNA extraction.
  • Integration of PCR with a paper-based lateral flow assay (LFA) for simultaneous detection on a single strip.
  • Implementation of threshold-guided PCR amplification and ImageJ-based analysis for signal quantification and cut-off determination.

Main Results:

  • The integrated PCR-LFA assay successfully detected representative Gram-negative and Gram-positive uropathogens within one hour.
  • The assay demonstrated selective detection of clinically significant bacterial loads (≥10^5 CFU/mL) using threshold-guided amplification.
  • ImageJ analysis provided quantitative T/C signal intensities to establish cut-off values for distinguishing pathogenic conditions.

Conclusions:

  • A novel, rapid, paper-based molecular diagnostic assay for UTIs has been successfully developed and demonstrated.
  • The threshold-driven PCR-LFA approach enhances interpretability and offers a potential adjunctive decision-support tool for UTI diagnosis.
  • This technology highlights the potential of paper-based platforms for accessible and adaptable point-of-care molecular diagnostics.