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Detection of Cancer-Associated Mutations Using Primer Exchange Reaction-Based Signal Amplification and Lateral Flow

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Summary

This study introduces a novel DNA self-assembly method using primer exchange reaction (PER) for sensitive detection of cancer mutations in liquid biopsies. The approach enhances signal amplification for improved point-of-care diagnostics.

Keywords:
DNA self‐assemblyRNAcirculating tumor DNAlateral flow assayliquid biopsymutation detectionprimer exchange reactionsignal amplification

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Nanotechnology

Background:

  • Sensitive detection of cancer-associated nucleic acids with single-nucleotide mutations is crucial for early diagnosis and personalized medicine.
  • Current point-of-care (POC) diagnostic methods face challenges in detecting low-abundance nucleic acid fragments, especially those with single-nucleotide variations.
  • Non-invasive liquid biopsy approaches offer significant potential but require highly sensitive and specific detection technologies.

Purpose of the Study:

  • To develop a programmable DNA self-assembly strategy for sensitive and specific colorimetric detection of cancer-specific DNA and RNA fragments.
  • To enhance signal amplification for improved detection limits on lateral flow assays (LFAs).
  • To validate the method's performance in detecting clinically relevant cancer mutations in complex biological samples.

Main Methods:

  • Utilized a primer exchange reaction (PER) for isothermal signal amplification.
  • Developed a DNA self-assembly strategy using PER-generated DNA concatemers functionalized with multiple FITC-labeled imager strands.
  • Integrated the system with gold nanoparticle-based lateral flow assays (LFAs) for colorimetric detection.

Main Results:

  • Achieved a limit of detection as low as 16 pM for a synthetic P53 oncogene fragment, a 16-fold improvement over controls.
  • Demonstrated reliable distinction of single-nucleotide mutations at 10% relative abundance within a wild-type background.
  • Successfully detected mutant fragments in serum, saliva, breast cancer cell line RNA, and circulating tumor DNA (ctDNA) from patient plasma, including PIK3CA and P53 mutations.

Conclusions:

  • The PER-based self-assembly system offers a robust and sensitive platform for mutation-specific nucleic acid detection using LFAs.
  • The method shows strong potential for translation into laboratory research and POC diagnostic workflows for cancer and other genetic disorders.
  • This approach significantly advances the capability for sensitive detection of low-abundance nucleic acids in liquid biopsies.