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A multiplexed electrochemical quantitative polymerase chain reaction platform for single-base mutation analysis.

Yang Wang1, Hong Sun2, Gaolian Xu3

  • 1Beijing Advanced Innovation Center for Biomedical Engineering, Beijing, 102433, China.

Biosensors & Bioelectronics
|July 8, 2022
PubMed
Summary

This study presents an electrochemical quantitative polymerase chain reaction (E-PCR) platform for detecting ultra-low abundance single-base mutations (SbM). The novel method achieves high sensitivity and specificity, enabling precise SbM analysis in precious samples.

Keywords:
E-PCR platformLNA-Mediated PCRMultiplexed detectionSingle-based mutation

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Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Molecular Diagnostics

Background:

  • Detecting ultra-low abundance single-base mutations (SbM) is challenging due to limitations in multiplexing, sensitivity, and quantification accuracy.
  • Existing methods struggle with distinguishing rare mutations from abundant wild-type alleles in precious samples.

Purpose of the Study:

  • To develop a highly sensitive and multiplexed electrochemical quantitative polymerase chain reaction (E-PCR) platform for accurate SbM detection.
  • To enable precise single-nucleotide discrimination and quantification of SbM in limited samples.

Main Methods:

  • Developed a locked nucleic acid (LNA)-mediated multiplexed PCR system for selective amplification of SbM genes and suppression of wild-type alleles.
  • Utilized sequence-specific hairpin probes on a reduced graphene oxide-gold nanoparticles functionalized electrode for simultaneous amplicon detection.
  • Integrated LNA-mediated PCR upstream of electrochemical detection to enhance sensitivity.

Main Results:

  • Achieved a 2-orders-of-magnitude improvement in the limit of detection (LOD), reaching as low as 5 copies μL-1.
  • Demonstrated ultra-sensitive and specific detection of 0.05% SbM against 10,000 copies of wild-type alleles.
  • The platform showed high adaptability for expanded multiplexed detection.

Conclusions:

  • The developed E-PCR platform offers a universal tool for multiplexed SbM identification with unprecedented sensitivity and specificity.
  • This technology is well-suited for analyzing precious samples and has potential for broad applications in genetic analysis.
  • The LNA-mediated approach significantly enhances the detection capabilities for ultra-rare mutations.