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Rapid, Highly Sensitive, and Label-Free Pathogen Assay System Using a Solid-Phase Self-Interference Recombinase

Xiangyu Jin1, Rongxin Fu1, Wenli Du1

  • 1Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China.

Analytical Chemistry
|February 2, 2022
PubMed
Summary

A novel solid-phase self-interference RPA chip (SiSA-chip) combined with hyperspectral interferometry offers a rapid, sensitive, and label-free method for pathogen identification. This advanced assay system can detect Plasmodium falciparum DNA with high specificity and efficiency within 20 minutes.

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Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Parasitology

Background:

  • Recombinase polymerase amplification (RPA) is crucial for pathogen identification, but existing label-free detection methods often lack sensitivity, specificity, efficiency, or simplicity.
  • Developing advanced diagnostic tools is essential for rapid and accurate detection of infectious diseases.

Purpose of the Study:

  • To develop a rapid, highly sensitive, and label-free pathogen assay system.
  • To utilize a solid-phase self-interference RPA chip (SiSA-chip) coupled with hyperspectral interferometry for enhanced detection.

Main Methods:

  • The study employed SiSA-chips for amplifying and selectively capturing RPA amplicons, preventing interference from primer dimers.
  • Hyperspectral interferometry was used to analyze the optical length changes of the SiSA-chips, quantifying RPA detection results.
  • The assay was tested for detecting 18S rRNA gene of Plasmodium falciparum and performing single nucleotide polymorphism (SNP) genotyping of the dhfr gene.

Main Results:

  • The assay demonstrated a limit of detection as low as six copies of the target 18S rRNA gene within 20 minutes.
  • A strong linear relationship was observed between detection results and target gene concentration (R² = 0.9903).
  • The system achieved high specificity, distinguishing as little as 1% of mutant gene from wild-type loci for SNP genotyping.

Conclusions:

  • The developed SiSA-chip and hyperspectral interferometry system provides a highly efficient, sensitive, specific, and low-cost pathogen DNA identification method.
  • This assay system is promising for rapid pathogen identification and diagnosis of infectious diseases, including SNP genotyping.