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One-Pot Ligation-Recombinase Polymerase Amplification-Clustered Regularly Interspaced Short Palindromic

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A new ligation-RPA-CRISPR/Cas12a (LRCC) method enables sensitive detection of microRNAs (miRNAs) for early cancer diagnosis. This streamlined assay offers improved accuracy and point-of-care testing performance.

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Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Nanotechnology

Background:

  • Sensitive microRNA (miRNA) detection is crucial for early cancer diagnosis.
  • Existing methods like RPA-CRISPR/Cas12a are not ideal for short-stranded miRNAs.
  • There is a need for improved point-of-care diagnostic tools.

Purpose of the Study:

  • To develop a novel, sensitive, and streamlined miRNA detection method.
  • To enhance the efficiency and accuracy of nucleic acid detection for clinical applications.
  • To enable trimode detection for improved diagnostic capabilities.

Main Methods:

  • Developed ligation-RPA-CRISPR/Cas12a (LRCC) by integrating T4 ligase with RPA-CRISPR/Cas12a.
  • Utilized a glycerol-enhanced one-pot reaction strategy and lateral flow assay (LFA).
  • Employed "three-in-one" Au-Pt nanostars (Au@Pt NSs) for colorimetric, photothermal, and SERS detection, stabilized by "click" chemistry.

Main Results:

  • The glycerol-enhanced one-pot reaction showed significantly higher catalytic efficiency.
  • Achieved ultra-low detection limits: 23.6 fM (colorimetric), 2.19 fM (photothermal), and 72.29 aM (SERS).
  • Demonstrated trimode detection with enhanced accuracy and improved probe capture efficiency.

Conclusions:

  • The LRCC strategy offers a sensitive, rapid, and efficient method for miRNA detection.
  • The developed assay has strong practical applicability for point-of-care testing.
  • This approach is crucial for advancing early disease diagnosis based on miRNA biomarkers.