Enhancing Point-of-Care Diagnosis of African Swine Fever Virus (ASFV) DNA with the CRISPR-Cas12a-Assisted Triplex Amplified Assay

Anal Chem. 2024 Apr 2;96(13):5178-5187. doi: 10.1021/acs.analchem.3c05364. Epub 2024 Mar 18.

Abstract

Accurate, ultrasensitive, and point-of-care (POC) diagnosis of the African swine fever virus (ASFV) remains imperative to prevent its spread and limit the losses incurred. Herein, we propose a CRISPR-Cas12a-assisted triplex amplified colorimetric assay for ASFV DNA detection with ultrahigh sensitivity and specificity. The specific recognition of recombinase aided amplification (RAA)-amplified ASFV DNA could activate the Cas12a/crRNA/ASFV DNA complex, leading to the digestion of the linker DNA (bio-L1) on magnetic beads (MBs), thereby preventing its binding of gold nanoparticles (AuNPs) network. After magnetic separation, the release of AuNPs network comprising a substantial quantity of AuNPs could lead to a discernible alteration in color and significantly amplify the plasmonic signal, which could be read by spectrophotometers or smartphones. By combining the RAA, CRISPR/Cas12a-assisted cleavage, and AuNPs network-mediated colorimetric amplification together, the assay could detect as low as 0.1 copies/μL ASFV DNA within 1 h. The assay showed an accuracy of 100% for the detection of ASFV DNA in 16 swine tissue fluid samples, demonstrating its potential for on-site diagnosis of ASFV.

MeSH terms

  • African Swine Fever Virus* / genetics
  • Animals
  • CRISPR-Cas Systems / genetics
  • Gold
  • Hydrolases
  • Metal Nanoparticles*
  • Nucleic Acid Amplification Techniques
  • Point-of-Care Systems
  • Recombinases
  • Sensitivity and Specificity
  • Swine

Substances

  • Gold
  • Hydrolases
  • Recombinases