Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay

Acta Biochim Biophys Sin (Shanghai). 2020 Dec 29;52(12):1413-1419. doi: 10.1093/abbs/gmaa135.

Abstract

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.

Keywords: African swine fever; African swine fever virus; CRISPR-Cas12a; DNA endonuclease-targeted CRISPR trans reporter assay; recombinase polymerase amplification.

Publication types

  • Comparative Study

MeSH terms

  • African Swine Fever / blood
  • African Swine Fever / diagnosis*
  • African Swine Fever / virology
  • African Swine Fever Virus / genetics*
  • Animals
  • Bacterial Proteins / biosynthesis
  • Bacterial Proteins / isolation & purification
  • CRISPR-Associated Proteins / biosynthesis
  • CRISPR-Associated Proteins / isolation & purification
  • CRISPR-Cas Systems
  • China
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • DNA, Viral / genetics
  • Deoxyribonuclease I / genetics
  • Endodeoxyribonucleases / biosynthesis
  • Endodeoxyribonucleases / isolation & purification
  • Fluorescence
  • Limit of Detection
  • Molecular Diagnostic Techniques / methods*
  • Nucleic Acid Amplification Techniques / methods*
  • Real-Time Polymerase Chain Reaction
  • Recombinases / metabolism
  • Sensitivity and Specificity
  • Swine / blood*

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • DNA, Viral
  • Recombinases
  • Cas12a protein
  • Endodeoxyribonucleases
  • Deoxyribonuclease I