Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus

PLoS One. 2021 Jul 19;16(7):e0254815. doi: 10.1371/journal.pone.0254815. eCollection 2021.

Abstract

African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • African Swine Fever / diagnosis*
  • African Swine Fever / genetics
  • African Swine Fever / virology
  • African Swine Fever Virus / genetics
  • African Swine Fever Virus / isolation & purification*
  • Animals
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Cas Systems
  • DNA-Directed DNA Polymerase / chemistry
  • Endodeoxyribonucleases / chemistry
  • Endodeoxyribonucleases / genetics*
  • Molecular Diagnostic Techniques
  • Point-of-Care Systems
  • Recombinases / chemistry
  • Swine
  • Swine Diseases / diagnosis*
  • Swine Diseases / genetics
  • Swine Diseases / pathology
  • Swine Diseases / virology

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Recombinases
  • DNA-Directed DNA Polymerase
  • Cas12a protein
  • Endodeoxyribonucleases

Grants and funding

This work was supported by the National Key Research and Development Program (2019YFC1604600), Beijing Advanced Innovation Center for Soft Matter Science and Engineering, the Natural Science Foundation of HeBei province (19226631D), and Outstanding Talent Introduction Program from College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.