Point-of-care testing diagnosis of African swine fever virus by targeting multiple genes with enzymatic recombinase amplification and CRISPR/Cas12a System

Front Cell Infect Microbiol. 2024 Dec 4:14:1474825. doi: 10.3389/fcimb.2024.1474825. eCollection 2024.

Abstract

African swine fever virus (ASFV) infection is causing devastating outbreaks globally; pig farming has suffered severe economic losses due to the ASFV. Currently, strict biosecurity control measures can mitigate the incidence of ASF. Rapid, cost-effective, and sensitive detection of ASFV can significantly reduce disease transmission and mortality. CRISPR/Cas-associated proteins can detect polymorphisms with high specificity and sensitivity, making them ideal for detecting pathogens. In this study, based on CRISPR/Cas12a integrated with enzymatic recombinase amplification (ERA) technology, a CRISPR/Cas12a detection system capable of identifying ASFV E183L, K205R, and C962R gene sequences has been developed. The ERA-CRISPR/Cas12a detection system detected ASFV precisely without cross-reactivity with other porcine pathogen templates and with a sensitivity detection limit of 10 copies per reaction; it takes 60 minutes to complete the detection process. In combination with this integrated ERA pre-amplification and Cas12a/crRNA cutting assay, it provides a rapid, straightforward, sensitive, and specific method for ASFV detection in the field.

Keywords: African swine fever virus; CRISPR/Cas12a; diagnosis; enzymatic recombinase amplification; point-of-care testing.

MeSH terms

  • African Swine Fever Virus* / genetics
  • African Swine Fever Virus* / isolation & purification
  • African Swine Fever* / diagnosis
  • African Swine Fever* / virology
  • Animals
  • Bacterial Proteins
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Cas Systems*
  • Endodeoxyribonucleases / genetics
  • Molecular Diagnostic Techniques / methods
  • Nucleic Acid Amplification Techniques / methods
  • Point-of-Care Testing*
  • Recombinases* / genetics
  • Recombinases* / metabolism
  • Sensitivity and Specificity*
  • Swine

Substances

  • Recombinases
  • Cas12a protein
  • CRISPR-Associated Proteins
  • Endodeoxyribonucleases
  • Bacterial Proteins

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study received financial support from various sources, including a grant from the key project of Jiangsu Province’s Key Research and Development plan (modern Agriculture) (BE2020407), the funding of Swine Infectious Diseases Division (NSF2023TC01), the project of Jiangsu Agri-animal Husbandry Vocational College (NSF2022CB25), the Qing Lan Project of Jiangsu Province, the Natural Science Research Project of Higher Education of Jiangsu Province (2020220375), and the Taizhou Science and Technology Support Project (Agriculture) (TN202314).