Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Biotechnology

Background:

• African swine fever (ASF) is a highly contagious viral disease caused by the African swine fever virus (ASFV).
• ASF presents severe clinical signs including high fever, hemorrhages, and high mortality rates, significantly threatening the global swine industry and food security.
• Effective quarantine and control strategies are essential to prevent ASFV outbreaks and mitigate economic losses.

Purpose of the Study:

• To develop and validate a novel nucleic acid detection method for diagnosing African swine fever (ASF).
• To enhance the sensitivity and specificity of ASFV detection compared to existing diagnostic techniques.
• To assess the potential of the developed method for clinical applications in swine disease management.

Main Methods:

• Development of a recombinase polymerase amplification (RPA)-CRISPR-based nucleic acid detection system.
• Utilizing CRISPR-lwCas13a (formerly C2c2) for collateral cleavage and fluorescence signal detection via quantitative real-time PCR (qPCR).
• Integration of a lateral flow strip for rapid, on-site ASFV detection.

Main Results:

• The RPA-CRISPR method demonstrated high sensitivity, capable of detecting single copies of ASFV plasmid and genomic DNA.
• The diagnostic sensitivity was comparable to or exceeded traditional qPCR methods.
• The method showed 100% coincidence rate in diagnosing clinical ASF DNA samples (both positive and negative) without cross-reactivity with other porcine viruses.

Conclusions:

• The developed RPA-CRISPR nucleic acid detection method is a highly sensitive and specific tool for diagnosing African swine fever virus (ASFV).
• This innovative diagnostic approach holds significant potential for effective clinical quarantine and control of ASFV, thereby safeguarding the swine industry and global food security.