Establishment of a simple, sensitive, and specific ASFV detection method based on Pyrococcus furiosus argonaute

Area of Science:

• Veterinary diagnostics
• Molecular biology
• Pathogen detection

Background:

• African swine fever (ASF) poses a significant threat to the swine industry, necessitating effective control strategies.
• Early and accurate detection of the African swine fever virus (ASFV) is critical for disease prevention and management.

Purpose of the Study:

• To develop and evaluate a novel, highly sensitive, and specific nucleic acid assay for ASFV detection.
• To introduce a one-pot assay combining recombinase-aided amplification and restriction endonuclease-assisted Pyrococcus furiosus argonaute (PfAgo) detection.

Main Methods:

• Development of a one-pot assay termed REPD (recombinase-aided amplification and restriction endonuclease-assisted PfAgo detection).
• Assessment of REPD's sensitivity, specificity, and cross-reactivity against other pathogens.
• Validation of REPD using clinical samples compared to real-time PCR.

Main Results:

• The developed one-pot REPD assay demonstrated the ability to detect a single copy of ASFV nucleic acid.
• The assay exhibited no cross-reactivity with other tested pathogens, confirming its specificity.
• Detection results from clinical samples using REPD were 100% consistent with real-time PCR analysis.

Conclusions:

• The one-pot REPD assay is a convenient, sensitive, and specific method for ASFV detection.
• This novel assay shows potential for broad adaptability in detecting various pathogens.