One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a

Area of Science:

• Veterinary diagnostics
• Molecular biology
• Biotechnology

Background:

• African swine fever virus (ASFV) and Capripoxvirus (CaPV) cause significant economic losses in the livestock industry.
• Reliable and rapid detection methods are crucial for controlling these viral outbreaks.

Purpose of the Study:

• To develop a rapid, ultra-sensitive, and one-pot DNA detection platform for ASFV and CaPV.
• To combine the advantages of recombinase polymerase amplification (RPA) and CRISPR/Cas12a for simplified and accurate diagnostics.

Main Methods:

• Developed a one-pot RPA-Cas12a (OpRCas) platform integrating RPA and CRISPR/Cas12a reactions.
• Optimized reagent addition to prevent incompatibility and aerosol contamination.
• Validated the platform using a portable fluorometer and lateral flow assay (LFA) strips for point-of-care (POC) testing.

Main Results:

• The OpRCas platform demonstrated 10 to 100 times higher sensitivity than qPCR.
• Achieved a limit of detection as low as 1.2 × 10⁻⁶ ng/µL for ASFV and 7.7 × 10⁻⁵ ng/µL for CaPV.
• Showed 93.3% consistency with qPCR for clinical sample analysis, confirming its utility for POC testing.

Conclusions:

• The OpRCas platform provides an easy-handling, ultra-sensitive, and rapid method for ASFV and CaPV detection.
• The platform's one-pot reaction design simplifies operation and reduces costs.
• OpRCas is suitable for both quantitative testing and point-of-care diagnostics in livestock disease surveillance.