RPA-CRISPR/Cas12a-based detection of Pasteurella multocida: establishment and initial application

Area of Science:

• Veterinary Microbiology
• Molecular Diagnostics
• Livestock Disease Management

Background:

• Pasteurella multocida (Pm) causes significant respiratory disease and economic losses in sheep.
• Current diagnostic methods for Pm are often slow, complex, and not field-deployable.

Purpose of the Study:

• To develop a rapid, visual, and sensitive diagnostic assay for Pasteurella multocida (Pm) in sheep.
• To overcome limitations of existing diagnostic tools for field application.

Main Methods:

• Developed a diagnostic assay integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology.
• Targeted the kmt1 gene specific to Pm and optimized reaction parameters.
• Validated specificity against common respiratory pathogens and sensitivity using plasmid dilutions.

Main Results:

• Achieved a detection limit of 5 × 10⁻¹ copies/μL within 30 minutes.
• The RPA-CRISPR/Cas12a assay detected Pm in 40.20% of clinical samples, 4.1 times higher than PCR.
• Demonstrated high sensitivity and specificity for Pm detection.

Conclusions:

• The developed RPA-CRISPR/Cas12a assay provides a rapid, sensitive, and instrument-free method for Pm detection.
• This assay is a promising tool for frontline clinical settings in sheep farming.
• Facilitates timely diagnosis and management of Pasteurella multocida infections in sheep.