CRISPR/Cas13a-based rapid detection method for porcine deltacoronavirus

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Animal Health

Background:

• Porcine deltacoronavirus (PDCoV) causes significant economic losses in pig farming due to acute diarrhea and vomiting in piglets.
• Clinical symptoms of PDCoV are similar to other porcine enteroviruses, making accurate detection crucial for disease management.
• Current qPCR detection methods for PDCoV are time-consuming, require specialized equipment, and are not readily accessible for many farms.

Purpose of the Study:

• To develop a rapid, convenient, and sensitive detection method for Porcine deltacoronavirus (PDCoV).
• To combine Recombinase Polymerase Isothermal Amplification (RPA) with CRISPR/Cas13a technology for PDCoV detection.

Main Methods:

• Designed specific RPA primers and crRNA for PDCoV detection.
• Amplified target nucleic acids using RPA and detected them via fluorescent CRISPR/Cas13a.
• Evaluated the sensitivity and specificity of the RPA-CRISPR/Cas13a assay against qPCR.

Main Results:

• The RPA-CRISPR/Cas13a assay provided results within 90 minutes.
• Achieved a minimum detection limit of 5.7 × 10^1 copies/μL for PDCoV.
• Demonstrated high specificity, with no cross-reactivity observed with other common porcine enteroviruses.

Conclusions:

• The developed RPA-CRISPR/Cas13a method is a highly sensitive, specific, and rapid diagnostic tool for PDCoV.
• This assay offers a convenient alternative to traditional methods, facilitating timely PDCoV monitoring and control in swine populations.