Field-deployable porcine epidemic diarrhea virus diagnostics utilizing CRISPR-Cas13a

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Biotechnology

Background:

• Porcine epidemic diarrhoea virus (PEDV) causes significant economic losses in swine farming.
• Rapid and accurate detection methods are essential for controlling PEDV outbreaks.
• Existing diagnostic techniques may require specialized equipment or longer turnaround times.

Purpose of the Study:

• To develop a novel, rapid, and sensitive diagnostic approach for PEDV detection.
• To integrate recombinase polymerase amplification (RPA) with the CRISPR-Cas13a system for PEDV diagnosis.
• To evaluate the performance of the developed assay in terms of sensitivity, specificity, and applicability to clinical samples.

Main Methods:

• Development of a recombinase polymerase amplification (RPA) assay targeting PEDV.
• Utilizing the CRISPR-Cas13a system for signal amplification and detection of target recognition.
• Validation of the assay using synthetic PEDV DNA standards and clinical swine samples.
• Comparison of the assay's performance with reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Main Results:

• The RPA-based CRISPR-Cas13a assay successfully detected as low as 10 copies/µL of PEDV DNA standard within 40 minutes at 37°C.
• The assay demonstrated high specificity, distinguishing PEDV from other relevant pathogens.
• Comparable performance to RT-qPCR was observed when applied to clinical samples.
• Visual detection using lateral flow strips or fluorescence was achieved, enabling field deployment.

Conclusions:

• The developed RPA-based CRISPR-Cas13a approach offers a reliable, sensitive, and specific method for PEDV detection.
• This assay is suitable for rapid, field-deployable diagnostics in endemic regions.
• The methodology holds potential for the detection of other viruses and applications in food safety inspections.