Detection of duck adenovirus 3 using RAA-CRISPR/Cas12a based lateral flow dipstick method

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Biosensing Technology

Background:

• Duck adenovirus 3 (DAdV-3) is an emerging pathogen causing significant morbidity in Muscovy ducklings.
• Current diagnostic methods for DAdV-3 are often time-consuming and require specialized laboratory equipment.
• There is a critical need for rapid, sensitive, and field-deployable diagnostic tools for DAdV-3.

Purpose of the Study:

• To establish a novel, rapid, and sensitive detection platform for Duck adenovirus 3 (DAdV-3).
• To develop a CRISPR/Cas12a-based assay coupled with a Lateral Flow Dipstick (LFD) for visual DAdV-3 detection.
• To evaluate the sensitivity, specificity, and practical applicability of the developed diagnostic platform.

Main Methods:

• Utilized Recombinase Aided Amplification (RAA) for isothermal amplification of DAdV-3 target genes.
• Employed CRISPR-Cas12a system to leverage its trans-cleavage activity on amplified products.
• Integrated the CRISPR-Cas12a system with a Lateral Flow Dipstick (LFD) for visual signal readout.

Main Results:

• The RAA-CRISPR/Cas12a-LFD platform achieved a low detection limit of 2.87 × 10^2 copies/μL for DAdV-3 plasmid.
• Demonstrated high specificity, with no cross-reactivity against common duck viruses like AIV, NDV, DTMUV, DPV, MDPV, DHAV-1, and DRV.
• Enabled visual detection of DAdV-3 within 1 hour and 20 minutes, suitable for on-site applications.

Conclusions:

• The developed RAA-CRISPR/Cas12a-LFD assay is a sensitive, specific, and rapid method for DAdV-3 detection.
• This platform offers a cost-effective and user-friendly alternative for on-site diagnostics, particularly beneficial for small-scale duck farms.
• The assay provides a valuable tool for DAdV-3 epidemiological surveillance and disease management in the poultry industry.