Development of RT-RAA-CRISPR/Cas12a-Based Rapid Visual Detection Assay for Pigeon Rotavirus A

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Avian Pathology

Background:

• Pigeon rotavirus A (PiRVA) is an emerging disease threatening the pigeon industry and public health.
• Accurate, rapid, and convenient detection methods are crucial for PiRVA monitoring and early diagnosis.

Purpose of the Study:

• To establish a novel, rapid detection method for PiRVA.
• To evaluate the specificity, sensitivity, and reproducibility of the new method.

Main Methods:

• Designed crRNA and reverse transcription recombinase-aided amplification (RT-RAA) primers based on the PiRVA VP6 gene.
• Developed a PiRVA detection assay by combining RT-RAA with the CRISPR/Cas12a system and lateral flow strips.

Main Results:

• The RT-RAA-CRISPR/Cas12a method specifically detected PiRVA without cross-reacting with other pigeon viruses.
• The minimum detection limit was 16.8 copies/μL with consistent intra- and inter-batch results.
• Analysis of 56 clinical samples showed higher positive rates for PiRVA in racing and domestic pigeons compared to PCR, indicating widespread infection.

Conclusions:

• The established RT-RAA-CRISPR/Cas12a method offers good specificity, sensitivity, and reproducibility for PiRVA detection.
• This visually detectable assay is suitable for field applications, supporting epidemiological surveillance and etiological research of PiRVA.