Specific and sensitive detection of bovine coronavirus using CRISPR-Cas13a combined with RT-RAA technology

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• CRISPR Technology

Background:

• Bovine coronavirus (BCoV) causes significant enteric and respiratory diseases in cattle, leading to substantial economic losses.
• Accurate and sensitive detection of BCoV is crucial for effective disease prevention and control strategies.

Purpose of the Study:

• To develop and validate a specific, sensitive, and stable nucleic acid detection assay for Bovine coronavirus (BCoV).
• To provide a novel tool for early-stage disease prevention and epidemiological monitoring of BCoV in cattle.

Main Methods:

• CRISPR/Cas13a technology was combined with reverse transcription recombinase-aided amplification (RT-RAA).
• Primers for RT-RAA and CRISPR RNA (crRNA) were designed targeting the conserved nucleocapsid (N) gene of BCoV.

Main Results:

• The assay demonstrated a low detection limit of 1.72 copies/μl for BCoV.
• No cross-reactivity was observed with 10 other common bovine pathogens.
• The assay showed high specificity and sensitivity in clinical samples, detecting BCoV at a 58.3% positive rate, significantly higher than RT-qPCR.
• Intra- and inter-batch coefficients of variation were below 4.98% and 4.58%, respectively, indicating assay stability.

Conclusions:

• A robust RT-RAA-CRISPR/Cas13a assay for BCoV detection has been successfully developed.
• This assay offers enhanced technical support for clinical diagnosis and epidemiological surveillance of BCoV in cattle and yak populations.