AUTHOR=Liang Zili , Luo Ruxing , He Qifu , Tang Cheng , Zhang Zhidong , Li Yanmin , Guo Zijing 

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

JOURNAL=Frontiers in Veterinary Science

VOLUME=Volume 11 - 2024

YEAR=2025

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2024.1473674

DOI=10.3389/fvets.2024.1473674

ISSN=2297-1769

ABSTRACT=IntroductionBovine coronavirus (BCoV) is an important pathogen of enteric and respiratory disease in cattle, resulting in huge economic losses to the beef and dairy industries worldwide. A specific and sensitive detection assay for BCoV is critical to the early-stage disease prevention and control.MethodsWe established a specific, sensitive, and stable assay for BCoV nucleic acid detection based on CRISPR/Cas13a combined with reverse transcription recombinase-aided amplification (RT-RAA) technology. The specific primers for RT-RAA and CRISPR RNA (crRNA) were designed in the conserved region of the BCoV nucleocapsid (N) gene.ResultsThe detection limit of the RT-RAA CRISPR/Cas13a assays for BCoV detection was 1.72 copies/μl, and there were no cross-reactions with the other 10 common bovine enteric and respiratory disease-associated pathogens. The coefficient of variations (CVs) of within and between batches were less than 4.98 and 4.58%, respectively. The RT-RAA-CRISPR/Cas13a assays work well in clinical samples of cattle and yak, the BCoV positive rate of 84 clinical samples detected by RT-RAA-CRISPR/Cas13a assays was 58.3% (49/84), it was notably higher than that of RT-qPCR (2.4%, 2/84; p < 0.001). The 49 positive samples detected by RT-RAA-CRISPR/Cas13a assays were further confirmed as BCoV by Sanger sequencing.DiscussionA specific, sensitive, and stable assay based on RT-RAA-CRISPR/Cas13a assays for BCoV was developed, providing new technical support for the clinical detection and epidemiological monitoring of BCoV.