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ORIGINAL RESEARCH article

Front. Vet. Sci.
Sec. Veterinary Experimental and Diagnostic Pathology
Volume 11 - 2024 | doi: 10.3389/fvets.2024.1473674

Specific and Sensitive Detection of Bovine Coronavirus Using CRISPR-Cas13a Combined with RT-RAA Technology

Provisionally accepted
Zili Liang Zili Liang 1Ruxing Luo Ruxing Luo 1Qifu He Qifu He 2Cheng Tang Cheng Tang 1Zhidong Zhang Zhidong Zhang 1Yanmin Li Yanmin Li 1Zijing Guo Zijing Guo 1*
  • 1 College of Animal Husbandry and Veterinary Medicine, Southwest Minzu University, Chengdu, Sichuan Province, China
  • 2 Wuhou District Health Hospital for Women&Children., Chengdu, China

The final, formatted version of the article will be published soon.

    Bovine 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. In this study, we 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. The 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. Taken together, a 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.

    Keywords: CRISPR/Cas13a, RT-RAA, Bovine coronavirus, diagnostic, Nucleic acid detection

    Received: 31 Jul 2024; Accepted: 19 Dec 2024.

    Copyright: © 2024 Liang, Luo, He, Tang, Zhang, Li and Guo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Zijing Guo, College of Animal Husbandry and Veterinary Medicine, Southwest Minzu University, Chengdu, 610041, Sichuan Province, China

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.