Liang Zili, Luo Ruxing, He Qifu, Tang Cheng, Zhang Zhidong, Li Yanmin, Guo Zijing
Key Laboratory of Animal Medicine at Southwest Minzu University of Sichuan Province, College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu, China.
Wuhou District Health Hospital for Women & Children, Chengdu, China.
Front Vet Sci. 2025 Jan 7;11:1473674. doi: 10.3389/fvets.2024.1473674. eCollection 2024.
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. A specific and sensitive detection assay for BCoV is critical to the early-stage disease prevention and control.
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; < 0.001). The 49 positive samples detected by RT-RAA-CRISPR/Cas13a assays were further confirmed as BCoV by Sanger sequencing.
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.
牛冠状病毒（BCoV）是牛肠道和呼吸道疾病的重要病原体，给全球牛肉和乳制品行业造成巨大经济损失。一种针对BCoV的特异性和灵敏性检测方法对于疾病的早期预防和控制至关重要。
我们基于CRISPR/Cas13a与逆转录重组酶辅助扩增（RT-RAA）技术建立了一种用于BCoV核酸检测的特异性、灵敏性和稳定性检测方法。在BCoV核衣壳（N）基因的保守区域设计了RT-RAA的特异性引物和CRISPR RNA（crRNA）。
用于BCoV检测的RT-RAA CRISPR/Cas13a检测方法的检测限为1.72拷贝/微升，与其他10种常见的牛肠道和呼吸道疾病相关病原体无交叉反应。批内和批间变异系数（CVs）分别小于4.98%和4.58%。RT-RAA-CRISPR/Cas13a检测方法在牛和牦牛的临床样本中效果良好，通过RT-RAA-CRISPR/Cas13a检测方法检测的84份临床样本中BCoV阳性率为58.3%（49/84），显著高于RT-qPCR（2.4%，2/84；<0.001）。通过RT-RAA-CRISPR/Cas13a检测方法检测出的49份阳性样本经桑格测序进一步确认为BCoV。
开发了一种基于RT-RAA-CRISPR/Cas13a检测方法的针对BCoV的特异性、灵敏性和稳定性检测方法，为BCoV的临床检测和流行病学监测提供了新的技术支持。
