Specific and sensitive detection of bovine coronavirus using CRISPR-Cas13a combined with RT-RAA technology
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.M...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-01-01
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| Series: | Frontiers in Veterinary Science |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2024.1473674/full |
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| Summary: | 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. |
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| ISSN: | 2297-1769 |