ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Veterinary and Zoonotic Infection
Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1621012
This article is part of the Research TopicCutting-edge diagnostics in livestock disease managementView all articles
Rapid and Accurate Detection Method for Bluetongue Virus Based on CRISPR-Cas13a Combined with RT-ERA
Provisionally accepted
- Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
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Bluetongue virus (BTV), a vector-borne pathogen of domestic and wild ruminants, poses substantial global threats to livestock health and trade. Conventional detection methods, such as RT-qPCR, remain constrained by reliance on specialized equipment and prolonged turnaround times, limiting their utility in field settings. To overcome these challenges, we developed an integrated isothermal amplification-CRISPR detection platform—Reverse Transcription-Enzymatic Recombinase Amplification coupled with CRISPR-Cas13a (RT-ERA/CRISPR-Cas13a)—enabling rapid, sensitive, specific and visual pan-serotype detection of BTV. The assay demonstrated a sensitivity of 20 RNA copies/reaction within 55 min using three readout modalities: fluorescence values, visual fluorescence signals, and lateral flow test strips. Specificity evaluation revealed no cross-reactivity with 9 non-target pathogens, including epidemiologically significant viruses such as EHDV, AKAV, and CHUV. Clinical validation using 263 field samples demonstrated that RT-ERA/CRISPR-Cas13a achieved clinical sensitivities of 100%, 100%, and 96% with fluorescence values, fluorescence signals, and lateral flow strips, respectively, while maintaining 100% clinical specificity via all modalities. Field adaptation using Nucleic Acid Release Reagent (NARR) simplified crude sample processing, delivering 97% clinical sensitivity and 100% clinical specificity even in the presence of inhibitors from unpurified samples. In conclusion, this work represents the first CRISPR-Cas13a-based platform for pan-serotype BTV detection, combining portability, cost-efficiency, and detective accuracy suitable for point-of-care deployments. By bridging the gap between high laboratory sensitivity and practical field applicability, this system enables real-time BTV surveillance and facilitates timely outbreak containment in resource-constrained agricultural and veterinary settings.
Keywords: Bluetongue virus, Crispr-cas13a, RT-ERA, on-site detection, visualization Reverse Transcription Enzymatic Recombinase
Received: 30 Apr 2025; Accepted: 06 Aug 2025.
Copyright: © 2025 Zhou, Yu, Shao, Gao, Xia and Qi. 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:
Changyou Xia, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Yinglin Qi, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
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