Feng Xiujuan, Li Yongchang, Li Shiyun, Zafar Iqra, Rizk Mohamed Abdo, Fu Xianyue, Cui Zeyun, Zhang Wei, Zhang Yang, Hu Ercha, Guo Qingyong, Chahan Bayin
Parasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, China.
Laboratory of Sustainable Animal Environment, Graduate School of Agriculture Science, Tohoku University, Sendai, Japan.
Front Microbiol. 2025 Aug 4;16:1647929. doi: 10.3389/fmicb.2025.1647929. eCollection 2025.
, a globally significant blood parasite in livestock, causes substantial economic losses in resource-limited regions by compromising animal health and hindering the development of the livestock industry. To address this, a rapid, reliable, and sensitive diagnostic assay integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a technology was developed. This assay enables visual interpretation through multiple detection modalities, including UV and blue light illumination. Among three primer pairs and two CRISPR RNA (crRNA) candidates screened, the F3/R3 primer set combined with crRNA1 demonstrated the best performance. The optimized ERA protocol achieved complete amplification within 20 min at 37°C. This assay exhibited high specificity for detection, with a sensitivity limit of 10 copies/μL, a 100-fold greater sensitivity than conventional PCR, while completing detection within 40 min. Validation of 51 bovine blood samples from a farm in Turpan, Xinjiang, revealed that PCR detected 12 positive cases (23.5% prevalence), whereas the ERA-CRISPR/Cas12a system identified 15 positive cases (29.4% prevalence). The enhanced detection capability of this integrated method provides crucial technical support for field applications in resource-limited settings, effectively addressing the urgent need for rapid and accurate diagnosis of bovine theileriosis.
是家畜中一种具有全球重要意义的血液寄生虫，通过损害动物健康和阻碍畜牧业发展，在资源有限的地区造成了巨大的经济损失。为了解决这一问题，开发了一种将酶促重组酶扩增（ERA）与CRISPR/Cas12a技术相结合的快速、可靠且灵敏的诊断检测方法。该检测方法能够通过多种检测方式进行视觉解读，包括紫外线和蓝光照射。在筛选的三对引物和两种CRISPR RNA（crRNA）候选物中，F3/R3引物组与crRNA1组合表现出最佳性能。优化后的ERA方案在37°C下20分钟内即可完成完全扩增。该检测方法对检测具有高度特异性，灵敏度极限为10拷贝/微升，比传统PCR灵敏度高100倍，同时能在40分钟内完成检测。对来自新疆吐鲁番一个农场的51份牛血样进行验证发现，PCR检测出12例阳性病例（患病率为23.5%），而ERA-CRISPR/Cas12a系统鉴定出15例阳性病例（患病率为29.4%）。这种集成方法增强的检测能力为资源有限环境下的现场应用提供了关键技术支持，有效满足了快速准确诊断牛泰勒虫病的迫切需求。
