Rapid and Visual On-Site Detection System for Epizootic Hemorrhagic Disease Virus Based on a Combination of CRISPR-Cas12a and RT-ERA

doi:10.1016/j.jia.2025.09.023

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Dong Zhou^{1, 2, 3*}, Junyong Guan^1*, Haibo Yu^{1, 2, 3}, Yuntong Shao^{1, 2, 3}, Changyou Xia^{1, 2, 3}, Caixia Gao^{1, 2, 3#}, Yinglin Qi^{1, 2, 3#}

¹ State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

² Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin 150069, China

³ National Poultry Laboratory Animal Resource Center, Harbin 150069, China

Highlights:

1. First development of a RT-ERA/CRISPR-Cas12a platform for rapid and visual detection of EHDV, enabling on-site diagnosis within 1 hour without specialized equipment.

2. High sensitivity and broad serotype coverage: The assay detects as low as 1.7 × 10¹ copies reaction^-1 and recognizes 8 EHDV serotypes (1, 2, 4–8, and 10), overcoming the limitation of serotype-specific antisera required in traditional methods.

3. Integration with HUDSON sample processing allows direct detection from crude samples without RNA extraction, making it suitable for field use in resource-limited settings and supporting point-of-care testing in ruminant populations.

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摘要

目的：流行性出血病（Epizootic Hemorrhagic Disease, EHD）是一种由流行性出血病病毒（EHDV）引起、经库蠓传播的虫媒传染病，感染野生及家养反刍动物，被世界动物卫生组织（WOAH）列为须通报动物疫病。近年来我国监测显示多个EHDV血清型在南方省份流行，且血清学阳性率极高，暴发风险严峻。然而，目前缺乏适用于现场、无需复杂仪器的快速检测技术。本研究旨在开发一种基于RT-ERA和CRISPR-Cas12a技术的EHDV核酸检测新方法，以实现对EHDV的高灵敏、高特异、快速且可视化的现场检测。

方法：本研究首先通过对EHDV不同血清型基因组序列进行比对分析，选定高度保守的S1基因片段作为检测靶标，并设计特异性crRNA。通过荧光检测法筛选并优化了CRISPR-Cas12a系统中的crRNA及Cas12a蛋白的最佳工作浓度。随后，针对该靶标设计了多对RT-ERA引物，通过筛选获得了最优扩增引物对（F6/R3）。将优化的RT-ERA扩增体系与CRISPR-Cas12a检测系统联用，构建了RT-ERA/CRISPR-Cas12a检测平台。通过使用梯度稀释的病毒RNA评估了该系统的检测灵敏度；通过检测蓝舌病病毒（BTV）、中山病毒（CHUV）等其他常见反刍动物病原体评估其特异性。最后，使用54份临床样本，分别经传统TRIzol提取法和HUDSON快速处理法处理样本后，将该检测系统与已建立的实时荧光RT-PCR方法进行比较，以评估其临床应用的灵敏度和特异性。

结果：本研究成功建立了EHDV的RT-ERA/CRISPR-Cas12a检测方法。优化的CRISPR-Cas12a系统在75 ng Cas12a蛋白和400 nM crRNA1条件下效果最佳。此外，最优RT-ERA引物对为F6/R3。该联用检测系统的灵敏度极高，荧光读值法和横向流动试纸条法的检测下限分别可达1.7 × 10¹拷贝/反应和1.7 × 10²拷贝/反应。特异性试验表明，该系统能有效检测EHDV-1, 2, 4-8, 10共8种血清型，而对BTV等其他病原体无一交叉反应。在54份临床样本检测中，基于TRIzol提取RNA的方法与实时荧光RT-PCR结果完全一致（灵敏度与特异性均为100%）；基于HUDSON快速处理的样本，其检测灵敏度为96%，特异性仍保持100%，可在无需核酸纯化的条件下实现快速检测。

Online: 23 September 2025

Fund:

This study was supported by the National Key Research and Development Program of China (Grant No. 2022YFF0710500, No. 2023YFF0724603), Key Research & Development Program of Heilongjiang province (Innovation Base) (JD2023SJ10) and Central Public-interest Scientific Institution Basal Research (1610302023003).

About author: Dong Zhou，Tel: +86+17852877096，E-mail: zhoudong0719@163.com； Junyong Guan, Tel: +86+15237393287, E-mail: guanzky@163.com; #Correspondence Yinglin Qi, Tel: +86+13897547758, E-mail: qiyinglin@caas.cn; Caixia Gao, Tel: +86+15114516823, E-mail: gaocaixia@caas.cn * These authors contributed equally to this study.

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