A one-pot Cas12i3/Cas13d-based assay for rapid portable identification of genotype I and II African swine fever viruses

doi:10.1016/j.jia.2025.02.013

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Zhe Wang¹^,², Haili Wang¹^,², Xinghui Yan^{3, 5}, Jin Li^{3, 5}, Yu Wang^{3, 5}, Guosong Qin³^,⁶^,⁷, Wenbo Sun⁸, Yanfang Wang^4#, Gaiping Zhang¹^,²^,^9#, Jianguo Zhao³^,⁵^,⁶^,^7#

¹Longhu Laboratory, Zhengzhou 450046, China

²Henan Key Laboratory of Immunobiology, Zhengzhou 450001, China

³Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

⁴State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

⁵Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China

⁶Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China

⁷Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China

⁸Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China

⁹School of Advanced Agricultural Sciences, Peking University, Beijing 100871, China

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摘要

非洲猪瘟（ASF）的爆发对我国生猪产业造成了巨大的经济损失。根据基因型的不同，国内流行的非洲猪瘟病毒（ASFV）可分为I型和II型两种。近期研究表明，基于基因II型ASFV为母本研发的减毒疫苗虽然能对同基因型的毒株攻毒产生良好的保护效果，但对基因I型毒株的保护效果不佳。因此，ASFV的快速基因分型方法不但是ASF诊断和流行病学调查的得力工具，更是未来弱毒疫苗使用的重要支撑技术。本文利用基因I型和II型ASFV在B646L基因第1569位处的单核苷酸多态性（SNP），建立了基于正交CRISPR-Cas12i3/Cas13d系统的一管法鉴别检测方案，命名为OBServe.v2，其中具有国内自主知识产权的基因编辑工具Cas12i3应用于核酸检测领域尚属首次报道。以市售qPCR试剂盒（青岛立见）作为参考方法，发现OBServe.v2的分析灵敏度为8拷贝每微升，对临床样本的诊断灵敏度和特异性均为100%，且与常见猪源病毒无交叉反应。进一步将OBServe.v2和PCR-测序方法的的基因分型的结果进行比对，发现两者的符合度为100%。综上所述，本研究建立了一个高灵敏、高特异，但成本较低、可现场操作的ASFV基因分型方法，具有良好的应用和推广前景。

Online: 12 February 2025

Fund:

This work was supported by the National Natural Science Fund for Distinguished Young Scholars (31925036 and 32025034), the Shandong Provincial Key R&D Program of China (2021LZGC001), the Biological Breeding-Major Projects (2023ZD04074, 2023ZD0404604), the Special Project of Longhu Laboratory (LHLab_ZD20230012), the Program of National Natural Science Foundation of China (32230100, 32330099 and 32201257), the National Key Research and Development Program of China (2020YFA0509503, 2022YFF0710703, 2021YFA0805902, 2022XAGG0121, 2022YFF1002803).

About author: #Correspondence Yanfang Wang, E-mail: wangyanfang@caas.cn; Gaiping Zhang, E-mail: zhanggaip@126.com; Jianguo Zhao, E-mail: zhaojg@ioz.ac.cn

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Cite this article:

Zhe Wang, Haili Wang, Xinghui Yan, Jin Li, Yu Wang, Guosong Qin, Wenbo Sun, Yanfang Wang, Gaiping Zhang, Jianguo Zhao. 2025. A one-pot Cas12i3/Cas13d-based assay for rapid portable identification of genotype I and II African swine fever viruses. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.02.013

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