您好，欢迎访问山东省农业科学院文献资源数据库平台

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus

文献类型： 外文期刊

作者： Luan, Haorui 1 ; Wang, Shujuan 3 ; Ju, Lin 1 ; Liu, Tong 1 ; Shi, Haoyue 1 ; Ge, Shengqiang 3 ; Jiang, Shijin 1 ; Wu, Jiaqiang 4 ; Peng, Jun 1 ;

作者机构： 1.Shandong Agr Univ, Coll Vet Med, Tai An, Peoples R China

2.Shandong Prov Key Lab Anim Biotechnol & Dis Contro, East China Sci Expt Stn Anim Pathogen Biol, Minist Agr & Rural Affairs, Tai An, Peoples R China

3.China Anim Hlth & Epidemiol Ctr, Qingdao, Peoples R China

4.Inst Anim Sci & Vet Med, Shandong Acad Agr Sci, Jinan, Peoples R China

关键词： African swine fever virus; KP177R gene; recombinase polymerase amplification; fluorescein-labeled; biotin-labeled; CRISPR/Cas12

期刊名称：FRONTIERS IN IMMUNOLOGY （ 影响因子：7.3； 五年影响因子：8.0 ）

ISSN： 1664-3224

年卷期： 2024 年 15 卷

页码：

收录情况： SCI

摘要： Introduction: Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. Methods: This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. Results: The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/mu L, and both assays were completed in 30 min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. Discussion: The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

  • 相关文献
作者其他论文 更多>>
置顶
购物车
反馈

© 京ICP备09089781号-29 主办单位：山东省农业科学院文献资源数据库平台

学术资源： 中国农业科学院 农业专业知识服务系统 农科联盟资源共建共享平台 中国农业科技文献与信息服务平台 中国农业期刊集成服务平台