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Integration of RPA and CRISPR-Cas13a collateral activity for one-step detection of DHAV-3: A biological macromolecule-enabled diagnostic platform

文献类型： 外文期刊

作者： Zhao, Saisai 1 ; Zhang, Jiahao 1 ; Yu, Shumin 1 ; He, Dalin 1 ; Li, Bing 2 ; Fan, Yaru 1 ; Liu, Guocheng 1 ; Tang, Yi 2 ; Diao, Youxiang 1 ;

作者机构： 1.Shandong Agr Univ, Coll Vet Med, Tai An 271018, Shandong Provin, Peoples R China

2.Chinese Acad Agr Sci, Inst Anim Sci, Beijing 10091, Peoples R China

关键词： CRISRPR/Cas13; RPA-CRISPR Cas13a; Lateral flow assay; Duck hepatitis a virus 3

期刊名称：INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES （ 影响因子：8.5； 五年影响因子：8.7 ）

ISSN： 0141-8130

年卷期： 2025 年 314 卷

页码：

收录情况： SCI

摘要： Duck hepatitis A virus type 3 (DHAV-3), which is families of the Picornaviridae, poses severe threats to waterfowl industries due to rapid antigenic evolution and limitations in conventional diagnostics. Herein, we engineered a CRISPR-Cas13a-mediated RNA detection system by leveraging the intrinsic HEPN domain-dependent collateral cleavage activity of Cas13a, synergistically integrated with recombinase polymerase amplification (RPA) to target DHAV-3 RNA. This biological macromolecule-driven platform achieved ultrasensitive detection (1 copies/ mu L) within 35 min through sequence-specific crRNA guidance and Cas13a-triggered fluorescent/lateral flow signal amplification. Rigorous validation against four avian pathogens (ARV, H9N2 AIV, TMUV, AstV) confirmed 100 % specificity, highlighting the precise macromolecular interactions between Cas13a and target RNA. Clinical evaluation of 30 field samples demonstrated complete concordance with RT-qPCR. By harnessing the programmable functionality of Cas13a and the thermostable enzymes in RPA, this study provides a novel paradigm for RNA-guided biological macromolecule applications in point-of-care diagnostics, bridging molecular mechanisms with agricultural biosecurity needs.

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