A rapid and portable dual-mode RAA-CRISPR/Cas12a system for one-pot detection of spring viraemia of carp virus (SVCV)

文献类型： 外文期刊

第一作者： Hu, Tian

作者： Hu, Tian;Hou, Zewei;Zhao, Yichen;Zhang, Ye;Si, Yufeng;Lu, Liqun;Wang, Hao;Hu, Tian;Hou, Zewei;Zhao, Yichen;Zhang, Ye;Lu, Liqun;Wang, Hao;Si, Yufeng

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关键词： Spring viraemia of carp virus; RAA-CRISPR/Cas12a; Lateral flow test strip; Rapid detection

期刊名称：AQUACULTURE REPORTS （ 影响因子：3.7； 五年影响因子：4.0 ）

ISSN： 2352-5134

年卷期： 2025 年 43 卷

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收录情况： SCI

摘要： Spring viraemia of carp virus (SVCV), a pathogen with significant economic impacts on global aquaculture, necessitates the development of rapid, sensitive diagnostic tools for effective disease management. This study presents two innovative detection methods that integrate recombinase-aided amplification (RAA) with CRISPR/ Cas12a technology: the One-Pot RAA-CRISPR/Cas12a fluorescence assay and the RAA-CRISPR/Cas12a Lateral Flow Dipstick (LFD) assay. By targeting the highly conserved glycoprotein (G) gene of SVCV, optimized RAA primers and CRISPR RNA (crRNA) were designed. Additionally, a sucrose-stabilized biphasic reaction system was developed to facilitate seamless one-pot amplification and detection while minimizing the risk and simplifying operational procedures. The fluorescence-based method exploits trans-cleavage activity of Cas12a to generate detectable fluorescent signals, while the LFD assay employs a FAM-biotin dual-labeled ssDNA probe for visual detection via colloidal gold accumulation on lateral flow strips. Sensitivity analysis revealed detection limits of 102 copies/mu L for the fluorescence assay and 100 copies/mu L for the LFD assay, representing a 10-1000-fold improvement over traditional nested PCR (103 copies/mu L). Specificity testing confirmed no cross-reactivity with common fish pathogens, such as KHV, CyHV-2, GCRV-I, GCRV-II, GCRV-III, or Aeromonas hydrophila. Clinical validation involving 30 samples yielded 100 % concordance with results obtained from the national standard PCR protocol. This portable, instrument-free platform streamlines on-site SVCV detection in aquaculture settings, particularly in resource-constrained environments, by reducing operational complexity and assay time to 90 min. The dual-modality design-offering both qualitative fluorescence and naked-eye colorimetric readouts-supports the critical objectives of early viral detection, rapid intervention, and biosecurity management, positioning it as a transformative tool for global aquatic disease surveillance.

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