一种基于 MIRA-CRISPR/EsCas13d的猪腺病毒 3型可视化快速检测方法

Development of a visual and rapid detection method for porcine adenovirus type 3 based on MIRA-CRISPR/EsCas13d

摘要:

背景 猪腺病毒3型(porcine adenovirus type 3, PAdV-3)在猪群中广泛传播，具有潜在的致病性和跨种传播风险，亟需建立快速、灵敏的现场诊断方法以加强早期发现与防控。 目的 构建一种操作简便、反应快速、结果可视化的一步法核酸检测平台，实现PAdV-3的现场快速检测。 方法 本平台基于多酶恒温快速核酸扩增(multi-enzyme isothermal rapid amplification, MIRA)与CRISPR/EsCas13d体系，将核酸扩增、体外转录及Cas13d介导的切割反应集成于单一反应体系中。通过优化引物与CRISPR RNA (crRNA)序列降低背景干扰，提高检测灵敏度；采用简易裂解法和冻干反应试剂提升系统稳定性与现场适用性。检测结果可通过qPCR仪读取、紫外光下管内荧光观察和侧向层析试纸条3种方式判断。 结果 优化后平台在常温或体温条件下均可稳定运行，检测限可达60 copies/μL，与RT-qPCR检测结果一致性达100%。整个检测流程最快可在35 min内完成。 结论 本研究建立的MIRA-CRISPR/EsCas13d一步法可视化核酸检测平台具备快速、灵敏、特异、便捷等优势，适用于猪腺病毒3型的现场检测，也为其他重大动物疫病的基层分子诊断提供了技术参考。

Abstract:

Background Porcine adenovirus type 3 (PAdV-3) is widely prevalent in swine populations and poses potential pathogenic and cross-species transmission risks, highlighting the urgent need for rapid and sensitive point-of-care diagnostic tools to support early detection and control. Objective This study aimed to establish a simple, rapid, and visual one-pot nucleic acid detection platform for point-of-care detection of PAdV-3. Methods The platform integrated multiple steps: multi-enzyme isothermal rapid amplification (MIRA), in vitro transcription, and CRISPR/EsCas13d-mediated cleavage, into a single reaction system. Primer and crRNA sequences were optimized to reduce background noise and improve sensitivity. A simple lysis protocol and lyophilized reagents were employed to enhance stability and field applicability. Detection outcomes could be interpreted by qPCR instrument readout, fluorescence visualization under UV light, or lateral flow strips. Results The optimized platform operated reliably under ambient or physiological temperatures, with a detection limit of 60 copies/μL and 100% concordance with RT-qPCR. The entire workflow could be completed within 35 min. Conclusion The MIRA-CRISPR/EsCas13d one-pot visual nucleic acid detection platform developed in this study is rapid, sensitive, specific, and user-friendly. It is suitable for field detection of PAdV-3 and provides a technical reference for molecular diagnosis of other major animal diseases in primary-level institutions.