基于CRISPR-Cas13a的免扩增电化学生物传感器快速检测牛病毒性腹泻病毒

A CRISPR-Cas13a-based amplification-free electrochemical biosensor for rapid detection of bovine viral diarrhea virus

摘要:

牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)是危害全球养牛业的重要病原体，可导致腹泻、发热及繁殖障碍，造成严重经济损失。建立快速、精准的BVDV检测方法对疫情防控具有重要意义。现有检测方法存在明显局限：PCR等核酸扩增技术需精密仪器且操作复杂；而CRISPR-Cas13a系统虽特异性强，却仍需核酸预扩增，导致检测流程繁琐和污染风险。为开发更简便高效的BVDV现场快速检测技术，本研究将CRISPR-Cas13a系统的特异性识别能力与电化学传感技术的高效信号转换特性相结合，建立了一种免核酸扩增的BVDV检测新方法。通过优化CRISPR RNA (crRNA)组合、反应缓冲液组分及Cas13a/crRNA浓度比等关键参数，该传感器检测限达3 090 copies/μL，较传统Cas13a荧光检测方法灵敏度提升4-5个数量级，可在35 min内完成从加样到结果输出的全过程。特异性实验表明，该方法仅对BVDV产生特异性检测信号，而对其他常见牛源病毒(包括对牛副流感病毒3型、牛呼吸道合胞病毒、蓝舌病病毒和口蹄疫病毒)均未产生可检测的交叉反应信号。22份临床样本验证结果显示，该传感器的特异性和灵敏度均达到100%。本研究建立的CRISPR-Cas13a电化学生物传感器具有免核酸扩增、操作简便等优势，为BVDV的现场快速检测提供了新型高效工具，在临床诊断和疫情防控中具有重要应用价值。

Abstract:

Bovine viral diarrhea virus (BVDV), a major pathogen in the global bovine industry, causes diarrhea, fever, and reproductive disorders, leading to substantial economic losses. Developing the methods for rapid and accurate detection of BVDV is crucial for epidemic control. Current detection methods have notable limitations. PCR-based nucleic acid amplification techniques rely on sophisticated instruments and complex procedures. CRISPR-Cas13a systems, despite their high specificity, still require nucleic acid pre-amplification, which results in cumbersome workflows and contamination risks. To establish a simpler and more efficient on-site detection method for BVDV, this study integrated the CRISPR-Cas13a system characterized by specific recognition with electrochemical sensing praised for efficient signal transduction to establish a novel nucleic acid amplification-free method for the detection of BVDV. Through optimization of key parameters, including CRISPR RNA (crRNA) combination, buffer components, and Cas13a/crRNA concentration ratio, the biosensor achieved a detection limit of 3 090 copies/μL—representing a 4-5 order of magnitude improvement in sensitivity compared with conventional Cas13a fluorescence-based detection—and completed the entire process from sample loading to result output within 35 min. Specificity tests demonstrated that the sensor exclusively detected BVDV without cross-reactivity to other common bovine viruses (bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bluetongue virus, and foot-and-mouth disease virus). Clinical validation with 22 samples demonstrated 100% specificity and sensitivity. The developed CRISPR-Cas13a-based electrochemical biosensor offers the advantages of being nucleic acid amplification-free and operationally simple, serving as a powerful new tool for rapid on-site BVDV detection with significant potential for veterinary diagnostics and epidemic prevention and control.