Pathogens significantly restrict the production of Brassica rapa (Brassica rapa L. ssp. Pekinensis), with climate change and evolving planting patterns exacerbating disease prevalence. Multi-channel rapid diagnostic methods in the field can facilitate the early detection and control of diseases in B. rapa. Here, we established a multi-channel lateral flow biosensor (LFB) combined with a CRISPR/Cas12a cleavage assay for the simultaneous detection of four B. rapa diseases. Key innovations of this study include: (1) High specificity and sensitivity, down to pathogen concentrations of 1.5 pg/μL—due to the optimization of crRNA secondary structure: the more stable the crRNA, the higher its detection sensitivity. (2) Optimized visual detection parameters. We identified ideal concentration ratios for the visual fluorescence detection system: 50 nM Cas12a, 50 nM crRNA, and 500 nM ssDNA fluorescent probe. Furthermore, the optimal concentrations of components on the LFB detection system were 3 μL SA-GNPs, 500 nM ssDNA test strip probe, 0.5 mg/mL biotin-BSA as the test line, and 1 mg/mL anti-FITC as the control line. (3) Field-Ready Cas-AIRPA Platform. We developed the on-site Cas-AIRPA platform for the simultaneous detection of B. rapa pathogens by combining rapid nucleic acid extraction and a four-channel lateral flow biosensor (4-LFB), which quickly provides disease-related information through a specific two-dimensional barcode. Analysis of B. rapa samples in the field confirmed the suitability of the Cas-AIRPA platform for rapid (~25 min) and simultaneous on-site detection of four diseases of B. rapa. This platform can also be adapted to detect other plant diseases in the field.

中文翻译：

---

一种基于 CRISPR 的多通道方法，用于现场快速、灵敏地检测甘蓝四种病害


病原体显着限制了 Brassica rapa （Brassica rapa L. ssp. Pekinensis） 的生产，气候变化和不断发展的种植模式加剧了疾病的流行。田间多通道快速诊断方法可促进白菜病害的早期发现和控制。在这里，我们建立了一个多通道侧向流生物传感器 （LFB） 结合 CRISPR/Cas12a 切割测定法，用于同时检测四种 B. rapa 疾病。本研究的主要创新包括：（1） 由于 crRNA 二级结构的优化，病原体浓度低至 1.5 pg/μL，特异性和灵敏度高：crRNA 越稳定，其检测灵敏度越高。（2） 优化视觉检测参数。我们确定了视觉荧光检测系统的理想浓度比：50 nM Cas12a、50 nM crRNA 和 500 nM ssDNA 荧光探针。此外，LFB 检测系统上组分的最佳浓度为 3 μL SA-GNPs、500 nM ssDNA 试纸探针、0.5 mg/mL 生物素-BSA 作为测试线，1 mg/mL 抗 FITC 作为对照线。（3） 现场就绪的 Cas-AIRPA 平台。我们开发了现场 Cas-AIRPA 平台，通过结合快速核酸提取和四通道侧向层析生物传感器 （4-LFB） 同时检测 B. rapa 病原体，该平台通过特定的二维条形码快速提供疾病相关信息。现场对 B. rapa 样品的分析证实了 Cas-AIRPA 平台适用于快速 （~25 min） 和同时现场检测 B. rapa 的四种病害。该平台还可以用于检测田间的其他植物病害。