Liu Xiaojing, Su Tongbing, Xin Xiaoyun, Li Peirong, Wang Weihong, Song Cancan, Zhao Xiuyun, Zhang Deshuang, Yu Yangjun, Wang Jiao, Li Ning, Wang Miao, Zhang Fenglan, Yu Shuancang
State Key Laboratory of Vegetable Biobreeding, Beijing Vegetable Research Center (BVRC), Beijing Academy of Agriculture and Forestry Science (BAAFS), No. 50, Zhanghua Road, Haidian District, Beijing 100097, China.
Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture, No. 50, Zhanghua Road, Haidian District, Beijing 100097, China.
Hortic Res. 2024 Dec 12;12(3):uhae351. doi: 10.1093/hr/uhae351. eCollection 2025 Mar.
Pathogens significantly restrict the production of ( L. ssp. Pekinensis), with climate change and evolving planting patterns exacerbating disease prevalence. Multichannel rapid diagnostic methods in the field can facilitate the early detection and control of diseases in . Here, we established a multichannel lateral flow biosensor (LFB) combined with a CRISPR/Cas12a cleavage assay for the simultaneous detection of four 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 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 2D barcode. Analysis of 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 . This platform can also be adapted to detect other plant diseases in the field.
病原体严重限制了（L. ssp. Pekinensis）的产量，气候变化和种植模式的演变加剧了疾病的流行。田间多通道快速诊断方法有助于早期发现和控制（L. ssp. Pekinensis）的疾病。在此，我们建立了一种多通道侧向流动生物传感器（LFB），结合CRISPR/Cas12a切割检测法，用于同时检测四种（L. ssp. Pekinensis）疾病。本研究的关键创新点包括：（1）高特异性和灵敏度，由于优化了crRNA二级结构，病原体浓度低至1.5 pg/μl：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检测系统上各组分的最佳浓度为3 μl SA-GNPs、500 nM ssDNA试纸条探针、0.5 mg/ml生物素-BSA作为检测线，1 mg/ml抗FITC作为对照线。（3）现场可用的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流动生物传感器（4-LFB）同时检测（L. ssp. Pekinensis）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。对田间（L. ssp. Pekinensis）样本的分析证实了Cas-AIRPA平台适用于快速（约25分钟）同时现场检测（L. ssp. Pekinensis）的四种疾病。该平台还可适用于田间检测其他植物疾病。
