A multi-channel CRISPR-based method for rapid, sensitive detection of four diseases of Brassica rapa in the field

IF 8.7 1区 农林科学 Q1 Agricultural and Biological Sciences
Xiaojing Liu, Tongbing Su, Xiaoyun Xin, Peirong Li, Weihong Wang, Cancan Song, Xiuyun Zhao, Deshuang Zhang, Yangjun Yu, Jiao Wang, Ning Li, Miao Wang, Fenglan Zhang, Shuancang Yu
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Abstract

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.
一种基于多通道crispr的快速、灵敏检测油菜四种病害的方法
病原菌显著限制了油菜(Brassica rapa L. ssp)的产量。随着气候变化和不断演变的种植模式加剧了疾病的流行。现场多通道快速诊断方法可促进rapa病害的早期发现和控制。在这里,我们建立了一种多通道横向流动生物传感器(LFB)结合CRISPR/Cas12a切割实验,用于同时检测四种rapa B.疾病。本研究的主要创新点包括:(1)特异性和灵敏度高,病原菌浓度可达1.5 pg/μ l——由于crRNA二级结构的优化,越稳定的crRNA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。LFB检测系统的最佳组分浓度为3 μL SA-GNPs, 500 nM ssDNA试纸探针,0.5 mg/mL生物素- bsa为检测线,1 mg/mL抗fitc为对照线。(3)现场就绪Cas-AIRPA平台。结合快速核酸提取和四通道横向流动生物传感器(4-LFB),通过特定的二维条形码快速提供疾病相关信息,开发了现场Cas-AIRPA平台,用于rapa病原菌的同时检测。现场rapa样品分析证实了Cas-AIRPA平台对rapa四种病害的快速(~25 min)现场同时检测的适用性。该平台还可用于现场检测其他植物病害。
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来源期刊
Horticulture Research
Horticulture Research Biochemistry, Genetics and Molecular Biology-Biochemistry
CiteScore
11.20
自引率
6.90%
发文量
367
审稿时长
20 weeks
期刊介绍: Horticulture Research, an open access journal affiliated with Nanjing Agricultural University, has achieved the prestigious ranking of number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2022. As a leading publication in the field, the journal is dedicated to disseminating original research articles, comprehensive reviews, insightful perspectives, thought-provoking comments, and valuable correspondence articles and letters to the editor. Its scope encompasses all vital aspects of horticultural plants and disciplines, such as biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.
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