National Center for the Molecular Characterization of Genetically Modified Organisms, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Garrison Forest School, Owings Mills, MD, 21117, USA.
Biosens Bioelectron. 2022 May 15;204:114076. doi: 10.1016/j.bios.2022.114076. Epub 2022 Feb 12.
Pathogenic disease is an important factor affecting rice growth, yield and quality, and the development and application of rapid diagnostic methods will contribute to the prevention and control of rice disease. Herein, we developed a novel protospacer adjacent motif (PAM)-free loop-mediated isothermal amplification (LAMP) assisted CRISPR/Cas12a cleavage (Cas-PfLAMP) assay for detection of three rice pathogens; Xanthomonas oryzae pv. Oryzae (XOO), rice stripe virus (RSV), and rice black-streaked dwarf virus (RBSDV). The Cas-PfLAMP assay showed high specificity due to doubly specific recognition of LAMP primer sets and FnCas12a/sgRNA, and high sensitivity down to 9 or 3 copies due to LAMP amplification and CRISPR/Cas12a trans cleavage activity. Furthermore, a visual on-spot Cas-PfLAMP platform was established for detection of rice pathogens by combining solid-phase nucleic acid extraction and a lateral flow strip (LFS) test. Analysis of rice leaf field samples confirmed the impressive performance of the Cas-PfLAMP platform, demonstrating its suitability for rapid (∼50 min) on-spot detection of rice diseases. The assay could also be extended to detection of other plant diseases, and other nucleic acid field tests.
病原病害是影响水稻生长、产量和品质的重要因素，快速诊断方法的发展和应用将有助于水稻病害的防治。在此，我们开发了一种新型的无原间隔基序（PAM）环介导等温扩增（LAMP）辅助 CRISPR/Cas12a 切割（Cas-PfLAMP）检测方法，用于检测三种水稻病原体：稻黄单胞菌（Xoo）、水稻条纹病毒（RSV）和水稻黑条矮缩病毒（RBSDV）。Cas-PfLAMP 检测方法由于 LAMP 引物对和 FnCas12a/sgRNA 的双重特异性识别，表现出高度的特异性，并且由于 LAMP 扩增和 CRISPR/Cas12a 转切割活性，灵敏度高达 9 或 3 拷贝。此外，通过固相核酸提取和侧向流条（LFS）试验建立了用于检测水稻病原体的现场可视化 Cas-PfLAMP 平台。对田间水稻叶片样本的分析证实了该 Cas-PfLAMP 平台的出色性能，表明其适合快速（约 50 分钟）现场检测水稻病害。该检测方法还可扩展用于检测其他植物疾病和其他核酸现场检测。
