Li Zhiting, Feng Wanzhen, Zhu Zaobing, Lu Shengdan, Lin Mingze, Dong Jiali, Wang Zhixin, Liu Fuxiu, Chen Qinghe
School of Breeding and Multiplication, School of Tropical Agriculture and Forestry, Hainan University, Sanya, China.
Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Ministry of Education, Hainan University, Haikou, China.
Front Microbiol. 2024 Jun 5;15:1390422. doi: 10.3389/fmicb.2024.1390422. eCollection 2024.
is a devastating plant pathogen that causes soybean root rot worldwide. Early on-site and accurate detection of the causal pathogen is critical for successful management. In this study, we have developed a novel and specific one-pot RPA/PCR-CRISPR/Cas12 assay for on-site detection (Cas-OPRAD) of root rot (). Compared to the traditional RPA/PCR detection methods, the Cas-OPRAD assay has significant detection performance. The Cas-OPRAD platform has excellent specificity to distinguish 33 from closely related oomycetes or fungal species. The PCR-Cas12a assay had a consistent detection limit of 100 pg. μL, while the RPA-Cas12a assay achieved a detection limit of 10 pg. μL. Furthermore, the Cas-OPRAD assay was equipped with a lateral flow assay for on-site diagnosis and enabled the visual detection of on the infected field soybean samples. This assay provides a simple, efficient, rapid (<1 h), and visual detection platform for diagnosing root rot based on the one-pot CRISPR/Cas12a assay. Our work provides important methods for early and accurate on-site detection of root rot in the field or customs fields.
是一种具有毁灭性的植物病原体，在全球范围内导致大豆根腐病。早期进行现场准确检测致病病原体对于成功防治至关重要。在本研究中，我们开发了一种新颖且特异的用于根腐病现场检测（Cas-OPRAD）的一锅式RPA/PCR-CRISPR/Cas12检测法。与传统的RPA/PCR检测方法相比，Cas-OPRAD检测法具有显著的检测性能。Cas-OPRAD平台对区分33种与密切相关的卵菌或真菌物种具有出色的特异性。PCR-Cas12a检测法的一致检测限为100 pg·μL，而RPA-Cas12a检测法的检测限达到10 pg·μL。此外，Cas-OPRAD检测法配备了用于现场诊断的侧向流动检测法，并能够在感染田间大豆样品上实现对的可视化检测。该检测法基于一锅式CRISPR/Cas12a检测法提供了一个简单、高效、快速（<1小时）且可视化的检测平台，用于诊断根腐病。我们的工作为在田间或海关现场早期准确检测根腐病提供了重要方法。
