Hussain Rafakat, Zhao Bing-Yu, Aarti Aarti, Yin Wei-Xiao, Luo Chao-Xi
National Key Laboratory for Germplasm Innovation Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan 430070, China.
Hubei Key Laboratory of Plant Pathology, Huazhong Agricultural University, Wuhan, China.
Plant Dis. 2025 Jun;109(6):1244-1253. doi: 10.1094/PDIS-09-24-1979-RE. Epub 2025 Jun 4.
The fungus is a devastating plant pathogen that causes gray mold rot in many fruits and vegetables, leading to substantial losses globally. The pathogen induces a broad range of symptoms, making it challenging to differentiate from other pathogens, especially when plants exhibit no symptoms during visual inspection. Therefore, an efficient early warning and rapid detection system is crucial for surveillance and monitoring plant health, as well as for quantitative pathogen risk assessment to reduce microbial risks. In this study, we developed a single-tube detection method for based on RPA/Cas12a (Bc-RPA/Cas12a), which can be performed under field conditions and visualized using mini-UV light and ordinary mobile phone light within 45 min at a constant temperature. The Bc-RPA/Cas12a assay can detect without cross-reactivity with other pathogens and without compromising sensitivity. The universality of the Bc-RPA/Cas12a assay was demonstrated through its adaptability across different geographical regions of China. Furthermore, field simulation assays revealed that fruits inoculated with the target pathogen showed fluorescence, whereas fruits inoculated with nontargeted pathogens and double-distilled water did not exhibit fluorescence. The findings suggest that this assay can be applied directly in field conditions for rapid and accurate detection of without the need for specialized equipment.
这种真菌是一种具有破坏性的植物病原体，会在许多水果和蔬菜上引发灰霉腐烂病，在全球范围内导致大量损失。该病原体引发的症状多种多样，这使得它很难与其他病原体区分开来，尤其是当植物在肉眼检查时没有症状的情况下。因此，一个高效的早期预警和快速检测系统对于监测和监控植物健康以及进行病原体风险定量评估以降低微生物风险至关重要。在本研究中，我们开发了一种基于RPA/Cas12a（Bc-RPA/Cas12a）的单管检测方法，该方法可在野外条件下进行，并能在恒温45分钟内使用小型紫外线灯和普通手机灯光进行可视化检测。Bc-RPA/Cas12a检测方法能够检测该真菌，且与其他病原体无交叉反应，同时不影响灵敏度。通过在中国不同地理区域的适用性，证明了Bc-RPA/Cas12a检测方法的通用性。此外，野外模拟试验表明，接种目标病原体的水果呈现荧光，而接种非目标病原体和双蒸水的水果则未显示荧光。研究结果表明，该检测方法无需专门设备即可直接应用于野外条件下，用于快速准确地检测该真菌。
