Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu, China.
Jiangsu Provincial Key Construction Laboratory of Special Biomass Resource Utilization, Nanjing Xiaozhuang University, Nanjing, China.
Front Cell Infect Microbiol. 2023 May 26;13:1208837. doi: 10.3389/fcimb.2023.1208837. eCollection 2023.
is among the most devastating pathogens of soybean (Glycine max) and severely impacts soybean production in several countries. The resulting disease can be difficult to diagnose and other Phytophthora species can also infect soybean. Accurate diagnosis is important for management of the disease caused by .
In this study, recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system were used for detection of . The assay was highly specific to .
The test results were positive for 29 isolates of , but negative for 64 isolates of 29 Phytophthora species, 7 Phytopythium and Pythium species, 32 fungal species, and 2 Bursaphelenchus species. The method was highly sensitive, detecting as little as 10 pg.µL of genomic DNA at 37°C in 20 min. The test results were visible under UV light and readout coming from fluorophores. In addition, was detected from natural inoculated hypocotyls of soybean seedlings using this novel assay. The rapidity and accuracy of the method were verified using 30 soybean rhizosphere samples.
In conclusion, the RPA-CRISPR/Cas12a detection assay developed here is sensitive, efficient, and convenient, and has potential for further development as a kit for monitoring root rot of soybean in the field.
是大豆（Glycine max）最具破坏性的病原体之一，严重影响了多个国家的大豆产量。由此产生的疾病可能难以诊断，其他疫霉菌也会感染大豆。准确的诊断对于管理由引起的疾病非常重要。
在本研究中，重组酶聚合酶扩增（RPA）与 CRISPR/Cas12a 系统结合用于检测。该检测方法对具有高度特异性。
该检测方法对 29 个分离株呈阳性，但对 29 种疫霉菌、7 种 Phytopythium 和 Pythium 种、32 种真菌种和 2 种 Bursaphelenchus 种的 64 个分离株呈阴性。该方法非常灵敏，在 37°C 下，20 分钟即可检测到低至 10 pg.µL 的基因组 DNA。在紫外光下可见检测结果，并可读取荧光团的读数。此外，该新型检测方法可从大豆幼苗的天然接种下胚轴中检测到。通过 30 个大豆根际样本验证了该方法的快速性和准确性。
总之，这里开发的 RPA-CRISPR/Cas12a 检测方法具有灵敏、高效和便捷的特点，有潜力进一步开发为田间监测大豆根腐病的试剂盒。
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