Elhamouly Neveen Atta, Atta Nehal, Liu Shiming, Tu Yuyao, Peng Deliang
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 1000193, China.
Department of Agricultural Botany, Faculty of Agriculture, Menoufia University, Shibin El-Kom, 32514, Egypt.
Sci Rep. 2025 Jul 1;15(1):21217. doi: 10.1038/s41598-025-04315-7.
Aphelenchoides besseyi is a highly prevalent plant parasitic nematode which has a substantial impact and poses an economic risk to soybean cultivation, with a reported 2017 outbreak resulting in significant yield losses of up to 60%. Therefore, more effective control of this nematode depends on early and accurate nucleic acid detection. One of the promising detection approaches is to combine the CRISPR technology with the isothermal RPA. However, incorporating the RPA amplicon with the CRISPR ingredients in a single pot remains a significant challenge due to their incompatibility. In the current research, we propose a visual nucleic acid detection technique that takes less than thirty minutes and is highly sensitive for detecting A. besseyi. First, we conduct the RPA amplification, then we perform the CRISPR reaction using either a portable thermal cup or our body heat temperature. We tested this new assay on forty-four soybean samples exhibiting GSFR syndrome symptoms, and it effectively detected samples containing the A. besseyi. We designed three different ways for data collection and visualization to suit the requirements of various environments. Our findings confirm that the suggested new low-instrumentation portable single-pot RPA-CRISPR assay is durable, specific, and has strong nucleic acid sensitivity in the open field.
贝西滑刃线虫是一种广泛存在的植物寄生线虫，对大豆种植有重大影响并构成经济风险，据报道2017年的疫情导致高达60%的显著产量损失。因此，更有效地控制这种线虫依赖于早期和准确的核酸检测。一种有前景的检测方法是将CRISPR技术与等温RPA相结合。然而，由于RPA扩增子与CRISPR成分不相容，在同一容器中混合它们仍然是一个重大挑战。在当前的研究中，我们提出了一种可视化核酸检测技术，该技术耗时不到30分钟，对检测贝西滑刃线虫具有高度敏感性。首先，我们进行RPA扩增，然后使用便携式热杯或体温进行CRISPR反应。我们在44个表现出GSFR综合征症状的大豆样本上测试了这种新方法，它有效地检测出了含有贝西滑刃线虫的样本。我们设计了三种不同的数据收集和可视化方式，以适应各种环境的要求。我们的研究结果证实，所建议的新型低仪器便携式单容器RPA-CRISPR检测方法在野外耐用、特异且具有很强的核酸敏感性。
