A novel assay incorporating CRISPR with RPA in a single pot for visual and accurate detection of Aphelenchoides besseyi in soybean

文献类型： 外文期刊

第一作者： Elhamouly, Neveen Atta

作者： Elhamouly, Neveen Atta;Liu, Shiming;Tu, Yuyao;Peng, Deliang;Elhamouly, Neveen Atta;Atta, Nehal

作者机构：

关键词： Aphelenchoides besseyi; GSFR syndrome; Soybean; RPA-LFD; RPA-CRISPR; Single-pot reaction; Visual detection

期刊名称：SCIENTIFIC REPORTS （ 影响因子：3.9； 五年影响因子：4.3 ）

ISSN： 2045-2322

年卷期： 2025 年 15 卷 1 期

页码：

收录情况： SCI

摘要： 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.

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