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Development of highly sensitive one-pot ERA-CRISPR assays for on-site detection of CaMV35S promoter and NOS terminator in genetically modified crops

文献类型： 外文期刊

作者： Liang, Qianqian 1 ; Wang, Xiaohui 1 ; Xie, Qingqing 1 ; Cha, Shaozhen 1 ; Lei, Jiawen 1 ; Cheng, Guojun 1 ; Yu, Guanghui 1 ; Wu, Yuhua 2 ; Zhang, Li 1 ;

作者机构： 1.South Cent Minzu Univ, Coll Life Sci, Hubei Prov Key Lab Protect & Applicat Special Plan, Wuhan 430074, Peoples R China

2.Chinese Acad Agr Sci, Oil Crops Res Inst, Key Lab Agr Genet Modified Organisms Traceabil, Minist Agr & Rural Affairs, Wuhan 430062, Peoples R China

关键词： Genetically modified organisms; CRISPR/Cas12a; Enzymatic recombinase amplification; One-pot; On-site detection

期刊名称：FOOD CONTROL （ 影响因子：6.3； 五年影响因子：6.1 ）

ISSN： 0956-7135

年卷期： 2025 年 170 卷

页码：

收录情况： SCI

摘要： The integration of nucleic acid isothermal amplification with CRISPR/Cas12a detection technologies has significantly advanced molecular diagnostics. However, the challenge of integrating both assays into a one-pot reaction to reduce operational complexity and the risk of aerosol contamination has persisted. In this study, we developed a one-pot assay that combines these two powerful tools to improve the detection of genetically modified organisms (GMOs). Targeting the widely used Cauliflower Mosaic Virus 35S promoter (P-CaMV 35S) and the nopaline synthase terminator (T-NOS) from Agrobacterium tumefaciens in genetically modified (GM) crops, we employed a definitive screening design (DSD) approach to optimize the balance between ERA amplification and Cas12a activity. This optimization was achieved by enhancing ERA amplification and precisely adjusting the concentrations of Cas12a and other reaction components, resulting in an efficient and streamlined process. The optimized one-pot ERA-CRISPR/Cas12a system achieved a detection sensitivity of 10 copies per reaction for both P-CaMV 35S and T-NOS within 40 min at 40 degrees C, and was capable of detecting GMO content as low as 0.1% in spiked samples. Moreover, with minimal equipment requirements, such as an LED blue light and a smartphone for result interpretation, this method is highly user-friendly. Combining rapid detection, high specificity, and operational simplicity, this system represents a significant advancement in supporting GMO regulation and global trade, and serves as a promising model for the development of future nucleic acid-based assays.

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