PCR-LFS-Based Rapid Detection Technology for CRISPR/Cas9 Gene-editing Elements and Its Application in Transgenic Crop Monitoring
GAO Jie-Yu1, DING Liu1, KONG Wei-Heng2, YOU Zheng-He1, LI Yun-Fei1, ZONG Kai1, SUN Juan-Juan1, YU Hua-Zheng1, HU Kang-Di3, HAN Fang1, ZENG De-Xin1,*, YU Xiao-Feng1,*
1 Technical Center of Hefei Customs/Anhui Provincial Key Laboratory of Food Safety Monitoring and Quality Control, Hefei 230022, China; 2 Science and Technology Research Center of China Customs, Beijing 100026, China; 3 School of Food and Biological engineering, Hefei University of Technology, Hefei 230601, China
Abstract：The widespread application of CRISPR/Cas9 gene editing technology in crop breeding has demonstrated significant potential for improving crop resistance, yield, and quality. However, this progress has also heightened the need for effective detection of gene-edited components. A significant number of crop samples containing non-excised exogenous CRISPR/Cas9 elements remain prevalent during early breeding stages and under specific conditions. However, existing detection methods are associated with limitations such as low sensitivity, time-consuming procedures, or dependence on sophisticated instrumentation. To meet the growing demand for rapid detection of CRISPR/Cas9-edited crops, a highly sensitive method integrating PCR with lateral flow strips (PCR-LFS) was developed in this study. Cas9-specific primers were designed and the amplification system was optimized, allowing rapid visual detection using colloidal gold-labeled test strips. A detection limit of 0.012% (by mass fraction) and sensitivity of 17.5 copies/μL were achieved, outperforming conventional PCR-capillary gel electrophoresis (0.1%) and quantitative PCR (1.75×104 copies/μL). Specificity testing confirmed that Cas9-positive tomato (Solanum lycopersicum) samples were accurately distinguished from non-transgenic crops without cross-reactivity. Practical validation demonstrated that reliable detection was achieved in mixed samples at a ratio of 1∶213, with high repeatability and stability. Compared with traditional techniques, this method eliminated the need for complex instruments, reduced detection time to 3 h, and was suitable for on-site screening and port inspections. It was suggested that future optimization of primer design and LFS procedures might extend the application to other gene-edited components. This study provides strong technical support for precise monitoring of genetically modified crops.
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