基于PCR-LFS的CRISPR/Cas9基因编辑元件快速检测技术及其在转基因作物监测中的应用OA

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.