Rapid on-site screening of genetically modified (GM) crops is crucial for effective biosafety regulation. To overcome the limitations of current detection methods, such as equipment dependency and operational complexity, this study developed a closed-tube detection system by integrating recombinase polymerase amplification (RPA) with split DNAzyme (MNAzyme). The system enables rapid, sensitive, and on-site detection of the GM soybean event DBN9004, supporting regulatory compliance and industrial safety management.

Using GM soybean DBN9004 and its non-GM counterpart Jack as experimental materials, we firstly identified event-specific sequences for target detection through bioinformatics analysis. Then a recombinant plasmid (9004P) was constructed as a standard template. An asymmetric RPA system was designed to efficiently amplify the target sequence while generating abundant single-stranded DNA (ssDNA) products for MNAzyme activation. Critical reaction parameters were systematically optimized, including reaction temperature (35-60 ℃), probe concentration (125-1000 nmol·L^-1), and RPA primer ratios (10000: 10000 nmol·L^-1-10000: 31.25 nmol·L^-1). Sensitivity assessment was evaluated using gradient-diluted plasmids (8×10^-1-8×10⁵ copies/μL), while specificity evaluation was verified against ten GM crop lines (GTS40-3-2, ZH10-6, etc.). Field samples (n=13) were tested and compared with qPCR results.

The method demonstrated exceptional sensitivity (8 copies/reaction), good repeatability (RSD=4.44%) and reproducibility (RSD=5.75%), absolute specificity for DBN9004 with no cross-reactivity against ten prevalent GM soybean varieties. Field testing demonstrated perfect concordance (100%) with qPCR results (n=13).

This study implemented an asymmetric RPA strategy to efficiently generate target-specific ssDNA amplicons. The resulting ssDNA products demonstrate specific binding affinity for pre-engineered split DNAzyme subunits (A/B), triggering their activation and subsequent continuous cleavage of fluorophore-quencher labeled substrate probes. Leveraging this molecular mechanism, we established a novel RPA-MNAzyme integrated platform for rapid and reliable detection of genetically modified soybean event DBN9004. By combining asymmetric RPA with MNAzyme cascade amplification, the method achieves dual-specificity recognition and signal enhancement. The closed-tube design prevents aerosol contamination, while the dual-mode output system accommodates both laboratory and on-site screening needs.