基于DNAzyme信号放大的转基因大豆DBN9004快速检测方法OA

[Objective]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.[Method]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(125-1 000 nmol·L-1),and RPA primer ratios(10 000﹕10 000 nmol·L-1-10 000﹕31.25 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.[Result]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).[Conclusion]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.