Development and Validation of Event-Specific PCR Method for the Quantification of Genetically Modified Soybean DBN8205

The genetically modified soybean DBN8205 event has been approved for a biosafety certification (for commercial application) in China, and its derived varieties are nearing commercial planting. This study aimed to establish an event-specific detection method for DBN8205 to support the implementation of biosafety regulations and the threshold labeling policy for genetically modified organisms.

Event-specific primers and probes were designed based on the unique molecular characteristics of the DBN8205 event. The optimal primer/probe set was selected by comparing the amplification curves and Ct values of multiple combinations. The performance of the DBN8205 event-specific PCR method, including specificity, limit of detection (LOD), limit of quantification (LOQ), dynamic range, and quantitative accuracy, was thoroughly evaluated on both real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) platforms. A collaborative trial was conducted across eight laboratories to validate the qPCR method, with the resulting data statistically analyzed to evaluate its repeatability and reproducibility.

The primer/probe combination DBN8205-QF/QR/QP was identified as optimal, producing a 120 bp amplicon specifically identified as optimal the DBN8205 event with high specificity. On the qPCR platform, the LOD and LOQ were 10 and 40 copies, respectively. The standard curve exhibited excellent linearity (R²>0.99) over a dynamic range from 40 to 8.2×10⁴ copies, allowing for accurate quantification of samples with DBN8205 content as low as 0.1%. The collaborative validation of eight laboratories confirmed that the qPCR method demonstrated good repeatability and reproducibility. On the ddPCR platform, the LOD and LOQ were identical to those of qPCR. The duplex ddPCR assay also showed a good linear correlation between measured and expected values within the range of 40 to 8.0×10⁴ copies and provided more precise quantification for low-concentration samples (0.1%) compared to qPCR. A t-test indicated no significant difference between the quantitative results obtained from qPCR and ddPCR, demonstrating good consistency between the two platforms.

The established DBN8205 event-specific PCR method enables unambiguous identification and accurate quantification of the DBN8205 event in products on both qPCR and ddPCR platforms.