Establishment of Digital PCR Method for Quantitative Detection of Genetically Modified Soybean WYN341GmC

To establish a quantitative detection method with high specificity and sensitivity based on droplet digital PCR (ddPCR), thereby providing reliable technical support for the precise quantification and safety supervision of genetically modified soybean WYN341GmC.

Using the event-specific junction sequence of genetically modified soybean WYN341GmC as the target, specific primers and probes were designed, and the reaction system parameters, including primer and probe concentrations, were optimized. The performance of the method was evaluated in terms of specificity, sensitivity, linear range, trueness, and precision. During method screening, five criteria were primarily considered: stable dual-channel droplet signals in the two-dimensional amplitude plots, clear separation between positive and negative droplets, minimal rain, a sufficient number of valid droplets, and an observed copy number ratio of WYN341GmC to Lectin close to the theoretical value. During method validation, droplet digital PCR, combined with serial dilution experiments using reference materials, was mainly employed to assess the limit of quantification (LOQ), limit of detection (LOD), trueness, and precision of the method.

A duplex ddPCR method for precise quantification was established using the event-specific sequence of WYN341GmC and the soybean endogenous reference gene Lectin as detection targets. Based on the comprehensive evaluation of the above screening criteria, the event-specific primer/probe set WYN341LB-QF2/LB-QR1/LB-QP1 and the endogenous reference primer/probe set LectinQF/QR/QP were ultimately selected for subsequent experiments. For the reaction system, the two primer/probe sets were used at the same concentration levels, with a primer concentration of 0.8 μmol·L^-1 and a probe concentration of 0.4 μmol·L^-1, and the annealing/extension temperature was optimized to 58 ℃. Under these ddPCR conditions, the transgene and endogenous reference droplets exhibited clear clustering and stable partitioning, and the method showed good precision. Performance validation demonstrated that the LOD of the method was 8 copies and the LOQ was 17 copies, indicating that the method was suitable for reliable quantification. Within the dynamic range of 17 to 2.08×10⁴ copies, the coefficients of determination (R²) of the regression curves were 1.000 for WYN341GmC and 0.999 for Lectin, demonstrating a strong linear relationship between the theoretical and measured template copy numbers and confirming the suitability of the method for quantitative detection within this range. When applied to blind soybean samples, the method enabled accurate quantification of WYN341GmC content, with the bias in value assignment controlled within 25%.

A duplex ddPCR method for the quantification of genetically modified soybean WYN341GmC was established. The detection targets, reaction system, and assay conditions were clearly defined, and the relevant methodological validation was completed. This method can be used on the ddPCR platform for the precise quantification of WYN341GmC event content.