IntroductionGenetically modified (GM) soybeans are the predominant GM crop, accounting for 48.9% of the global GM crop planting area and 73.7% of the global soybean planting area1. Given this prevalence, GM soybeans are likely to be used as raw materials in food processing. However, the safety of GM crops has long been a focal point of public concern, prompting many countries to implement quantitative labeling of GM ingredients in food2,3. To accurately determine the content of GM crops in food, standard reference materials for GM detection have been developed4,5. By integrating corresponding molecular biotechnologies and rigorous statistical methods, these reference materials have become a crucial foundation for GMO safety supervision, qualitative and quantitative testing of GMO products, and the research and standardization of detection methods6. They effectively enhance the scientific validity, reliability, and traceability of GMO detection results, thereby ensuring high-quality analytical data.Genomic DNA reference materials are one of the three primary types of standard materials used for testing genetically modified (GM) products7,8,9,10. They are typically extracted from tissues of GM and non-GM products, such as seeds and leaves. This type of reference material offers several advantages, including ease of large-scale preparation, excellent uniformity, precise quantification, and convenient application. As a result, they have been widely adopted in the detection of genetically modified organisms (GMOs). However, compared to plasmid reference materials and matrix reference materials, genomic DNA reference materials tend to be less stable and require more stringent conditions for storage and transportation11.Digital PCR (dPCR) technology has emerged as a powerful tool for the absolute quantification of nucleic acids. By directly measuring the copy number of DNA molecules, dPCR eliminates the need for a standard curve, thereby enhancing accuracy and reliability12,13,14,15,16. This feature makes it particularly suitable for quantifying the characteristic values of DNA standard substances, including genomic DNA reference materials.Transgenic soybean line Zhonghuang 6106 is a novel herbicide-tolerant soybean variety developed by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. It incorporates the GAT and G2-EPSPS genes, which confer herbicide tolerance. Currently, China has established corresponding qualitative testing standards for this variety17. However, there have been no reports on the development of nucleic acid reference materials for this specific transgenic soybean. Given this gap, the present study focuses on the development of a genomic DNA reference material for the herbicide-tolerant transgenic soybean Zhonghuang6106. The aim is to provide a scientific and effective basis for food-safety supervision and the detection of GMO components, thereby enhancing the accuracy and reliability of GMO testing and regulatory oversight.ResultsThe results of gDNA assessmentThe qualified gDNA was diluted to a concentration of 25 ng·µL−1 using a 0.1× TE solution. Subsequently, the gDNA from Zhonghuang 6106 soybean was mixed with that from its non-transgenic recipient (Zhonghuang 10) at a ratio of 1:20. Three aliquots were taken from the top, middle, and bottom of the mixture tube, respectively, yielding nine samples in total. The copy number concentration and copy number ratio of each DNA solution were then measured by dPCR to assess mixing uniformity. Results (Table S1) showed no significant differences in either metric among samples taken from different positions.Sanger sequencing was performed on the dPCR amplicons, and the resulting sequences were aligned—using DNAMAN v9.0 software—against the signature sequence of Zhonghuang 6106 and the corresponding specific regions of other genetically modified crops (Fig. S1). The results showed that the specific sequence of Zhonghuang 6106 displayed partial homology with the promoter regions of GM soybean MON89788,GM maize 3272, GM canola MON88302 and GM rice T1c-19. Importantly, the diagnostic primers for Zhonghuang 6106 were designed outside this region, ensuring the PCR assay’s specificity. To confirm specificity, real-time PCR specificity assays were performed on 100% samples of the above high-homology transformants; none produced detectable amplification (Fig. S2). Moreover, the amplified sequence of Zhonghuang 6106 did not align with any sequences from other GM crops. To further assess the amplification specificity of the Zhonghuang 6106 specific primers and probe in the dPCR system, the following templates were tested: gDNA from Zhonghuang 6106 soybean, GM soybean, GM rice, GM corn, GM cotton, GM canola, non-GM soybean (NTC), and ddH₂O (CK). Only the Zhonghuang 6106 gDNA produced positive droplets, whereas all other samples were negative (Fig. S3). This confirms that the dPCR system exhibits excellent specificity for Zhonghuang6106.The results of homogeneity assessmentForty-five randomly selected gDNA samples of Zhonghuang 6106 were analyzed using dPCR, and the measured copy values are presented in Table S2. The copy number ratio of Zhonghuang 6106 to Lectin was calculated based on the specific sequence and the copy number of the internal standard gene. The measured data were analyzed by one-way ANOVA (F-test), and the results showed that F \(s_{2}^{2}\), the uncertainty introduced by the homogeneity of copy number ratio was estimated as: ubb=sbb =\(\:\sqrt{{\:(}{{\text{}\text{s}}_{\text{1}}}^{\text{2}}\text{-}\:{{\text{s}}_{\text{2}}}^{\text{2}}\text{)/n}}\:\)=\(\:\sqrt{\frac{\text{6.60}\times{\text{10}}^{\text{-7}}\text{-5.56}\times{\text{10}}^{\text{-7}}}{\text{3}}}\)=0.00018. The corresponding relative uncertainty: urel(bb)=ubb/\(\:\stackrel{-}{x}\)=0.00018/0.05=0.0036. For the copy number concentration of Zhonghuang6106, the homogeneity uncertainty was: ubb=sbb =\(\:\sqrt{{\:(}{{\text{}\text{s}}_{\text{1}}}^{\text{2}}\text{-}\:{{\text{s}}_{\text{2}}}^{\text{2}}\text{)/n}}\:\)=\(\:\sqrt{\frac{\text{3.74}\times{\text{10}}^{\text{3}}\text{-2.97}\times{\text{10}}^{\text{3}}}{\text{3}}}\)=16.00. The relative uncertainty: urel(bb)=ubb/\(\:\stackrel{-}{x}\)=16.00/1.01 × 103=0.016. Therefore, the gDNA CRM of Zhonghuang 6106 exhibits excellent homogeneity.Table 1 Results of homogeneity analysis.Full size tableFig. 1Schematic diagram of homogeneity test results. Each data point represents the measured value from individual vials, with error bars indicating the standard error. The dashed red line denotes the certified value.Full size imageThe results of stability assessmentThe results of short-term stability assessmentThrough digital PCR analysis, it was found that after 10 days of storage at −20 °C, 4 °C, 25 °C, and 37 °C, the copy number ratio of Zhonghuang 6106 to Lectin remained within the acceptable range, indicating good short-term stability (Table S3, Fig. 2A-H). The slope was not significant, as determined by the condition\(\:\mid{\upbeta}\text{1}\mid{