Digital PCR-based characterization of certified genomic DNA reference materials from genetically modified papaya Huanong No. 1.

Reliable reference materials (RMs) are essential for the accurate, traceable, and quantitative detection of genetically modified (GM) crops and their products. In this study, leaves of GM papaya Huanong No. 1 (HN1) and non-GM papaya Tainong No. 2 (TN2) were used as raw materials to prepare genomic DNA (gDNA) certified reference materials (CRMs) at two different concentrations. Digital PCR (dPCR) was employed to evaluate their homogeneity and stability and to conduct collaborative characterization. The prepared 25% and 5% HN1 gDNA CRMs exhibited good homogeneity and stability. Through collaborative characterization and uncertainty assessment involving nine laboratories, three property values were established: the copy number concentration of the HN1 event-specific sequence, the copy number concentration of the papain gene, and the copy number ratio. For the 25% HN1 gDNA CRM, the three property values were 6.91 × 10⁴ copies/µL, 2.63 × 10⁵ copies/µL, and 2.64 × 10⁻¹, respectively. For the 5% HN1 gDNA CRM, the values were 1.27 × 10⁴ copies/µL, 2.50 × 10⁵ copies/µL, and 5.08 × 10⁻². The minimum sample intake required for the use of these CRMs is 2 µL. These materials provide accurate and reliable standards for both qualitative and quantitative detection of GM papaya HN1 and its processed products, while the established method offers a theoretical basis for the development of other CRMs.