Development of certified matrix-based reference material of genetically modified rice event TT51-1 for real-time PCR quantification

In 2009, the genetically modified (GM) rice event TT51-1 with an engineered insect resistance trait became the first GM rice event to be granted certification for safe production in China, and its derivative lines Bt 63 and Huahui No.1 are expected to be commercialized soon. The development of certified reference material (CRM) for TT51-1 is necessary to monitor and inspect the TT51-1 event and its derivates. In this work, we developed four matrix-based TT51-1 rice CRMs (TT51-1(a), TT51-1(b), TT51-1(c), and TT51-1(d)) with different TT51-1 mass fraction ratios by blending seed powders of homozygous TT51-1 and its recipient cultivar Minghui 63. The between-bottle homogeneity and the within-bottle homogeneity were tested, and good results were obtained. The potential degradation during transportation and shelf life were evaluated, and demonstrated an expiration period of at least 36 months. The characterization values of the four TT51-1 CRMs based on the mass fraction ratio were 1000.000 +/- 51.430 g/kg, 49.940 +/- 4.620 g/kg, 9.990 +/- 1.110 g/kg, and 4.990 +/- 0.620 g/kg, respectively. The characterization values based on the copy number ratio were certified by digital PCR analysis as 97.442 +/- 5.253 %, 4.851 +/- 0.486 %, 1.042 +/- 0.135 %, and 0.556 +/- 0.073 %, respectively. These results suggested that the TT51-1 matrix-based CRMs developed are of high quality and can be used as potential calibrators for TT51-1 GM rice inspection and monitoring.