Development of a certified genomic DNA reference material for detection and quantification of genetically modified rice KMD

摘要

Qualitative and quantitative detection of genetically modified products is inseparable from the application of reference materials (RMs). In this study, a batch of genomic DNA (gDNA) certified reference materials (CRMs) was developed using genetically modified rice Kemingdao (KMD) homozygotes as the raw material. The gDNA CRMs in this batch showed good homogeneity; the minimum sample intake was determined to be 2 mu L. The stability study showed that transportation by cold chain is preferable, no significant degradation trend was observed during a 12-month period when storing the gDNA CRMs at 4 degrees C and - 20 degrees C, and the number of freeze-thaw cycles cannot exceed 10. The property values of the copy number ratio of transgene and endogenous gene and the copy number concentration for gDNA CRMs were determined by a collaborative characterization of eight laboratories using the duplex KMD/PLDdroplet digital PCR (ddPCR) assays. The uncertainty components of characterization, potential between-unit heterogeneity, and potential degradation during long-term storage were combined to estimate the expanded uncertainty of the certified value with a coverage factorkof 2.0. The certified value of copy number ratio for KMD gDNA CRM is 0.99 +/- 0.05, and that of copy number concentration is (1.76 +/- 0.10) x 10(5)copies/mu L. Compared to the gDNA CRMs in availability, this batch of KMD gDNA CRMs is assigned accurate property values and can be directly used for qualitative and quantitative detection of GMOs as well as evaluation of the parameters of analytical methods with no need of further DNA concentration measurement.