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The implementation of genetically modified organism (GMO) labeling policies requires accurate quantitative methods to measure the GMO content in test samples. A Kemingdao/phospholipase D (KMD/PLD) duplex ddPCR method was established with rice genomic DNA (gDNA) of homozygous KMD as template by optimizing the annealing temperature and cycle number. Duplex ddPCR showed a linear response over the dynamic range from 68 to 175,000 copies, covering four orders of magnitude. The limit of detection (LOD) and limit of quantification (LOQ) for duplex ddPCR were determined to be 9 copies and 34 copies of the rice haploid genome, respectively. A very high dilution factor would result in unacceptable bias and coefficients of variation for determining copy number of the gDNA solution, and more than 1000 copies of the DNA template in one reaction is preferred to obtain accurate quantitative results by duplex PCR. Five blinded DNA samples with copy number ratio of 10%, 5%, 1%, 0.1%, and 0.05%, and three blinded real-life matrix samples with mass fraction of 5%, 1%, and 0.5% were quantified by duplex ddPCR, simplex ddPCR, and qPCR. These three methods all gave comparable GMO content and copy numbers within the required precision, but the duplex ddPCR showed the narrowest uncertainty interval and provided the highest precision in comparison to simplex ddPCR and qPCR. The ddPCR is a more appealing and reliable technology for the accurate quantification of GMO content than simplex ddPCR and qPCR considering the uncertainty and precision of quantitative results, the time consumption of generating droplets, and the cost of ddPCR reagents.

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 μL. 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 °C and − 20 °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/PLD droplet 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 factor k of 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) × 105 copies/μL. 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.

The enforcement of GMO labeling regulations requires validated analytical methods and certified reference materials (CRMs). The early labeling regulations stipulated that the GMO content should be expressed as percentage, but did not specify what unit this percentage referred to. Two reference systems, using mass fraction and copy number ratio as measurement units, individually, are established for GMO analysis using different metrological traceability chains. Three types of CRMs, powder CRMs certified for mass fractions, genomic DNA CRMs, and plasmid DNA CRMs certified for copy number ratios, were developed for calibration and quality control. The type, certification, and measurement unit commutability of current GMO CRMs are presented and discussed in this paper. Both existing reference systems are facing a metrological challenge, although later EU regulations specified that the measurement unit of GMO content must be expressed in mass fraction and recommended to convert one unit into another by introducing a conversion factor, further efforts are required to explore which reference system is more metrologically sound. The determination of conversion factor per CRM batch is recommended to be based on the pure CRMs produced from pure GM materials, which is expected to be the best choice for calibration of PCR measurement results.

Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison.

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 (TT51-1a, TT51-1b, TT51-1c, and TT51-1d) 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.