Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction
Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in real-time PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maize MON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio.
KeywordsPCR Maize Zea mays Genetically modified organism Biochips high-throughput screening Nucleic acids (DNA|RNA)
Certified reference material
Digital polymerase chain reaction
European reference material
Genetically modified organism
High-mobility-group protein A gene
Polymerase chain reaction
Quantitative polymerase chain reaction
Relative standard deviation
GM content in food and feed is strictly regulated in most countries around the world. Regulations (EC) No 1829/2003 and (EC) No 1830/2003 form the basis for GMO food and feed use and labelling in the EU . The presence of GMOs must be declared unless the presence of authorised GM material can be proven to be adventitious or technically unavoidable and levels are below the threshold specified for non-declaration of GMO presence. The threshold for declaration is 0.9% for approved GMOs and 0.5% for unapproved GMOs that are under assessment and have passed the safety review step of the approval process. The current interpretations of “adventitious” and “technically unavoidable” are generally explained by guidelines within the respective member states. The unit of measurement for the threshold mentioned in the European Regulation has been specified in a Commission Recommendation defining the GM content as “the percentage of GM DNA copy number in relation to target taxon specific DNA copy numbers, calculated in terms of haploid genomes (HG)” .
The ratio in terms of GM copies per HG differs from the GM percentage in mass ratio. A unique mathematical relationship between the two units does exist but depends on the species and the tissue analysed. An empirical relationship between GM mass percentage and the GM content expressed per HG estimated by real-time PCR has been discussed recently taking the experimental data from MON810 maize seeds as a practical example .
Detection and quantification methods have been developed based on the amplification of DNA copies extracted from the sample to be analysed. Real-time qPCR utilises optical measurement of generated amplicons to assess PCR amplifications. The initial template concentration is derived from the number of amplification cycles (Ct value) required for the optical signal to pass a threshold chosen for the measurement. In theory, PCR exponentially amplifies nucleic acids, and the number of amplification cycles and the amount of PCR amplicons should allow the computation of the starting quantity of the initial template. That starting quantity is calculated using a calibrator for which the initial amount of copies is ideally known and to which the signal of the unknown sample is compared.
For GM analysis, the copy number ratio between the amount of transgene copies and the amount of a reference gene which is present once per HG is calculated. Dilution series of purified plasmid DNA certified to contain one copy of each target per plasmid can be generated to produce a calibration curve for each of the two copies . Following this approach, demonstration must be made that the amplification kinetics of each target is the same in the material analysed as for the calibrator used. However, many factors complicate this approach, creating uncertainties and inaccuracies in the measurement. Indeed, (1) the initial amplification cycles may not be exponential , (2) the PCR amplification eventually plateaus after an unknown number of cycles, (3) the low initial concentrations of target DNA molecules may not allow amplification to detectable levels, (4) the PCR amplification efficiency in a sample of interest may be different from that of the calibrator and finally (5) the PCR amplification may be less efficient for one target DNA fragment compared to another target. These differences are mainly observed when the assays are not fully optimised or when the DNA solution still contains co-extracted impurities that affect the kinetics of the amplification process  or the DNA is degraded [7, 8].
Digital PCR overcomes several of these difficulties by transforming exponential data from conventional PCR to digital signals that simply indicate whether or not amplification has occurred after a defined number of cycles. dPCR involves distributing the PCR solution containing template nucleic acid molecules across a very large number of individual partitions prior to amplification. Following PCR amplification, a count of the proportion of partitions containing a detectable number of PCR amplicons can be used to estimate the total number of template DNA copies in the original DNA extract. Accurate quantification relies on the fact that after 40 to 45 amplification cycles, the number of false negatives (single DNA templates present in a partition which are not detected) is very low. The sources of uncertainties related to DNA quantification by dPCR have recently been studied .
In this paper, we used dPCR to quantify the amount of MON810 DNA copies in seeds, and in powders that were certified for their mass fraction as well as for their MON810 copy number ratio. MON810 was chosen as an example as it is the only reference material that has been certified for its mass fraction as well as for the ratio of MON810 copies per hmg copies to date. MON810 represents also the only GM crop currently cultivated in the EU making the choice of this GM variety even more relevant in a European context.
Materials and methods
Seeds from two MON810 hybrids, DK 513 and DKC57-84, which differ only in the transgenic locus originating from the female and male parents, respectively, were provided by Monsanto for the production of the of CRMs ERM-BF418 and ERM-BF418k series, respectively. All other materials tested were seed-powder-based CRMs (ERM-BF413a, ERM-BF413b, ERM-BF413d, ERM-BF413e, ERM-BF413f)  from the Institute for Reference Material and Measurements (IRMM, Geel, Belgium). The seeds were rinsed in water, and dried under vacuum at 30 °C. The dried seeds were then milled using a high-impact mill with a triangular ribbed open grinding track in order to obtain the ground base material. The high-impact mill was flushed with nitrogen gas throughout the milling process and milling was interrupted if the temperature rose above 40 °C. An additional vacuum drying at 30 °C was carried out to further reduce the water content of the once ground base material. The powders were ground a second time under the same conditions, followed by a second drying step under vacuum at 30 °C. For the second grinding step, a sieve insert with a mesh size of 0.5 mm was used. Slow feeding of the mill ensured that the whole base material passed the sieve, thus excluding selection during grinding. Each ground base material was mixed in a Dynamic CM-200 mixer (WAB, Basel, CH) for 30 min to improve equal distribution of the different parts of the maize tissues separated by the milling process.
Extraction of genomic DNA
Genomic DNA (gDNA) was extracted using the Wizard genomic DNA extraction protocol (Promega) from five individual 20 mg portions of GM maize (MON810) CRM, ERM-BF413 series (IRMM, Geel, Belgium) and the resultant DNA was pooled together. Six hundred microlitres of nuclei lysis solution was added to 20 mg of powder in a 1.7-mL Eppendorf tube and vortexed for 30 s. The tubes were incubated at 65 °C for 20 min. Four microlitres of RNase A solution was added and incubated at 37 °C for 15 min. Two hundred fifty microlitres of protein precipitation solution was then added and the contents were vigorously vortexed for 20 s followed by centrifugation at 14,000 rpm for 10 min. Six hundred microlitres of the supernatant containing DNA was transferred to a clean 1.7-mL Eppendorf tube containing 600 µL of isopropanol placed on ice. The contents were gently mixed by inverting the tubes for several times followed by centrifugation at 13,000 rpm for 5 min. The supernatant was discarded and the DNA pellet was washed with 70% ice cold ethanol by gently inverting the tubes several times followed by centrifugation at 13,000 rpm for 1 min (unless indicated all centrifugation steps were performed at room temperature). Excess ethanol was removed and the pellet was air-dried and dissolved in 30 µL of 1 × TE0.1 (10 mM Tris, 0.1 mM EDTA, pH 8.0). The tubes were incubated overnight at 4 °C to rehydrate the DNA. The five fractions were pooled and the quality and purity of the extracted gDNA was estimated by absorbance at 260 nm and by the PicoGreen® dsDNA quantification kit (Invitrogen).
Digestion of DNA
For enzyme digestion, 30 to 50 ng of MON810 gDNA was used in a total volume of 50 µL of the restriction digestion reaction mix containing the appropriate restriction enzyme buffer, 0.02 mg/mL RNase A and 40 unit of HinP1I (NEB, Arundel, Australia). The final volume was made up with nuclease-free water (Promega, Sydney, Australia) and incubated for 2 h at 65 °C for HinP1I digestion. The HinP1I enzyme was inactivated by incubating at 80 °C for 10 min. 5 µL of digested gDNA was analysed on a 1% agarose gel to confirm complete digestion.
Digital PCR analysis
Digital PCR was performed on the BioMark System (Fluidigm, South San Francisco) using the 12.765 digital arrays (Fluidigm). The digital array comprises twelve panels and each panel contains 765 individual partitions of approximately 6 nL volume each with a total volume per panel of approximately 4.6 µL (6 nL × 765) [11, 12]. The instrument software generates PCR amplification curves and real-time cycle threshold (Ct) values for each of the 9,180 chambers (765 × 12). Following amplification, digital raw data was processed by the BioMark dPCR Analysis software using a manually set threshold of 0.03 and target Ct range of 23 to 43.
Primers and probes used in this study
Final PCR concentration (nM)
(P) 6-FAM- AACATCCTTTGCCATTGCCCAGC - BHQ-1
The digital array thermocycling conditions on the BioMark System PCR consisted of a 10 min activation step at 95 °C, followed by 45 cycles of a two-step thermal profile involving 15 s at 95 °C for denaturation, and 60 s at 60 °C, for annealing and extension.
Results and discussion
The ERM-BF418k CRM series has been produced to replace the existing ERM-BF418 CRM series and will be released by IRMM. For the new ERM-BF418k CRM series, the MON810 transformation event was introduced from the male parent whereas for the ERM-BF418 CRM series the MON810 transformation event was introduced from the female parent. It was therefore necessary to test the MON810 copy number ratio in the two varieties DK 513 and DKC57-84 used for the production of the ERM-BF418 and ERM-BF418k, respectively.
Number of positive partitions for MON810 and hmg, estimated number of copies per panel and copy number ratio measured in two MON810 varieties in which the MON810 event originated from the female (DK 513) or from the male (DKC57-84) parent
Number positive partitions
Estimated number of copies per panel
Average number of copies per panel
Number positive partitions
Estimated number of copies per panel
Average number of copies per panel
Average MON810/hmg ratio (%)
In the paper of Zhang , the ratio of transgenic HGs to the total HG in maize seeds is calculated taking into account the parental origin of the transgenic allele as well as the impact of the maize endosperm DNA content which was reported earlier to vary between 36.3% and 59.4% in maize seeds according to varieties . The average relative DNA ratio of the endosperm to total gDNA measured on four independent seeds was found to be 36.3% (SD of 2.4%) and 42.1% (SD of 1.9%) for the varieties DK 512 and DK 585, respectively . Despite the fact that the relative DNA ratio of the endosperm to total gDNA proportion was not measured for the varieties DK 513 and DKC57-84 in the above study, one can calculate a theoretical MON810 copy number ratio of 57.1% and 41.1% for the female and male parent, respectively, under the assumption of a 40% DNA ratio of the endosperm to total gDNA. These two predicted values fit perfectly with the ratios measured by dPCR (Table 2).
The MON810 copy number ratio measured for the MON810 variety DK 513 can also be compared to the certified copy number ratio for the CRM ERM-BF413d. This 10 g/kg (1% m/m) powder mixture has been produced from whole seeds of non-modified maize (variety DK 512) and GM MON810 maize (variety DK 513). To support EU legislation, CRM ERM-BF413d was also certified for the DNA copy number ratio of the MON810 event-specific plant/P35S junction region and a single copy target of the hmg measured by qPCR using the same PCR targets as those in this study but calibrated with a MON810 maize plasmid DNA ERM-AD413 . This DNA copy number ratio was certified to be 0.57% ± 0.17% (expanded uncertainty using a coverage factor k = 2) which is entirely consistent with the expected ratio since the pure MON810 powder (variety DK 513) used to prepare the 1% m/m CRM contained an average of 57 copies (SD of six copies) of MON810 per 100 copies of hmg when measured by dPCR (Table 2).
Estimated number of MON810 and hmg copies and MON810/hmg copy number ratio measured in the ERM-BF413 series by dPCR (n = 5)
Mass fraction of DK 513
Target DNA concentration in DNA extract (copies per μL)
MON810/hmg copy number ratio (%)
Certified value (g/kg)
Expanded uncertainty (g/kg)
The level of repeatability of positive partitions among the five replicate panels within the same digital arrays appears to be very adequate. The relative standard deviation (RSD) of the MON810/hmg ratio as shown in Tables 2 and 3 is reported as the square root of the sum of the squares of the relative standard deviations obtained for each estimated target. The ERM-BF413a and ERM-BF413b samples contained only low amounts of MON810 with an average of five and 16 copies MON810/μL DNA extract, respectively. Consequently, these two samples produced the highest RSDs for the copy number ratio of 26.8% and 22.6%, respectively. The RSDs for the copy number ratio in the remaining samples were between 6.8% and 15.5%.
This paper describes for the first time the absolute quantification of GM target using a dPCR apparatus and digital arrays. This technology relies on the spatial randomness of the positive partitions as demonstrated earlier  and on the ability to reliably detect DNA molecules present at a very low number of copies as was the case for the blank material tested in this study. Digital PCR is an alternative technique for quantifying gene copy number which may provide more accurate measurements than other approaches currently available as it is not dependant on amplification efficiency. Of particular importance, dPCR measurements are made independent of any calibrator and, therefore, this technique has the potential to be considered as a primary method for use in certification of nucleic acid reference materials. The measurement principle has a high metrological quality. However, the absolute number of DNA molecules estimated is a measure of those molecules that can be amplified during the PCR. The number of amplifiable DNA target sequences is not necessarily equal to the total number of target molecules initially present in the reaction solution as it is difficult to prove that all target DNA sequences have been amplified after 40 or 45 cycles. However, this limitation also exists with real-time qPCR analysis. Taking into account its specific uncertainties, dPCR has the potential to be used for the assignment of copy number ratios to reference materials during certification. Current costs of the digital arrays may however hinder the use of dPCR for routine analysis of GM samples.
We thank S. Trapmann and H. Emons (IRMM) for their very useful review and constructive criticism of the manuscript.
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