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Use of reference materials
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As Calibrators
For metrological traceability
As QC control
To ensure international comparability
– Only common reference point for GM analysis
– Only common item internationally and
independent from technology used
Test methods
Cause
DNA
PCR (polymerase
chain reaction)
based on DNA
analysis (the genes)
 all
Means
Protein
Protein-based
techniques (ELISA,
lateral-flow devices) 
most of current
AgBiotech traits
Desired effect
RR-tolerance
Based on phenotype
(Bioassay) 
herbicide tolerances
Choice of reference materials for GMOs
• Artificial materials
– DNA: carries information of desired trait
• Genomic DNA
• Plasmids
• PCR amplicons
– Proteins: will produce the desired trait in plants
• Native plant proteins
• Bacterial proteins
• Peptides
• ‘Real-life’ materials
– Seeds: carry desired trait
– Grain: carries desired trait
Proteins as reference materials
• Protein based methods are best used in
unprocessed materials
• Native proteins
– Proteins as expressed in plants
– Expression level (amount produced in plants) is
influenced by environment
• Bacterial protein/peptides
– Very pure materials
– Differ from native protein
• Affinity of antibodies
• Epitopes still identical?
• No labeling regulation sets limits in units of
protein quantities
DNA-based standards
• Genomic DNA
– Native DNA
– Stability and purity are critical parameters
– Correlation to regulations (weight-%) is unclear
• Plasmids, PCR amplicons
– Easy to produce
– Differences in amplification efficiency
• Purity
• Length
• Circular/linear
– Correlation to regulations (weight-%) is unclear
Seed grain based reference materials
• Seed is commercialized commodity
• Grain is the first food product
• BUT
– Genetics depend on plant
• Soybeans carry trait on both chromosomes (homozygous) 
all grain will be 100% GMO
• Corn carries trait only on one of two chromosomes
(heterozygous): hybrid  Grain will only be 75% GMO
XX x XX  XX + 2XX + XX (still 50% on DNA level)
• Tissue-specific genetics
• Seed based reference materials require huge
amount of materials (1000 kernels ~ 300g)
• Seed based materials are produced for weight-%
Whole seed kernels as reference materials
• Purity of negative and positive material is critical
• No such thing as 0% or a 100% (sampling error)
• Probability that pool contains at least one positive kernel
Seed Bulk
Size
100
200
250
300
500
600
1000
Conventional reference material purity level
99.99%
99.9%
99%
1%
10%
63%
2%
18%
87%
2%
22%
92%
3%
26%
95%
5%
39%
99%
6%
45%
100%
10%
63%
100%
Seed pools of 1% as target concentration
300
GM Trait
Ref. material
impurity
4.0%
Non-GM
Ref. material
Impurity
0.01%
4.0%
0.10%
2.0%
0.01%
2.0%
0.10%
1.0%
0.01%
1.0%
0.10%
0.5%
0.01%
0.5%
0.10%
2-4 GM
seeds
100*
(98, 100)
97
(93, 99)
100
(99, 100)
97
(93, 99)
100
(99, 100)
97
(93, 99)
100
(100, 100)
97
(93, 99)
Pool Size
600
5-7 GM
seeds
98
(95, 100)
89
(84, 94)
100
(98, 100)
89
(83, 94)
100
(99, 100)
89
(83, 94)
100
(99, 100)
88
(83, 93)
1000
8-12 GM seeds
100
(98, 100)
94
(90, 98)
100
(99, 100)
93
(89, 97)
100
(100, 100)
93
(88, 97)
100
(100, 100)
93
(88, 96)
Seed kernels vs. flour
• Kernel based reference materials
– Are closest to reality
– Require extreme pure materials
– Require excessive amount of seeds
• Flour based reference materials
–
–
–
–
Derived from seeds: commercial commodity
Require large amount of pure materials
Are not always easy to produce, especially for oilseeds
Process history and small and uniform particles are
critical issue
– Stability of analyte is critical
– Are convenient to handle
Flour-based materials
• Are closest to reality (materials on the market)
• Analyte is likely to be affected by processing
• Different production conditions may easily lead to
differences in measurement results
• Reproducibility is key requirement
• Are best suited to promote international
comparability of measurements results
• Sampling considerations apply here as well
– Particle size distribution needs to be known and
adjusted to the recommended quantities (function of
concentration)
Interpretation of test results
• How to relate test results to labeling provisions?
• Test results
– Protein in absolute quantities
– DNA in absolute or relative quantities
• Labeling provisions
– Labeling provisions in %
– No threshold (China)
– No units explicitly given (Europe 1%)
prevailing assumption weight-%
under dispute
– Weight-% (Australia)
More challenges
• Biological factors:
– Protein:
• Expression level
• Degradation
– DNA
• Zygosity/Hybrid status
• (Selective) Degradation
(Specific) Inhibition
• Tissue specific issues: apoptosis, tissue-specific genetic
factors, endoreduplication
• Measurement uncertainty
– Exponential systems
– Measurement unit (amount of DNA, %-DNA)
Stacks
• Breeding stack:
– two parent plants carry different trait
– Through allelic recombination, two traits may be on
one chromosome
• Breeding stacks cannot be distinguished in flour
from a mixture of individual events, only by single
kernel analysis (e.g., YieldgardPlus)
• Vector stack:
– Plant is transformed twice with different traits
– Gene cassette contains two traits (one transformation)
• Vector stacks are new events  easily
distinguishable by PCR (e.g., BT11)
Need for standardization
• International harmonization of validated detection
methods is needed
• Validation shall encompass all analytical
procedures (incl. extraction)
• Validation protocols for DNA extraction need to
be established
• Validation shall occur according to international
guidelines (e.g, Harmonized protocol)
• Laboratories should work in compliance with
ISO 17025
Reference materials
• Reference materials
– Build consistent basis for global harmonization
– Provide basis of comparability of measurement results
• Reference materials need to be harmonized on an
international level
– Duplication is redundant and should be avoided
– Process conditions may affect the analyte in a reference
material
• Bureau of weight and measurements (BIPM) has
set up a mutual recognition agreement amongst
different Institutes
Conclusions
• Reference materials will continue to be the
cornerstone to international comparability of
measurement results
• The MRA of BIPM should provide an adequate
measure to cross-recognize reference materials
and avoid redundancy and duplication
• It is important that reference materials are as close
as possible to the matrix/analyte to true samples
• We believe that only seed-based reference
materials are an objective and science-based
approach