Use of reference materials

• 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

DNA

Protein

RR-toleranceDesired effect

Cause

Means

PCR (polymerase chain reaction) based on DNA analysis (the genes) all

Protein-based techniques (ELISA,

lateral-flow devices) most of current AgBiotech traits

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% GMOXX 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 kernelSeed Bulk Conventional reference material purity level

Size 99.99% 99.9% 99% 100 1% 10% 63% 200 2% 18% 87% 250 2% 22% 92% 300 3% 26% 95% 500 5% 39% 99% 600 6% 45% 100%

1000 10% 63% 100%

Seed pools of 1% as target concentration

Pool Size 300 600 1000

GM Trait Ref. material

impurity

Non-GM Ref. material

Impurity

2-4 GM

seeds

5-7 GM

seeds

8-12 GM seeds

4.0%

0.01% 100* (98, 100)

98 (95, 100)

100 (98, 100)

4.0%

0.10% 97 (93, 99)

89 (84, 94)

94 (90, 98)

2.0% 0.01% 100 (99, 100)

100 (98, 100)

100 (99, 100)

2.0% 0.10% 97 (93, 99)

89 (83, 94)

93 (89, 97)

1.0% 0.01% 100 (99, 100)

100 (99, 100)

100 (100, 100)

1.0% 0.10% 97 (93, 99)

89 (83, 94)

93 (88, 97)

0.5% 0.01% 100 (100, 100)

100 (99, 100)

100 (100, 100)

0.5% 0.10% 97 (93, 99)

88 (83, 93)

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