IRMM information - European Reference Materials

Report EUR 20273 EN 

CERTIFICATION REPORT 

The certification of dry-mixed soya powder with 

different mass fractions of Roundup Ready TM Soya 

Certified Reference Materials ERM BF410a, 

BF410b, BF410c, BF410d, BF410e, BF410f


IRMM Information 

Reference Materials 

The Certification of Reference Materials 

of Dry-mixed Soya Powder with different 

Mass Fractions of Roundup Ready TM Soya 

IRMM-410S

Mission 

The mission of IRMM is to promote a common European measurement system in 

support of EU policies, especially internal market, environment, health and consumer 

protection standards.


European Commission 

IRMM information 

REFERENCE MATERIALS 

The Certification of Reference Materials 

of Dry-Mixed Soya Powder with different 

Mass Fractions of Roundup ReadyTM Soya 

IRMM-410S 

(IRMM-410S-0/IRMM-410S-1/IRMM-410S-2/IRMM-410S-3/IRMM-410S-4/IRMM-410S-5) 

S. Trapmann, P. Catalani, P. Conneely, P. Corbisier, D. Gancberg, E. Hannes, 

L. Le Guern, G. N. Kramer, J. Prokisch, P. Robouch, H. Schimmel, R. Zeleny and 

J. Pauwels 

European Commission, Joint Research Centre 

Institute for Reference Materials and Measurements (IRMM), Geel (BE) 

G. Van den Eede, F. Weighardt, M. Mazzara and E. Anklam 

European Commission, Joint Research Centre 

Institute for Health and Consumer Protection (IHCP), Ispra (IT)

Legal Notice 

Neither the European Commission nor any person acting on behalf of the Commission is responsible 

for the use which might be made of the following information. 

Reproduction is authorized provided the source is acknowledged 

http://www.jrc.cec.eu.int/ 

© European Communities, 2002 

Printed in 2002

SUMMARY 

This report describes the preparation and certification of dry-mixed soya powder CRMs (IRMM- 

410S-0) with different mass fractions of genetically modified Roundup Ready TM soya powder 

(Certified reference materials IRMM-410S-0, IRMM-410S-1, IRMM-410S-2, IRMM-410S-3, 

IRMM-410S-4 and IRMM-410S-5). The CRMs were produced in 2001 by the Joint Research 

Centres of the European Commission, the Institute for Reference Materials and Measurements 

(IRMM) in Geel, Belgium. The CRMs are intended for the validation and calibration of methods for 

the detection of genetically modified food. The Roundup Ready TM concentration of IRMM-410S 

was verified with the help of DNA based and protein based detection methods. The CRMs are 

available in the form of glass bottles containing 1 g of soya powder packed under argon atmosphere. 

Seeds of non-modified soya (line A1900) and Roundup Ready TM (event 40-3-2, line AG5602 RR) 

both produced by Asgrow (Faribault, MO, USA) and delivered by Monsanto (St.Louis, MO, 

USA) were rinsed with demineralised water, additionally drained and dried at 30°C in order to 

minimise dust contamination from other crops. The materials were prepared by quantitative Turbula 

and dry-mixing of non-modified soya powder and 100% Roundup Ready TM GMO soya powder. It 

is recommended to use samplings not smaller than 100 mg. Bottles should be stored dry and cool 

(+4 °C) and kept in the dark.

TABLE OF CONTENT 

1. Introduction 1 

2. CRM Preparation 1 

2.1 Characterisation of the starting materials 1 

2.2 Production of powdery base materials 5 

2.3 Homogeneity study for dry-mixed maize powder 5 

2.4 Quantitative preparation of GMO/non-GMO mixtures 6 

2.5 Additional sieving and grinding steps 7 

2.6 Bottling 8 

2.7 Production control 8 

2.8 Minimum sample intake for analysis 10 

3. Certified mass fractions of GMO 10 

4. Verification of Roundup Ready TM mixtures 11 

4.1 Verification with DNA based methods 11 

4.2 Verification with protein based methods 15 

4.3 Comparison of results obtained with both methods 16 

4.4 Results 17 

5. References and acknowledgements 17 

5.1 References 17 

5.2 Acknowledgements 17 

6. Annex 19

1 Introduction 

Genetic modification of agricultural products is becoming an increasingly important item and 

authorisations to market such products are more and more under discussion. However, in the EU 

such authorisations are being combined with the obligation to declare food containing ingredients 

from genetically modified plants [1]. This enforces the necessity to develop suitable detection 

methods, and subsequently the use of certified reference materials. Therefore, the preparation of 

CRMs of mixtures of genetically modified (GMO) and non-genetically modified organisms has been 

started to allow proper validation of screening methods for the detection of genetic modification in 

agricultural and food products. 

A set of CRMs of soya powder with different mass fractions (0, 0.1, 0.5, 1, 2, 5 %) of dried 

powder of genetically modified soya (Roundup Ready TM ) was produced by IRMM. The six CRMs 

(IRMM-410S-0, IRMM-410S-1, IRMM-410S-2, IRMM-410S-3, IRMM-410S-4 and IRMM- 

410S-5) are available from IRMM and its authorised distributors [2]. 

IRMM-410S is replacing IRMM-410R, which was sold out in 2001. In contrary to IRMM-410R, 

IRMM-410S has been produced with the help of dry-mixing techniques in order to minimise DNA 

and protein degradation occurring during the production. 

2 CRM Preparation 

2.1 Characterisation of the starting materials 

For the preparation of the CRMs, 80 kg non-modified soya (Agrow line A1900) delivered by 

Monsanto (St.Louis, MO, USA) and 20 kg GMO Roundup Ready TM (event 40-3-2, Asgrow line 

AG5602 RR, [3]) delivered by Monsanto (St.Louis, MO, USA) of seed quality were supplied to 

IRMM. 

The delivered non-GMO seed batches have been tested at Monsanto (St. Louis, MO, USA) for 

their purity using ELISA and PCR techniques. Sixty pools of 50 seeds, each, of the conventional 

non-GMO soybean, line A1900, were qualitatively tested by immunoassay for CP4 EPSPS 

protein. All 60 pools of seed tested negative for CP4 EPSPS, which gave a 95% confidence that 

this batch of soybean seed is greater than or equal to 99.9% free of seed expressing the CP4 

EPSPS protein. 

Roundup Ready TM (RR) soybean 40-3-2 event-specific and 61-67-1 construct-specific multiplex 

PCR with aat (asparatate aminotransferase) internal control was used to confirm the identity and 

purity of soybean line A1900. For the A1900 seed, three pools of 50 seeds tested negative by 

PCR for both 40-3-2 and 61-67-1 and positive for the aat internal control. This confirmed the 

immunoassay results. 

Additionally the purity of the non-GMO base material has been tested after washing of the seeds 

and the first grinding (as described in chapter 2.2), in order also to test for remaining surface 

contaminations. GMO contaminations in the non-GMO material close to the detection limit (LOD) 

of the EPSPS real time-PCR (rt-PCR) method were measured (table 1). The LOD was calculated 

equal to (3.3*σ)/S, with σ representing the standard deviation of a defined GMO percentage and S 

the slope of the calibration curve. The defined GMO percentage was the lowest GMO percentage 

for which the amplification efficiency was optimal. The efficiency of the amplification was 

1

determined on basis on the slope of the regression line between the GMO percentage and the Ctvalues, 

which should not be lower than the theoretical value of 3,322. The LODs have been 

established on genomic plant DNA and are 0.019% for the EPSPS specific PCR, 0.124 % for 35S 

PCR and 0.09 % for the NOS PCR. 

Table 1: Quantitation of GMO contaminations in the non-GMO base material with 35S 

and EPSPS specific real-time PCR, determination in triplicate using 20 mg/extraction 

Non-GMO raw material 

35S PCR 1 

NOS PCR 1 

Number of 

measurements 

2 

Concentration of GMO 

contamination 2 

[n] [%] stdev 

5 0.006 0.003 

5 0.007 0.005 

EPSPS specific PCR 5 0.015 0.012 

1 Percentage is given as an indicative value since it is lower than the LOD for the 35S. 

2 Primers used can be found in the annex. 

For the determination of the purity and confirmation of the uniform zygosity of the GMO RR starting 

material 50 randomly chosen seeds were germinated on moistened paper in a growing chamber 

under identical conditions during 15 days. 50-mg pieces from young leaves were sampled from 46 

plants. The plant tissues were disrupted in a lysis buffer using the Mixer Mill MM 300 (Retsch, 

Haan, DE) and 3-mm tungsten carbide beads (Qiagen, Hilden, DE). Total DNA was extracted 

using the DNeasy® 96 Plant kit (Qiagen, Hilden, DE). The total DNA was analysed on a 0.7 % 

agarose gel and quantified using the PicoGreen® dsDNA quantitation kit (Molecular Probes 

Europe, Leiden, NL). The average DNA yield was 8.2 ± 1.26 µg/50 mg tissue. On basis of the 

agarose gel, DNA showing a high level degradation was not further taken into account for real time 

PCR analysis. The detection of RR soybeans was performed following the TaqMan® Universal 

PCR Master Mix protocol (Applied Biosystem, Foster City, CA, USA). A primer pair specific for 

the modified EPSPS gene (transgenic construct between the cauliflower mosaic virus 35S promotor 

and the Petunia hybrida 5-enolpyruvylshikimate-3-phosphate synthase signal peptide) was used 

together with a reporter-dye labelled with FAM. The threshold cycle values (CT) obtained after the 

PCR on the 46 plants (figure 1) were compared to a calibration curve obtained with 100% RR 

DNA diluted in 0% RR DNA.

Figure 1: Real-time PCR quantiation of the EPSPS gene from 25 young leaves in 

triplicate. The fluorescent signals below the threshold base line are those obtained with non-GMO 

control soybeans, without template or with an 0.2% SDS-inactivated polymerase. 

Figure 2: Real-time PCR quantitation of the lectin gene from 25 young leaves in triplicate. 

The calculated percentage of GMO was then corrected using primers targeting the soybean lectin 

gene as an additional estimation of total soybean DNA. The calculated average percentage of RR 

soybeans was 105.51% ± 11.40 (n=46) and no samples had a percentage value below 87.3 %. It 

could be concluded, that all plants were therefore homozygous (table 2). 

3

Table 2: Molecular composition and determination of uniform zygosity of the Roundup 

Ready TM starting material used for the production of IRMM-410S 

PCR method performed 

and primers used 

EPSPS specific real-time PCR 

(RRS-F primer and RRS-R primer) 1 

1 primer sequences can be found in the annex 

Number of 

plants tested 

4 

Number of 

heterozygous 

kernels 

Number of 

homozygous 

kernels 

46 0 46 

In order to determine the total DNA content of GMO and non-GMO starting material, the DNA 

was extracted using two different plant DNA extraction kits from Macherey-Nagel (Düren, DE) 

and from Qiagen (Hilden, DE) and using a CTAB method [4]. The DNA was afterwards quantified 

with the help of UV photometry at 260 nm (BioPhotometer from Eppendorf, Hamburg, DE) and 

with the Picogreen assay (Molecular Probe, Leiden, NL) in a spectrofluorometer (Fluostar Galaxy 

from BMG Labtechnologies GmbH, Offenburg, DE). After correction for the water content of the 

material, no significant differences in DNA extractability in the base material has been observed 

(table 3). 

Table 3: Ratio of DNA content of GMO and non-GMO powdery base material, corrected 

for the water content, determined after the first grinding procedure (as described in 

chapter 2.2) 

Extraction 

Method 

Detection 

method 

Macherey- UV260nm 

Nagel Plant kit Photometry 

Macherey- Fluorimetry 

Nagel Plant kit (Picogreen) 

CTAB [4] UV260nm 

Photometry 

CTAB [4] Fluorimetry 

(Picogreen) 

DNeasy Plant UV260nm 

Mini Kit Photometry 

DNeasy Plant Fluorimetry 

Mini Kit (Picogreen) 

n DNA content of Roundup Ready TM soya powder 

DNA content of non-GMO soya powder 

6 1.18 ± 0.09 

6 1.09 ± 0.11 

6 1.08 ± 0.57 

6 1.27 ± 0.32 

6 0.87 ± 0.37 

6 1.00 ± 0.29

2.2 Production of powdery base materials 

Prior to the production of IRMM-410 the kernels of the non-GMO batch and GMO batch were 

separately thoroughly mixed. 

During the production of the GMO and non-GMO powdery base materials each material was 

treated separately. Cross contamination and contamination with foreign DNA were avoided with 

the help of glove box systems, clean cells, disposable lab clothing and treatment of all contact 

surfaces prior to exposure to the base materials with a DNA destroying solution. 

The kernels used for production were rinsed in demineralised water and after draining dried under 

vacuum at 30°C for 25 hours. The dried starting materials were then ground using a fine impact mill 

(track mill). The ground materials were collected in plastic bags and placed in polyethylene 

containers. Particle size analysis and water analysis proved that both base materials had a similar 

particle size distributions and water contents. 

2.3 Homogeneity study for dry-mixed soya powder 

Prior to the production a homogeneity study for dry-mixed soybean powders has carried out, 

mixing Au spiked soybean powder with non-spiked, non-GMO soybean powder. All powders 

used in the homogeneity study have been processed in the same way as described for the base 

materials in chapter 2.2. A 10 % dilution was produced first and afterwards diluted two times 

further to reach concentration of 1 and 0.1 % Au spiked in non-spiked soya. During the analysis the 

Au concentration of the three mixtures was determined with the help of neutron activation analysis 

(NAA). NAA proved the homogeneity of the dry-mixed soya powder (table 4) and the adequacy 

of the dry-mixing technology for the preparation of the non-GMO/GMO soybean mixtures. 

Table 4: Homogeneity study on dry-mixed Au-spiked soya powder with non-spiked 

soybean powder, results of Au determination by neutron activation analysis (NAA) with a 

sample intake of 50 mg 

Material Mixture parts (%) Results NAA 

Au-spiked Non-spiked Au content RSD 

(ppm) 

(%) 

Au-spiked soya 100 0 1182 0.8 

Non-spiked soya 0 100 0.007 26.8 

10 % mixture 10 80 120 2.8 

1 % mixture 1 98 12.1 5.2 

0.1 % mixture 0.1 99.8 1.50 6.1 

During the dry-mixing study it has been observed, that ground soybean powder contains a fraction 

of particles > 500 µm independently of the grinding techniques applied (table 5). The fraction > 500 

µm proved to consist mostly of seed covers. The flexibility of the skinny material allowed the 

particles to fold and even pass inserted sieves. 

Soybean powder ground more than once with a track mill formed a sticky powder which either 

blocks the mill or in the case of cryo-grinding was not suitable for the dry-mixing. It has therefore 

5

een decided to reduce the larger particles in an additional sieving and grinding step (see chapter 

2.5). In order to have a whole meal soybean reference material, no fractions have been removed 

during the production. 

Table 5: Particle sizes of soybean powder after application of various grinding techniques 

determined by sieving test 

Particle 

size 

Soybean powder 

after 1 st fine impact 

mill grinding 

(without sieve insert) 


after 2 nd fine impact 

mill grinding 

(without sieve insert) 

6 


after cryo-grinding 

(with 500 µm sieve 

insert) 

[µm] [weight %] 

< 63 21 3 1 

63-90 40 42 14 

90-125 12 17 44 

125-180 9 30 4 

180-250 7 2 0 

250-355 6 3 0 

355-500 3 1 0 

> 500 2 2 1 

2.4 Quantitative preparation of GMO/non-GMO mixtures 

Mixtures containing 0.1, 0.5, 1, 2, and 5 % Roundup Ready TM soybean powder in non-genetically 

modified soya powder and the 0 % RR soybean powder were prepared quantitatively using a drymixing 

method. Water contents of the ground GMO and non-GMO powders, used as base 

materials, were determined in threefold by Karl Fischer titration in order to correct for the water 

content of the material (table 6). A 10 % GMO mix was produced first using 100 % GMO and 

non-GMO base material. All lower concentrations were achieved by further dilution with non- 

GMO maize powder. The dilution factor was kept below 10 as this factor has been tested in the 

homogeneity study. Powders were weighed using calibrated balances. Weighing details are given as 

well in table 6.

Table 6: Water content and weighing details of powdery base materials used for the 

production of IRMM-410S 

IRMM 

Water content 

-410S 

of base material Mass after water correction 

GMO GMO base material non-GMO base GMO non- Total 

content used 

material used base GMO weight of 

material base mixture 

used material 

used 

(%) GMO GMO GMO non- non- non- (g) (g) (g) 

(%) water 

content 

(%) 

water 

content 

n =3 

(stdev) 

GMO 

(%) 

GMO 

water 

content 

(%) 

GMO 

water 

content 

n =3 

(stdev) 

10 100 6.70 0.26 0 5.70* 0.26 403.85 3596.15 4000.00 

5 10 5.27 0.25 0 5.70* 0.26 1995.45 2004.55 4000.00 

2 10 5.27 0.25 0 5.70* 0.26 797.09 3202.91 4000.00 

1 10 5.27 0.25 0 5.37* 0.21 399.62 3600.38 4000.00 

0.5 5 5.13 0.15 0 5.37* 0.21 399.62 3600.91 4000.00 

0.1 1 4.97 0.21 0 5.70* 0.21 397.23 3602.77 4000.00 

* Two containers with non-GMO base material with slightly different water content have been used. 

The calculated weight fractions were in a first step manually pre-mixed in container and afterwards 

turbula mixed. The whole material was then transferred into a dry-mixing device and mixed for 2 

min. 

The standard uncertainty on the dried GMO powder mass fractions of the CRM preparations (0.1, 

0.5, 1, 2 and 5 % GMO) is estimated at 0.1 % relative. 

2.5 Additional sieving and grinding steps 

In order to reduce the fraction of particles > 500 µm, the material once ground in a fine impact mill 

has been sieved for around 1.5 h (mesh size 500 µm) and the fraction > 500 µm ground for a 

second time using a laboratory grinder. After grinding the material was sieved again and the 

remaining fraction ground repetitively (in total up to 6 times) until a fraction too small for the 

laboratory grinder remained (table 7). As the grinding of such small volumes leads to an enormous 

temperature increase, which might damage the material, the grinding chamber has been cooled with 

circulating cooling liquid down to 5°C. 

7

Table 7: Additional sieving and grinding steps 

Material GMO 

content 

Amount of 

material sieved 

(500 µm mesh size) 

8 

Material 

exposed to 

additional 

grinding steps 

Material 

remaining 

IRMM-410S-0 0 % 4000 g 188 g 5 g 

IRMM-410S-1 0.1 % 4000 g 181 g 4 g 

IRMM-410S-2 0.5 % 4000 g 185 g 6 g 

IRMM-410S-3 1 % batch A 4000 g 190 g 7 g 

1 % batch B* 4000 g 200 g 5 g 

1 % batch C* 4000 g 194 g 8 g 

1 % batch D* 4000 g 140 g 5 g 



* Additional batches of 1 % material have been produced for future production of intentionally degraded 

material. 

The remaining fraction of 0.1-0.2 weight % mainly contained of skinny material originating from the 

seed cover of soya. The materials took up some water during the cooled grinding procedure and 

were therefore vacuum dried for around 8 h before any other application. As the goal was to 

produce a whole seed GMO CRM, the remaining fraction has been added to the rest of the batch 

and dry-mixed once more. 

2.6 Bottling 

The dry-mixed products were bottled in well-cleaned 10-mL brown glass vials using an automatic 

sampling device. The first 30 filled bottles of each batch were discarded as an additional measure 

against carry over contamination. Bottles were manually closed with rubber stoppers. Before final 

closure of the vials they were placed in the freeze-dryer, filled with argon and closed using the 

hydraulically moveable device of the freeze-dryer. All vials were sealed with aluminium caps to 

prevent opening of rubber stoppers during storage and transport. 

2.7 Production control 

The water content of the dry powders was determined by Karl Fischer titration and amounted 

typically to values in the range of 0.7 to 1.8 %. Additionally to the water content the water activity 

of the final product has been measured (table 8). 

In order to determine the particle size distribution a sieving test following ISO 3310-1 using sieves 

with meshes of 63, 90, 125, 180, 250, 355 and 500 µm has been carried out. 10 times 10 g of the 

1% powder has been used, proving that 99.8% of the final product < 500 µm with a typical 

average particle size between 63 and 180 µm (table 9 and 10). 

The fraction of particles > 500 µm from the 10 sieving tests have been collected and merged for a 

final sieving test using sieves with meshes of 710 and 1000 µm (table 10).

Table 8: Water content and water activity of IRMM-410S 

Water content 

Water activity 

CRM Certified 

[%] 

[g/100g] n Mean std dev n avr. std dev 

IRMM-410S-0 0.0 5 1.09 0.13 5 < 0.06 - 






Table 9: Particle size distribution of IRMM-410S, determined by sieving test 

CRM Certified 

Particle size distribution 

[g/100g] n 

Fraction 

[µm] 

[weight %] std dev 

< 63 0 0.0 

> 63, < 90 28 10.2 

> 90, < 125 41 9.2 

IRMM-410S-1 0.10 10 > 125, < 180 12 2.0 

> 180, < 250 9 1.0 

> 250, < 355 7 1.2 

> 355, < 500 4 0.5 

> 500 0* 0.0 

* residual particles with a size > 500 µm are due to their total weight < 0.1g (m= 18.23 mg, s= 8.16 mg) not 

registered by the method (see table 10) 

Table 10: Particle size distribution of IRMM-410S, determined by sieving test, merged 

fractions of particles > 500 µm from 10 analysis (see table 9) 

CRM Certified 

Particle size distribution 

[g/100g] n 

Fraction 

[µm] 

[mg] [weight %] 

> 500 174.5 0.175 

IRMM-410S-1 0.10 1 > 500, < 710 2.6 0.003 

> 710, < 1000 0 0 

9

The contribution of the particle size to the uncertainty of the certified reference material has been 

estimated for the DNA based methods with the help of a programme and DNA extractability data 

obtained for the various particle size fractions [6]. It could be concluded, that the influence of the 

particle size distribution on the uncertainty of the material is < 5 % if an sample intake of 100 mg is 

used and no GMO concentration equal to or above 0.1 % are produced. 

Table 11: Uncertainty contribution calculated for a sample intake of 100 mg [6] 

Particle size 

[µm] 

C DNA 

[µg/g] 

GMO content 

[%] 

10 

Soya bean powder 

ground once in 

a track mill 

RSD [%] 

< 63 1170 0.1 % 4.62 

63-90 954 0.5 % 2.65 

90-125 806 1 % 1.66 

125-180 219 2 % 0.96 

180-250 163 5 % 0.74 

250-355 215 100% 0.20 

355-500 299 

500-1500 526 

2.8 Minimum sample intake for analysis 

The efficiency of the dry-mixing of soya powders was studied in a pilot trial carried out with Auspiked 

soya (chapter 2.3). 

For the recommended sample intake of 100 mg per analysis, one may estimate on the basis of the 

particle size distribution (average particle size < 125 µm) of the mixtures that this number is larger 

than 10 5 , which means that even for IRMM-410S-1 with 0.1% GMO content the number of GMO 

particles is larger than 100. On this basis uncertainties due to sample inhomogeneity were estimated 

(chapter 3). 

3 Certified mass fractions of GMO 

The materials IRMM-410S-0, IRMM-410S-1, IRMM-410S-2, IRMM-410S-3, IRMM-410S-4 

and IRMM-410S-5 form a set of 6 reference materials certified for the mass fraction of Roundup 

Ready TM soya powder. The certified mass fractions are based on quantitative dry-mixing of nonmodified 

soybean powder with RR soybean powder. Referring to the particle size distribution it is 

advised to use sample intakes not smaller than 100 mg. Taking into account the uncertainties of the 

weighing and of the humidity contents of the starting materials, uncertainties for the certified mass 

fractions at 100 mg level were estimated (table 12).

It must however be emphasised that due to the relatively large uncertainty inherent to quantitation of 

the total DNA content, the DNA/dry powder mass fraction of different lots soya kernels cannot be 

determined with the highest precision. Therefore, the ratio GMO-DNA/non-GMO-DNA in the 

reference materials may significantly deviate from the certified powder mass ratio values. 

Table 12: Uncertainty estimation (in g GMO dry powder per 100 g dry powder) 

CRM, 

certified 

content 

IRMM- 

410S-0 


410S-1 


410S-2 


410S-3 


410S-4 


410S-5 

(u1) 1 

(u2) 2 

Standard 

Uncertainty 

(u3) 3 

Combined 

uncertainty 

Expanded 

uncertainty 

All six materials were analysed for consistency and not with the aim to quantify the gravimetrically 

prepared materials of verified purity. 

For the verification study a Roundup Ready 

11 

TM specific real-time PCR method based on the 

amplification of part of the EPSPS sequence [5] and an event specific method based on the 

amplification of the 35S-junction [7] has been used. Additionally a real-time PCR GMO screening 

method based on the detection of the CaMV 35S promoter [5] and Agrobacterium tumefaciens 

NOS terminator has been applied. The amplified PCR products are measured cycle-by-cycle with 

(u4) 4 

(u5) 5 

(uc) (U = 2 * uc) 

< 0.03 - - - 0.0115 - 0.0115 0.03 

0.10 0.0001 0.0003 0.009 0.0115 0.0033 0.0242 0.05 

0.50 0.0005 0.0015 0.020 0.0115 0.0165 0.05 0.10 

1.0 0.001 0.003 0.029 0.0115 0.033 0.0775 0.2 

2.0 0.002 0.006 0.041 0.0115 0.066 0.1265 0.3 

5.0 0.005 0.015 0.064 0.0115 0.165 0.2605 0.6 

1 mass determination uncertainty estimated at 0.1% (see chapter 2.4) 

2 humidity content stdev < 0.3 % (see table 7) 

3 inhomogeneity at 100 mg level with an average particle size of 125 µm 

4 purity of non-GMO base < 0.02 % (see chapter 2.1) 

5 purity of GMO base material 96.7 % (see chapter 2.1) 

4 Verification of Roundup Ready TM mixtures 

The verification study was carried out using DNA-based detection methods like polymerase chain 

reaction (PCR) and protein-based detection methods like enzyme-linked immunosorbent assay 

(ELISA). 

4.1 Verification with DNA based methods

target specific reporter and quencher dyes, which lead to an increased fluorescence. The number of 

cycles (Ct-value) which are required to pass a fluorescence threshold correlates with the amount of 

target DNA in the starting sample. Results obtained can be found in table 13-15. The detection and 

quantitation limits of the EPSPS method and the CaMV 35S and Agrobacterium tumefaciens 

NOS screening methods have been established by dilution of DNA extracted from the 100% 

powder in 0% DNA extracted from verified non-GMO plants (table 17). The calibration curves 

can be found in figure 3 and demonstrate the low specificity of the 35S screening method in soya 

material. The PCR products were also analysed by a 2% agarose gel electrophoresis (1xTBE) and 

stained with ethidium bromide to confirm the expected amplicon size. 

Table 13: Quantitation of Roundup Ready TM -DNA with an EPSPS specific real-time PCR 

[5], DNA extracted with DNeasy Plant Mini, determination in triplicate using 20 

mg/extraction. 

12

CRM 

Certified 

GMO 

concentration 

Number of 

measurements 

13 

TM 1 

Roundup Ready 

Concentration 

[%] [n] [%] std dev 

IRMM-410S-0 < 0.03 5 0.02 0.01 

IRMM-410S-1 0.10 ± 0.05 5 0.13 0.05 





1 primers used can be found in the annex 

Table 14: Quantitation of Roundup Ready TM -DNA with 35S junction real-time PCR [7], 

DNA extracted with DNeasy Plant Mini, determination in triplicate using 20 


CRM 

Certified 

GMO 

Concentration 

Number of 

measurements 

TM 1 

Roundup Ready 

Concentration 

[%] [n] [%] std dev 

IRMM-410S-0 < 0.03 2 (0.006) 2 

(0.003) 







2 value below LOD (see table 17)

Table 15: Quantitation of Roundup Ready TM -DNA with 35S screening real-time PCR, 



CRM 

Certified 

GMO 

Concentration 

Number of 

measurements 

14 

Roundup Ready TM 

Concentration 1 

[%] [n] [%] std dev 


(0.01) 







2 value below LOD (see table 17) 

Table 16: Quantitation of Roundup Ready TM -DNA with NOS screening real-time PCR, 



CRM 

Certified 

GMO 

Concentration 

Number of 

measurements 


Concentration 1 

[%] [n] [%] std dev 


(0.005) 







2 value below LOD (see table 17)

Table 17: Limit of detection (LOD) and limit of quantitation (LOQ) of the real-time PCR 

methods used for the verification, established by dilution of DNA extracted from 100 % 

powder in 0 % DNA extracted from verified non-GMO plants 

Real-time PCR method LOD [% GMO] LOQ [% GMO] 

EPSPS (table 13, [5]) 0.02 0.06 

35S junction (table 14, [7]) 1 

15 

0.02 - 

35S screening (table 15, [5]) 0.13 0.38 

NOS screening (table 16) 0.009 0.028 

Threshold value [Ct] 

1 theoretical LOD and LOQ on soya [8] based on 20 genome copies 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

0.001 0.01 0.1 1 10 

Figure 3: Calibration curves for the EPSPS specific () and 35S screening () method 

established by dilution of DNA extracted from the 100% powder in 0% DNA extracted 

from verified non-GMO plants. The threshold values (cycle numbers) have been plotted 

against the log concentration of the GMO standard DNA in percent. 

4.2 Verification with protein based methods 

GMO concentration [%] 

Additionally all six materials were analysed with an ELISA test detecting the CP4 EPSPS protein 

(GMO ✓Soya Test Kit®, Strategic Diagnostics Inc., Newark, Delaware, USA). The system has 

been calibrated with non-GMO and 100% GMO material. The applied CP4 EPSPS protein

ELISA has a quantitative range between 0.3 and 2.5 %, the samples containing 5 % GMO were 

therefore diluted. Results obtained can be found in table 18. 

Table 18: EPSPS protein ELISA (GMO ✓Soya Test Kit®, Strategic Diagnostics Inc., 

Newark, Delaware, USA) at a sample intake level of 0.5 g, data analysed with linear curve 

fit 

CRM 

Certified GMO 

concentration 

Number of 

measurements 

16 


Concentration 

[%] [n] [%] std dev 

IRMM-410S-0 < 0.03 5 not detectable - 

IRMM-410S-1 0.10 ± 0.05 5 not detectable - 





4.3 Comparison of results obtained with both methods 

Results above the LOD obtained with the DNA based methods (table 13-16) and results obtained 

with the protein-based method (table 18) are compared in figure 4. 

GMO concentration determined by ELISA [%] 

6 

5 

4 

3 

2 

1 

0 

0 

0 1 2 3 4 5 6 7 

Certified GMO concentration [%] 

6 

5 

4 

3 

2 

1 

GMO concentration determined by PCR [%]

Figure 4: Quantitation of the Roundup Ready TM by ELISA () and by real-time PCR 

targeting the EPSPS (), the CaMV 35S-junction () the CaMV 35S () and the NOS 

() sequences. 

4.4 Results 

Quantitation of the GMO content of six mixtures of Roundup Ready TM soybean powder by DNA 

and protein based methods proved the consistency of CRM IRMM-410S (tables 13-16, 18 and 

figure 4). The results proved the consistency of the produced CRMs. The determination of the 

absolute GMO concentration has been detected correctly with the help of protein and DNA based 

methods when the systems have been calibrated with the 100% GMO material. 

One has to be careful to draw quantitative conclusions from measurements of unknown samples as 

DNA and/or protein based GMO quantitation may depend on varieties. 

5 References and acknowledgements 

5.1 References 

[1] EC No1139/98 on the labelling of certain foodstuffs produced from genetically modified 

organisms as amended by EC regulation 49/2000 and No 50/2000 

[2] Catalogue and sales conditions for certified reference materials: 

http://www.irmm.jrc.be/mrm.html 

[3] http://64.26.172.90/docroot/decdocs/gts40-3-2-update.pdf 

[4] Pietsch K., Waiblinger H.U., Brodmann P. and Wurz A. (1997): Screeningverfahren zur 

Identifizierung 'genteschnisch veränderter' pflanzlicher Lebensmittel, Deutsche Lebensmittel- 

Rundschau 2, 35-38. 

[5] Molekularbiologische Methoden (2001), in: Swiss Food Manual (Schweizerisches 

Lebensmittelbuch), Bundesamt für Gesundheit (eds.), Eidgenössische Drucksachen- und 

Materialzentrale, Bern 

[6] Prokisch J., Zeleny R., Trapmann S., Le Guern L., Schimmel H., Kramer G.N. and Pauwels J. 

(2001): Estimation of the Minimum Uncertainty for a GMO Containing maize sample candidate 

certified reference material, Fresenius J. Anal. Chem. 370 (7), 935-939. 

[7] Taveniers I.,Windels P., Van Bockstaele E. and De Loose M. (2001): Use of cloned DNA 

fragments for event-specific quantification of genetically modified organisms in pure and mixed food 

products, Eur. Food Res. Technol., 213: 417-424. 

[8] Kay S., Van den Eede G. (2001): The limits of GMO detection, Nature Biotechnology, 19(5), 

405. 

5.2 Acknowledgements 

Kim Magin and her staff from Monsanto (St.Louis, MO, USA) for the purity checks carried out on 

the non-GMO starting material. 

17

Seong Kon Lee from the National Agricultural Science & Technology Institute in Korea took care 

of the purity checks of the non-GMO shipment units during his stay at IRMM. 

Isabelle Taverniers from the Centre for Agricultural Research (CLO, Melle, BE) provided the 

Roundup Ready TM 35S-junction primers and probe. 

18

Annex I 

CaMV 35S real-time PCR (table 1, 13), [5] 

Primer: 

TM-35S-1: 5’-gcc tct gcc gac agt ggt-3’ 

TM-35S-2: 5’-aag acg tgg ttg gaa cgt ctt c-3’ 

Probe: 

35S-FAM: 5’-(FAM)-caa aga tgg acc ccc acc cac g-(TAMRA)-3’ 

Amplicon size: 83 bp 

CaMV NOS real-time PCR (table 1, 16), [unpublished method] 

Primer: 

Nost5'F: 5’-ttg gca ata aag ttt ctt aag att gaa t-3’ 

Nost5'R: 5'-aca tgc tta acg taa ttc aac aga aat t -3’ 

Probe: 

NOS-FAM: 5’-(FAM)-ctg ttg ccg gtc ttg cga tga tta tca t-(TAMRA)-3’ 


Soya specific lectin real-time PCR (table 14), [5] 

Primer: 

TM-Lectin-F: 5’-tcc acc ccc atc cac att t-3’ 

TM-Lectin-R: 5’-ggc ata gaa ggt gaa gtt gaa gga-3’ 

Probe: 

Lectin-FAM: 5’-(FAM)-aac cgg tag cgt tgc cag ctt cg-(TAMRA)-3’ 

EPSPS specific real time PCR (table 1, 14), [5] 

Primer: 

RRS-F: 5’-gcc atg ttg tta att tgt gcc at-3’ 

RRS-R: 5’-gaa gtt cat ttc att tgg aga gga c-3’ 

Probe: 

RRS-FAM: 5’-(FAM)-ctt gaa aga tct gct aga gtc agc ttg tca gcg-(TAMRA)- 3’ 


Junction Roundup Ready TM specific real time PCR (table 16), [7] 

Primer: 

35S4-JUNC: 5'-ctt tga aga cgt ggt tgg aa-3' 

RRS3-JUNC: 5'-aaa gga agg tgg ctc cta ca-3' 

Probe: 

TQ-JUN1: 5'-(TET)-aac gat ggc ctt tcc ttt atc gca at-(TAMRA)-3' 


19


EUR 20273 EN - Institute for Reference Materials and Measurements - 

The certification of Reference Materials of Dry-mixed Soya Powder with different Mass 

Fractions of Roundup Ready TM Soya - IRMM-410S 

Editors: S. Trapmann, P. Catalani, P. Conneely, P. Corbisier, D. Gancberg, E. Hannes, 

L. Le Guern, G. N. Kramer, J. Prokisch, P. Robouch, H. Schimmel, R. Zeleny and J. Pauwels, 

G. Van den Eede, F. Weighardt, M. Mazzara and E. Anklam 

Luxembourg: Office for Official Publications of the European Communities 

2002 - 21 pp. - 21.0 x 29.7 cm 

IRMM Information Series 

ISBN 92-894-3725-1 

Contact 


Joint Research Centre 

Institute for Reference Materials and Measurements 

Retieseweg 

B-2440 Geel 

Tel. +32 (0) 14 571 211 

Fax. +32 (0) 14 584 273 

http://www.irmm.jrc.be

The mission of the JRC is to provide customer-driven scientific and technical support for 

the conception, development, implementation and monitoring of EU policies. As a 

service of the European Commission, the JRC functions as a reference centre of 

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common interest of the Member States, while being independent of special interests, 

whether private or national. 

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ISBN 92-894-3725-1 

ISBN 92-894-3725-1 

15 LA-NA-20-273-EN-C

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