IMAGEMO REPORT
In the frame of this project a PCR based method for GM soy and maize was developed and
validated. For this purpose a 35S primer set was designed by our laboratory. In order to evaluate
the test Certified Reference Materials and commercial samples were analyzed.
The first step was the establishment of a sample pretreatment protocol (Annex I). When this was
completed, validation of the assay was performed (Annex II). Finally commercial samples were
screened for the presence of GM Soya and GM Maize. Samples positive for the presence of 35S
were analyzed by REAL TIME PCR (TaqMan™) for quantification of GM percentage. The same
samples were provided to partner No8 in order to quantify them by protein based techniques
IMAGEMO
ENGENE S.A REPORT
Annex I
PROTOCOL FOR GMO SCREENING
Sample Preparation
a) Mixture and weigh of sample
Kernel samples
All kernels are thoroughly mixed for 2 minutes and 10 kernels are randomly chosen from 5
different spots of the sample. The 10 randomly chosen kernels are weighed and 100 multiply their
weigh in order to estimate the weigh of 1000 kernels.
A sub-sample equal to 1000 kernels is taken from 5 different spots of the sample.
Raw material/processed foods
If sample is raw material or processed food then it is thoroughly mixed for 2 minutes and a subsample of 100 g is taken from 5 different spots of the sample.
b) Production of powdery based material.
A Thermomix mixer for 2 min in full speed grinds the sub samples. This procedure is repeated 3
times.
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ENGENE S.A REPORT
Sample Analysis
Powdry samples
2 analysts analyze each sample independently. Each analyst analyses two sub samples of 100
mg.
Lecithin, oil samples
2 analysts analyze each sample independently. Each analyst analyses 6 sub samples of 2 gr.
Extracted DNA from 6 sub samples is dissolved in one vial.
IMAGEMO
ENGENE S.A REPORT
Step 1. Extraction of DNA
DNA extraction from powdery based material (CTAB method)
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100 mg sample material are placed in an 1.5 ml eppendorf tube
500 µl of CTAB extraction buffer is added
the sample is incubated at 65°C for at least 30 min
centrifuge for 10 min at 16.000 g
transfer supernatant to a new reaction tube
add 200 µl chloroform and mix 30 s
centrifuge the mixture for 10 min (until phases separate) at 12.000 g
transfer the upper (aqueous) phase into a new reaction vessel
add two volumes CTAB precipitation solution and mix well
incubate 60 min at room temperature
centrifuge for 10 min at 14.500 g
discard the supernatant
dissolve the precipitate by adding 350 µl NaCl solution
add 350 µl chloroform and mix 30 s
centrifuge the mixture for 10 min (until phases separate) at 12.000 g
transfer the upper (aqueous) phase into a new reaction vessel
add 0.8 volume parts of isopropanol, mix and store for 15 min at room temperature
pellet the DNA by a 10 min centrifugation step at 12.000 g
discard the supernatant
wash the pellet with 500 µl of 70% Ethanol
precipitate the DNA with centrifugation for 10 min and 12.000 g decant the supernatant
carefully and discard it
dry DNA and resuspend the pellet e.g. in 100 µl of water or a suitable PCR buffer
Solutions
Extraction (CTAB) buffer solution (pH= 8.0):
CTAB precipitation solution:
CTAB (20 g/ l), NaCl (1.4 mol/ l), TRIS-base/ HCl
(0.1 mol/ l) and Na2-EDTA (20 mmol/ l) adjusted
with HCl
CTAB (5 g/ l), NaCl (0.04 mol/ l)
NaCl solution :
1.2 mol/ l
IMAGEMO
ENGENE S.A REPORT
DNA extraction from lecithin-oils
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2.0 g of sample lecithin are placed in a sterile 50 ml Falcon tube
add 10 ml n-Hexane and 1 ml of extraction buffer and mix well
centrifuge the samples for 15-20 min at 3000-5000 g for separation of organic and aqueous
phases
(if no separation of organic and aqueous phases was achieved add another 500 µl of extraction
buffer, mix well and centrifuge again)
the aqueous phase (at least 500 µl) is together with the interphase transfered into a new 2 ml
eppendorf tube
add 1 volume of chloroform and mix well
centrifuge for 10 min at 15.000 g
transfer the (upper) aqueous phase and add 2 µl of glycogen
add 0.8 volumes of isopropanol and mix well
incubate for 30 min at room temperature
pellet the DNA by a 10 min centrifugation step at 12.000 g
wash the pellet with 500 µl of 70 % ethanol
dry the DNA pellet and resuspend the pellet e.g. in 100 µl of water or a suitable PCR buffer
clean the extracted DNA using a Microspin S300 (Pharmacia Biotech. Uppsala, Sweden)
according to the manufacturers instructions
Solutions
Extraction (CTAB) buffer solution (pH= 8.0):
IMAGEMO
CTAB (20 g/ l), NaCl (1.4 mol/ l), TRIS-base/ HCl
(0.1 mol/ l) and Na2-EDTA (20 mmol/ l) adjusted
with HCl
ENGENE S.A REPORT
Step 2. Control PCR reaction
This PCR reaction amplifies a gene that is present naturally in all varieties of the plant and the
positive result verifies the absence of inhibitors.
MAIZE SAMPLES
Reference gene for maize : invertase
PCR conditions using Genius Techne PCR thermocycler system for 5μL of each PCR reaction
[5μL DNA solution, 1 U AmpliTaq Polymerase, 1.65mM Mg2+, 160μM of each dNTP and 0.6μM
of each primer, buffer for polymarase] are proposed as follows:
94° C για 10 min
94 °C για 20 sec
54 °C για 40 sec
x 35
72 °C για 40 sec
72 °C για 3 min
SOYA SAMPLES
Reference gene for soya : Lectin lec1
PCR conditions using Genius Techne PCR thermocycler system for 5μL of each PCR reaction
[5μL DNA solution, 1 U AmpliTaq Polymerase, 1.65mM Mg2+, 160μM of each dNTP and 0.6μM
of each primer, buffer for polymarase] are proposed as follows:
95° C για 10 min
94 °C για 30 sec
62 °C για 30 sec
x 35
72 °C για 45 sec
72 °C για 3 min
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ENGENE S.A REPORT
Step 3. Electrophoresis of PCR product
The products of control PCR are analyzed by agarose gel electrophoresis. Gel concentration is 2%.
The electrophoresis is carried out in room temperature at a voltage not exceeding 5V/cm of
electrode spacing). To determine the size of DNA fragments, DNA molecular marker is analysed
in parallel.
After completing the electrophoresis the gel is incubated, the gel is incubated for 15-30 minutes in
ethidium bromide solution. DNA becomes visible in UV light at 312 nm and the gel is
documented by a photo.
Samples that give positive PCR control reaction are further analysed for the presence of 35S
promoter and NOS terminator.
Step 4. Specific PCR reaction
This reaction amplifies a target sequence that is present only in GMO.
First PCR : 35S promoter
PCR conditions using Genius Techne PCR thermocycler system for 5μL of each PCR reaction
[5μL DNA solution, 1 U AmpliTaq Polymerase, 1.65mM Mg2+, 160μM of each dNTP and 0.6μM
of each primer, buffer for polymarase] are proposed as follows:
94° C για 3 min
94 °C για 20 sec
70 °C για 40 sec
x 35
72 °C για 60 sec
72 °C για 3 min
IMAGEMO
ENGENE S.A REPORT
Second PCR reaction : NOS terminator
PCR conditions using Genius Techne PCR thermocycler system for 5μL of each PCR reaction
[5μL DNA solution, 1 U AmpliTaq Polymerase, 2 mM Mg2+, 160μM of each dNTP and 0.6μM of
each primer, buffer for polymarase] are proposed as follows:
94° C για 10 min
94 °C για 25 sec
64 °C για 30 sec
x 50
72 °C για 45 sec
72 °C για 3 min
In specific PCR reaction 0 % GMO reference material and 0,1% GMO reference material
are analysed in parallel with the 4 sub samples of the unknown sample.
Step 5. Electrophoresis of PCR product
The products of 35S PCR and NOS PCR are analyzed by agarose gel electrophoresis. Gel
concentration is 2%. The electrophoresis is carried out in room temperature at a voltage not
exceeding 5V/cm of electrode spacing). To determine the size of DNA fragments, DNA
molecular marker is analysed in parallel.
After completing the electrophoresis the gel is incubated, the gel is incubated for 15-30 minutes in
ethidium bromide solution. DNA becomes visible in UV light at 312 nm and the gel is
documented by a photo.
IMAGEMO
ENGENE S.A REPORT
WORK FLOW
1. DNA extraction
Unknown
sample (x 4)
0 % GMO reference
sample
1 % GMO reference
sample
Extraction sample
The last one does not contain any sample
material and it is used to verify that all
reagents used are free from contamination.
2. Control PCR reaction
This PCR reaction amplifies a gene that is present naturally in all varieties of the plant and the
positive result verifies the absence of inhibitors.
Unknown
0 % GMO
sample (x 4) reference sample
Positive
Positive
0,1 % GMO
reference sample
1 % GMO
reference sample
Extraction
control
Positive
Positive
Negative
3. Specific PCR reaction
This reaction amplifies a target sequence that is present only in GMO.
Unknown
0 % GMO
sample (x 4) reference sample
?
IMAGEMO
Negative
0,1 % GMO
reference sample
1 % GMO
reference sample
Extraction
control
Positive
Positive
Negative
ENGENE S.A REPORT
Annex II
VALIDATION OF THE METHOD FOR GM SOY and MAIZE
The objective of the validation of an analytical PCR method is to demonstrate that the successive
procedures of sample extraction, preparation and analysis will yield acceptable, accurate precise
and reproducible results for a given analyte in a specified matrix. The process of validation allows
the independent use of methods and results, which are comparable among its other.
For the validation of a qualitative analytical test system Specificity/selectivity, sensitivity (matrix
effects/inhibition, limit of detection LOD, accuracy /precision) and robustness have to be
established. For this purpose the following experiments were performed.
IMAGEMO
ENGENE S.A REPORT
VALIDATION OF THE METHOD FOR GM SOY
Α. DNA Extraction
DNA extraction was performed by to independent analysts (M&Λ) in triplicate samples of 100
mg.
The following Certified Reference Materials were analyzed.

0 % RR soya (7550)

0,1 % RR soya (7486)
Β. Control PCR Reaction for the detection of lec1
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Μ
4.
Εx control Λ
5.
0 % RR (7550) Μ1
6.
0 % RR (7550) Μ2
7.
0 % RR (7550) Μ3
8.
0 % RR (7550) Λ1
9.
0 % RR (7550) Λ2
10. 0 % RR (7550) Λ3
11. 0,1 % RR (7486) Μ1
12. 0,1 % RR (7486) Μ2
13. 0,1 % RR (7486) Μ3
14. 0,1 % RR (7486) Λ1
15. 0,1 % RR (7486) Λ2
16. 0,1 % RR (7486) Λ3
IMAGEMO
ENGENE S.A REPORT
C. PCR Reaction for the detection of 35S promoter
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Μ
4.
Εx control Λ
5.
0,1 % RR (7486) Μ1
6.
0,1 % RR (7486) Μ1
7.
0,1 % RR (7486) Μ2
8.
0,1 % RR (7486) Μ2
9.
0,1 % RR (7486) Μ3
10. 0,1 % RR (7486) Μ3
11. 0,1 % RR (7486) Λ1
12. 0,1 % RR (7486) Λ1
13. 0,1 % RR (7486) Λ2
14. 0,1 % RR (7486) Λ2
15. 0,1 % RR (7486) Λ3
16
0,1 % RR (7486)
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Δ
4.
Εx control Λ
5.
0 % RR (7550) Μ1
6.
0 % RR (7550) Μ1
7.
0 % RR (7550) Μ2
8.
0 % RR (7550) Μ2
9.
0 % RR (7550) Μ3
10. 0 % RR (7550) Μ3
11. 0 % RR (7550) Λ1
12. 0 % RR (7550) Λ1
13. 0 % RR (7550) Λ2
14. 0 % RR (7550) Λ2
15. 0 % RR (7550) Λ3
16. 0% Bt-176 (0627) Λ3
IMAGEMO
ENGENE S.A REPORT
C. PCR Reaction for the detection of NOS terminator
1.
100 bp ladder DNA
2.
H2O
3.
Εx control M
4.
Εx control Λ
5.
0,1 % RR (7486) M1
6.
0,1 % RR (7486) M1
7.
0,1 % RR (7486) M2
8.
0,1 % RR (7486) M2
9.
0,1 % RR (7486) M3
10. 0,1 % RR (7486) M3
11. 0,1 % RR (7486) Λ1
12. 0,1 % RR (7486) Λ1
13. 0,1 % RR (7486) Λ2
14. 0,1 % RR (7486) Λ2
15. 0,1 % RR (7486) Λ3
16
0,1 % RR (7486) Λ3
1.
100 bp ladder DNA
2.
H2O
3.
Εx control M
4.
Εx control Λ
5.
0 % RR (7550) M1
6.
0 % RR (7550) M1
7.
0 % RR (7550) M2
8.
0 % RR (7550) M2
9.
0 % RR (7550) M3
10. 0 % RR (7550) M3
11. 0 % RR (7550) Λ1
12. 0 % RR (7550) Λ1
13. 0 % RR (7550) Λ2
14. 0 % RR (7550) Λ2
15. 0 % RR (7550) Λ3
16. 0% Bt-176 (0627) Λ3
IMAGEMO
ENGENE S.A REPORT
RESULTS
1) 6 DNA extractions were performed in triplicate by 2 analysts from samples:
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0 % RR soya (7550)
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0,1 % RR soya (7486)
For each DNA extract 2 PCR control reactions were performed in order to verify the absence of
inhibitors.
SAMPLE
CONTROL PCR RESULT
REFERENCE MATERIALS
positive
Η2Ο (PCR blind control)
negative
Extraction Control (M)
negative
Extraction Control (Λ)
negative
2) For each DNA extract of 0% RR soya (7550) και 0,1 % RR soya (7486) 2 PCR reactions for
the presence of 35S were performed.
SAMPLE
35S PCR RESULT
Η2Ο (PCR blind control)
negative
Extraction Control (M)
negative
Extraction Control (Λ)
negative
6 x 2 PCR reactions 0 % RR soya (7550)
12 negative
6 x 2 PCR reactions 0,1 % RR soya (7486)
12 positive
3) For each DNA extract of 0% RR soya (7550) και 0,1 % RR soya (7486) 2 PCR reactions for
the presence of NOS were performed.
SAMPLE
35S PCR RESULT
Η2Ο (PCR blind control)
negative
Extraction Control (M)
negative
Extraction Control (Λ)
negative
6 x 2 PCR reactions 0 % RR soya (7550)
IMAGEMO
12 negative
ENGENE S.A REPORT
6 x 2 PCR reactions 0,1 % RR soya (7486)
12 positive
According to these results the following parameters were estimated:
SENSITIVITY: 100 %
SENSITIVITY: 100 %
DETECTION LIMIT: 0,1% για 35S και ΝΟS
 Matrix effects
Each DNA extraction is followed by a control PCR reaction. This PCR reaction amplifies a gene
that is present naturally in all varieties of the plant and the positive result verifies the absence of
inhibitors. The Reference gene for soy is Lectin lec1.
Samples submitted to the assay (control PCR reaction positive) are listed to Table 1.
Table 1. Samples submitted to Control PCR reaction
Sample
Control PCR reaction
Lecithin
Positive
Soya kernels
Positive
Cake mix
Positive
Soy burger
Positive
Soy Flour
Positive
Fish Feed
Positive
Animal Feed
Positive
Cookies
Positive
 Repeatability
Inter laboratory variation was demonstrated by
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DNA extraction performed by three different analysts
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PCR reaction was set up by three different analysts
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2 different centrifuges were used for DNA extraction
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2 different Thermal Cycler were used for PCR reaction.
In each case all results were the expected ones.
IMAGEMO
ENGENE S.A REPORT
 Reproducibility
Reproducibility o the method was performed by official ring trials. In each case all results were
the expected ones.
 Robustness
1) Incubation temperature
Α. DNA Extraction
DNA extraction was performed by to independent analysts (M&Λ) in duplicate samples of 50
mg.
The following Certified Reference Materials were analyzed.

0 % RR soya (7550)

0,1 % RR soya (7486)
Incubation temperature with lysis buffer
Sample
Αnalyst
0 % RR (7550)
0,1 RR ( 7486)
1696 (Commercial Soy flour)
Μ
70 °C
70 °C
60 °C
Λ
60 °C
60 °C
70 °C
Β. Control PCR Reaction for the detection of lec1
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
0 % RR (7550) Λ1
6.
0 % RR (7550) Λ2
7.
0 % RR (7550) Μ1
8.
0 % RR (7550) Μ2
9.
0,1 % RR (7486) Λ1
10. 0,1 % RR (7486) Λ2
11. 0,1 % RR (7486) Μ1
12. 0,1 % RR (7486) Μ2
13. 1696 Λ1
14. 1696 Λ2
15. 1696Μ1
16. 1696Μ2
IMAGEMO
ENGENE S.A REPORT
C. PCR Reaction for the detection of 35S promoter
1.
100 bp ladder DNA
2.
H2O
3.
0 % RR (7550) Λ1
4.
0 % RR (7550) Λ2
5.
0 % RR (7550) Μ1
6.
0 % RR (7550) Μ2
7.
0,1 % RR (7486) Λ1
8.
0,1 % RR (7486) Λ2
9.
0,1 % RR (7486) Μ1
10. 0,1 % RR (7486) Μ2
11. 1696 Λ1
12. 1696 Λ2
13. 1696Μ1
14. 1696Μ2
15. Εx control Λ
16. Εx control Μ
C. PCR Reaction for the detection of NOS terminator
1.
100 bp ladder DNA
2.
H 2O
3.
Εx control Λ
4.
Εx control Μ
5.
0 % RR (7550) Λ1
6.
0 % RR (7550) Λ2
7.
0 % RR (7550) Μ1
8.
0 % RR (7550) Μ2
9.
0,1 % RR (7486) Λ1
10. 0,1 % RR (7486) Λ2
11. 0,1 % RR (7486) Μ1
12. 0,1 % RR (7486) Μ2
13. 1696 Λ1
14. 1696 Λ2
15. 1696Μ1
16. 1696Μ2
IMAGEMO
ENGENE S.A REPORT
2) Incubation time
Α. DNA Extraction
DNA extraction was performed by to independent analysts (M&Λ) in duplicate samples of 50
mg.
The following Certified Reference Materials were analyzed.

0 % RR soya (7550)

0,1 % RR soya (7486)
Incubation time with lysis buffer
Sample
Αnalyst
0 % RR (7550)
0,1 RR ( 7486)
1696 (Commercial Soy flour)
Μ
45 min
45 min
20 min
Λ.
20 min
20 min
45 min
Β. Control PCR Reaction for the detection of lec1
1.
100 bp ladder DNA
2.
H 2O
3.
0 % RR (7550) Λ1
4.
0 % RR (7550) Λ2
5.
0 % RR (7550) Μ1
6.
0 % RR (7550) Μ2
7.
0,1 % RR (7486) Λ1
8.
0,1 % RR (7486) Λ2
9.
0,1 % RR (7486) Μ1
10. 0,1 % RR (7486) Μ2
11. 1696 Λ1
12. 1696 Λ2
13. 1696Μ1
14. 1696Μ2
15. Εx control Λ
16. Εx control Μ
IMAGEMO
ENGENE S.A REPORT
C. PCR Reaction for the detection of 35S promoter
1.
100 bp ladder DNA
2.
0 % RR (7550) Λ1
3.
0 % RR (7550) Λ2
4.
0 % RR (7550) Μ1
5.
0 % RR (7550) Μ2
6.
0,1 % RR (7486) Λ1
7.
0,1 % RR (7486) Λ2
8.
0,1 % RR (7486) Μ1
9.
0,1 % RR (7486) Μ2
10. 1696 Λ1
11. 1696 Λ2
12. 1696Μ1
13. 1696Μ2
14. H2O
15. Εx control Λ
16. Εx control Μ
C. PCR Reaction for the detection of NOS termonator
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
0 % RR (7550) Λ1
6.
0 % RR (7550) Λ2
7.
0 % RR (7550) Μ1
8.
0 % RR (7550) Μ2
9.
0,1 % RR (7486) Λ1
10. 0,1 % RR (7486) Λ2
11. 0,1 % RR (7486) Μ1
12. 0,1 % RR (7486) Μ2
13. 1696 Λ1
14. 1696 Λ2
15. 1696Μ1
16. 1696Μ2
IMAGEMO
ENGENE S.A REPORT
VALIDATION OF THE METHOD FOR GM MAIZE
Α. DNA Extraction
DNA extraction was performed by to independent analysts (M&Λ) in triplicate samples of 100
mg.
The following Certified Reference Materials were analyzed.

0 % Bt-176 (0627)

0,1 Bt-176 (0687)

1% Bt-11 (0318)
Β. Control PCR Reaction for the detection of Invertase
17. 100 bp ladder DNA
1.
100 bp ladder DNA
18. H2O
2.
0,1 % Bt-176 (0687) Μ1
19. Εx control Μ
3.
0,1 % Bt-176 (0687) Μ2
20. Εx control Λ
4.
0,1 % Bt-176 (0687) Μ3
21. 0 % Bt-176 (0627) Μ1
5.
0,1 % Bt-176 (0687) Λ1
22. 0 % Bt-176 (0627) Μ2
6.
0,1 % Bt-176 (0687) Λ2
23. 0 % Bt-176 (0627) Μ3
7.
0,1 % Bt-176 (0687) Λ3
24. 0 % Bt-176 (0627) Λ1
25. 0 % Bt-176 (0627) Λ2
26. 0 % Bt-176 (0627) Λ3
27. 1% Bt-11 (0318) Μ1
28. 1% Bt-11 (0318) Μ2
29. 1% Bt-11 (0318) Μ3
30. 1% Bt-11 (0318) Λ1
31. 1%Bt-11 (0318) Λ2
32. 1% Bt-11 (0318) Λ3
IMAGEMO
ENGENE S.A REPORT
C. PCR Reaction for the detection of 35S promoter
16. 100 bp ladder DNA
17. H2O
18. Εx control Δ
19. Εx control Λ
20. 0,1 Bt-176 (0687) Μ1
21. 0,1 Bt-176 (0687) Μ1
22. 0,1 Bt-176 (0687) Μ2
23. 0,1 Bt-176 (0687) Μ2
24. 0,1 Bt-176 (0687) Μ3
25. 0,1 Bt-176 (0687) Μ3
26. 0,1 Βt-176 (0687) Λ1
27. 0,1 Bt-176 (0687) Λ1
28. 0,1 Bt-176 (0687) Λ2
29. 0,1 Bt-176 (0687) Λ2
30. 0,1 Bt-176 (0687) Λ3
16. 0,1 Bt-176 (0687) Λ3
16. 100 bp ladder DNA
17. H2O
18. Εx control Δ
19. Εx control Λ
20. 0% Bt-176 (0627) Μ1
21. 0% Bt-176 (0627) Μ1
22. 0% Bt-176 (0627) Μ2
23. 0% Bt-176 (0627) Μ2
24. 0 % Bt-176 (0627) Μ3
25. 0% Bt-176 (0627) Μ3
26. 0% Βt-176 (0627) Λ1
27. 0% Bt-176 (0627) Λ1
28. 0% Bt-176 (0627) Λ2
29. 0% Bt-176 (0627) Λ2
30. 0% Bt-176 (0627) Λ3
16. 0% Bt-176 (0627) Λ3
IMAGEMO
ENGENE S.A REPORT
C. PCR Reaction for the detection of NOS termonator
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
1% Bt-11 (0318) Λ1
6.
1% Bt-11 (0318) Λ1
7.
1% Bt-11 (0318) Λ2
8.
1% Bt-11 (0318) Λ2
9.
1% Bt-11 (0318) Λ3
10. 1% Bt-11 (0318) Λ3
11. 1% Βt-11 (0318) Μ1
12. 1% Bt-11 (0318) Μ1
13. 1% Bt-11 (0318) Μ2
14. 1% Bt-11 (0318) Μ2
15. 1% Bt-11 (0318) Μ3
16. 1% Bt-11 (0318) Μ3
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
0% Bt-176 (0627) Λ1
6.
0% Bt-176 (0627) Λ1
7.
0% Bt-176 (0627) Λ2
8.
0% Bt-176 (0627) Λ2
9.
0 % Bt-176 (0627) Λ3
10. 0% Bt-176 (0627) Λ3
11. 0% Βt-176 (0627) Μ1
12. 0% Bt-176 (0627) Μ1
13. 0% Bt-176 (0627) Μ2
14. 0% Bt-176 (0627) Μ2
15. 0% Bt-176 (0627) Μ3
16. 0% Bt-176 (0627) Μ3
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ENGENE S.A REPORT
RESULTS
1) 6 DNA extractions were performed in triplicate by 2 analysts from samples:

0 % Bt-176 (0627)

0,1 Bt-176 (0687)

1% Bt-11 (0318)
For each DNA extract 2 PCR control reactions were performed in order to verify the absence of
inhibitors.
SAMPLE
CONTROL PCR RESULT
REFERENCE MATERIALS
positive
Η2Ο (PCR blind control)
negative
Extraction Control (M)
negative
Extraction Control (Λ)
negative
2) For each DNA extract of 0% RR soya (7550) και 0,1 % RR soya (7486) 2 PCR reactions for
the presence of 35S were performed.
SAMPLE
35S PCR RESULT
Η2Ο (PCR blind control)
negative
Extraction Control (M)
negative
Extraction Control (Λ)
negative
6 x 2 PCR reactions 0 % Bt-176 (0627)
12 negative
6 x 2 PCR reactions 0,1 % Bt-176 (0687)
12 positive
3) For each DNA extract of 0% RR soya (7550) και 0,1 % RR soya (7486) 2 PCR reactions for
the presence of NOS were performed.
SAMPLE
35S PCR RESULT
Η2Ο (PCR blind control)
negative
Extraction Control (M)
negative
Extraction Control (Λ)
negative
6 x 2 PCR reactions 0 % Bt-176 (0627)
IMAGEMO
12 negative
ENGENE S.A REPORT
6 x 2 PCR reactions 1 % Bt-11 (0318)
12 positive
According to these results the following parameters were estimated:
SENSITIVITY: 100 %
SENSITIVITY: 100 %
DETECTION LIMIT: 0,1% για 35S και ΝΟS
 Matrix effects
Each DNA extraction is followed by a control PCR reaction. This PCR reaction amplifies a gene
that is present naturally in all varieties of the plant and the positive result verifies the absence of
inhibitors. The Reference gene for maize is invertase.
Samples submitted to the assay (control PCR reaction positive) are listed to Table 2.
Table 2. Samples submitted to Control PCR reaction
Sample
Control PCR reaction
Corn Flakes
Positive
Maize kernels
Positive
Cream Crackers
Positive
Biscuits
Positive
Fish Feed
Positive
Animal Feed
Positive
Corn flour
Positive
 Repeatability
Inter laboratory variation was demonstrated by
-
DNA extraction performed by three different analysts
-
PCR reaction was set up by three different analysts
-
2 different centrifuges were used for DNA extraction
-
2 different Thermal Cycler were used for PCR reaction.
In each case all results were the expected ones.
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 Reproducibility
Reproducibility o the method was performed by official ring trials. In each case all results were
the expected ones.
 Robustness
1) Incubation temperature
Α. DNA Extraction
DNA extraction was performed by to independent analysts (M&Λ) in duplicate samples of 50
mg.
The following Certified Reference Materials were analyzed.

0 % ΜΟΝ (0392)

0,1 ΜΟΝ (0405)

1% ΜΟΝ (0417)

2394 (maize flour)
Incubation temperature with lysis buffer
Sample
Αnalyst
0 % ΜΟΝ (0392)
0,1 ΜΟΝ (0405)
Μ
70 °C
70 °C
60 °C
Λ
60 °C
60 °C
70 °C
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Commercial Maize flour
ENGENE S.A REPORT
Β. Control PCR Reaction for the detection of Invertase
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
0 % ΜΟΝ (0392) Λ1
6.
0 % ΜΟΝ (0392) Λ2
7.
0 % ΜΟΝ (0392) Μ1
8.
0 % ΜΟΝ (0392) Μ2
9.
0,1% ΜON (0405) Λ1
10. 0,1% ΜON (0405) Λ2
11. 0,1% ΜON (0405) Μ1
12. 0,1% ΜON (0405) Μ2
13. 2394 Λ1
14. 2394 Λ2
15. 2394 Μ1
16. 2394 Μ2
C. PCR Reaction for the detection of 35S promoter
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
0 % ΜΟΝ (0392) Λ1
6.
0 % ΜΟΝ (0392) Λ2
7.
0 % ΜΟΝ (0392) Μ1
8.
0 % ΜΟΝ (0392) Μ2
9.
0,1% ΜON (0405) Λ1
10. 0,1% ΜON (0405) Λ2
11. 0,1% ΜON (0405) Μ1
12. 0,1% ΜON (0405) Μ2
13. 2394 Λ1
14. 2394 Λ2
15. 2394 Μ1
16. 2394 Μ2
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C. PCR Reaction for the detection of NOS
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
0 % ΜΟΝ (0392) Λ1
6.
0 % ΜΟΝ (0392) Λ2
7.
0 % ΜΟΝ (0392) Μ1
8.
0 % ΜΟΝ (0392) Μ2
9.
0,1% ΜON (0405) Λ1
10. 0,1% ΜON (0405) Λ2
11. 0,1% ΜON (0405) Μ1
12. 0,1% ΜON (0405) Μ2
13. 2394Λ1
14. 2394 Λ2
15. 2394 Μ1
16. 2394 Μ2
2) Incubation time
Α. DNA Extraction
DNA extraction was performed by to independent analysts (M&Λ) in duplicate samples of 50
mg.
The following Certified Reference Materials were analyzed.

0 % ΜΟΝ (0392)

0,1 ΜΟΝ (0405)

1% ΜΟΝ (0417)

2394 (Commercial Maize Flour)
Incubation time with lysis buffer
Sample
Αnalyst
0 % ΜΟΝ (0392)
0,1 ΜΟΝ (0405)
Μ
45 min
45 min
20 min
Λ.
20 min
20 min
45 min
IMAGEMO
Commercial Maize flour
ENGENE S.A REPORT
Β. Control PCR Reaction for the detection of Invertase
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
0 % ΜΟΝ (0392) Λ1
6.
0 % ΜΟΝ (0392) Λ2
7.
0 % ΜΟΝ (0392) Μ1
8.
0 % ΜΟΝ (0392) Μ2
9.
0,1% ΜON (0405) Λ1
10. 0,1% ΜON (0405) Λ2
11. 0,1% ΜON (0405) Μ1
12. 0,1% ΜON (0405) Μ2
13. 2394 Λ1
14. 2394 Λ2
15. 2394 Μ1
16. 2394 Μ2
C. PCR Reaction for the detection of 35S promoter
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
0 % ΜΟΝ (0392) Λ1
6.
0 % ΜΟΝ (0392) Λ2
7.
0 % ΜΟΝ (0392) Μ1
8.
0 % ΜΟΝ (0392) Μ2
9.
0,1% ΜON (0405) Λ1
10. 0,1% ΜON (0405) Λ2
11. 0,1% ΜON (0405) Μ1
12. 0,1% ΜON (0405) Μ2
13. 2394 Λ1
14. 2394 Λ2
15. 2394 Μ1
16. 2394 Μ2
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C. PCR Reaction for the detection of NOS
1.
100 bp ladder DNA
2.
H2O
3.
Εx control Λ
4.
Εx control Μ
5.
0 % ΜΟΝ (0392) Λ1
6.
0 % ΜΟΝ (0392) Λ2
7.
0 % ΜΟΝ (0392) Μ1
8.
0 % ΜΟΝ (0392) Μ2
9.
0,1% ΜON (0405) Λ1
10. 0,1% ΜON (0405) Λ2
11. 0,1% ΜON (0405) Μ1
12. 0,1% ΜON (0405) Μ2
13. 2394 Λ1
14. 2394 Λ2
15. 2394 Μ1
16. 2394 Μ2
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