Event-specific method for the quantification of soybean line A2704-12 using real-time PCR validation report.
Date: 2007-12-17
Institutions: Institute for Health and Consumer Protection
Subjects: biotechnology, consumer protection, evaluation method, genetically modified organism, research method, soya bean

Event-specific Method for the Quantification of 
Soybean Line A2704-12 Using Real-time PCR 

Validation Report and Protocol 
Soybean Seeds Sampling and DNA Extraction  

M. Mazzara, C. Delobel, E. Grazioli, S. Larcher, C. Savini and G. Van den Eede 

14 May 2007

Method development: 
Bayer CropScience GmbH 

Method validation: 
Community Reference Laboratory for GM Food and Feed (CRL-GMFF) 
Biotechnology & GMOs Unit 

Institute for Health and Consumer Protection 
2007 

EUR 22910 EN

 
 
 
 
 
 
 
 
 
 
 
 
 
The mission of the Institute for Health and Consumer Protection is to provide scientific support to the 
development and implementation of EU policies related to health and consumer protection. 

The Institute for Health and Consumer Protection carries out research to improve the understanding of 
potential health risks posed by chemical, physical and biological agents from various sources to which 
consumers are exposed. 

European Commission 
Directorate-General Joint Research Centre 
Institute for Health and Consumer Protection 

Contact information 
Address: Biotechnology and GMOs Unit  
E-mail: JRC-BGMO@ec.europa.eu
Tel.: +39 0332 786706 
Fax: +39 0332 786159 

http://ihcp.jrc.ec.europa.eu/
http://www.jrc.ec.europa.eu/

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EUR 22910 EN 
ISBN 978-92-79-06928-4 
ISSN 1018-5593 
Luxembourg: Office for Official Publications of the European Communities 

© European Communities, 2007 

Reproduction is authorised provided the source is acknowledged 

Printed in Italy 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CRLVL13/05VR 

Event-specific Method for the Quantification 
of Soybean Line A2704-12 Using 
Real-time PCR 

Validation Report 

14 May 2007 

Directorate General-Joint Research Centre 
Institute for Health and Consumer Protection  
Biotechnology & GMOs Unit 

Executive Summary 

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF), established 
by  Regulation  (EC)  No  1829/2003,  in  collaboration  with  the  European  Network  of  GMO 
Laboratories  (ENGL),  has  carried  out  a  collaborative  study  to  assess  the  performance  of  a 
quantitative event-specific method to detect and quantify the A2704-12 transformation event in 
soybean  DNA  (unique  identifier  ACS-GMØØ5-3).  The  collaborative  trial  was  conducted 
according to internationally accepted guidelines (1, 2). 

In  accordance  with  Regulation  (EC)  No  1829/2003  of  22  September  2003  on  genetically 
modified food and feed and Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for 
the  implementation  of  Regulation  (EC)  No  1829/2003,  Bayer  CropScience  provided  the 
detection method and the samples (genomic DNA extracted from wild-type and 100% soybean 
A2704-12  event).  The  JRC  prepared  the  validation  samples  (calibration  samples  and  blind 
samples at unknown GM percentage [DNA/DNA]). The international collaborative trial involved 
thirteen laboratories from ten European countries. 

The results of the international collaborative trial met the ENGL performance requirements and 
the scientific understanding about satisfactory method performance. Therefore, the CRL-GMFF 
considers the method validated as fit for the purpose of regulatory compliance. 

The results of the collaborative study are made publicly available at http://gmo-crl.jrc.it/.  

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Report on Steps 1-3 of the Validation Process 

Bayer  CropScience  submitted  the  detection  method  and  control  samples  for  soybean  event 
A2704-12  (unique  identifier  ACS-GMØØ5-3)  under  Article  5  and  17  of  Regulation  (EC)  No 
1829/2003 of the European Parliament and of the Council “on genetically modified food and 
feed”.  

The Community Reference Laboratory for GM Food and Feed (CRL-GMFF), following reception 
of  the  documentation  and  material,  including  control  samples,  (step  1  of  the  validation 
process)  carried  out  the  scientific  assessment  of  documentation  and  data  (step  2)  in 
accordance  to  Commission  Regulation  (EC)  No  641/2004  “on  detailed  rules  for  the 
implementation  of  Regulation  (EC)  No  1829/2003  of  the  European  Parliament  and  of  the 
Council as regards the application for the authorisation of new genetically modified food and 
feed,  the  notification  of  existing  products  and  adventitious  or  technically  unavoidable 
presence  of  genetically  modified  material  which  has  benefited  from  a  favourable  risk 
evaluation”  and  according  to  its  operational  procedures  (“Description  of  the  CRL-GMFF 
Validation Process”, http://gmo-crl.jrc.it/guidancedocs.htm). 

The  scientific  assessment  focused  on  the  method  performance  characteristics  evaluated 
against the method acceptance criteria set out by the European Network of GMO Laboratories 
and listed in the “Definition of Minimum Performance Requirements for Analytical Methods of 
GMO  Testing”  (http://gmo-crl.jrc.it/guidancedocs.htm)  (see  Annex  1  for  a  summary  of 
method acceptance criteria and method performance requirements). During step 2 and step 3 
(scientific  assessment  of  documentation  and  data  and  experimental  testing  of  the  samples 
and  methods,  respectively)  four  scientific  assessments  were  performed  and  requests  of 
complementary  information  addressed  to  the  applicant.  Upon  reception  of  complementary 
information,  the  scientific  evaluation  of  the  detection  method  for  event  A2704-12  was 
positively concluded in May 2006.  

In  May  2006,  the  CRL-GMFF  verified  experimentally  the  method  characteristics  (step  3, 
experimental testing of the samples and methods) by quantifying five blind GM levels within 
the  range  0.1%-3.3%  on  a  copy  number  basis.  The  experiments  were  performed  in 
repeatability  conditions  and  demonstrated  that  the  PCR  efficiency,  linearity,  accuracy  and 
precision  of  the  quantifications  were  within  the  limits  established  by  the  ENGL.  The  DNA 
extraction module of the method was tested on samples of food and feed.  

A Technical Report summarising the results of tests carried out by the CRL-GMFF (step 3) is 

available on request. 

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Content 

1. 

INTRODUCTION ................................................................................................ 5 

2. 

LIST OF PARTICIPATING LABORATORIES ....................................................... 6 

3.  MATERIALS ....................................................................................................... 7 

4.  EXPERIMENTAL DESIGN................................................................................... 8 

5.  METHOD ............................................................................................................ 8 

DESCRIPTION OF OPERATIONAL STEPS FOLLOWED

...............................................................8

6.  DEVIATIONS REPORTED................................................................................... 9 

7.  SUMMARY OF RESULTS .................................................................................... 9 

PCR EFFICIENCY AND LINEARITY
GMO QUANTIFICATION

....................................................................................9
.............................................................................................. 10

8.  METHOD PERFORMANCE REQUIREMENTS ..................................................... 12 

9.  CONCLUSIONS ................................................................................................ 13 

10.  QUALITY ASSURANCE..................................................................................... 13 

11.  REFERENCES ................................................................................................... 13 

12.  ANNEX  1:  METHOD  ACCEPTANCE  CRITERIA  AND  METHOD  PERFORMANCE 
REQUIREMENTS  AS  SET  BY  THE  EUROPEAN  NETWORK  OF  GMO 
LABORATORIES (ENGL) .................................................................................. 14 

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1. 

Introduction 

Bayer  CropScience  submitted  the  detection  method  and  control  samples  for  soybean  event 
A2704-12  (unique  identifier  ACS-GMØØ5-3)  under  Article  5  and  17  of  Regulation  (EC)  No 
1829/2003  of  the  European  Parliament  and  of  the  Council  “on  genetically  modified  food  and 
feed”. 

The  Directorate  General-Joint  Research  Centre  (JRC,  Biotechnology  and  GMOs  Unit  of  the 
Institute  for  Health  and  Consumer  Protection)  as  Community  Reference  Laboratory  for  GM 
Food  and  Feed  (see  Regulation  EC  No  1829/2003)  organised  the  international  collaborative 
study for the event-specific method for the detection and quantification of A2704-12. The study 
involved  thirteen  laboratories,  all  members  of  the  European  Network  of  GMO  Laboratories 
(ENGL). 

Upon  reception  of  method,  samples  and  related  data  (step  1),  the  JRC  carried  out  the 
assessment of the documentation (step 2) and the in-house evaluation of the method (step 3), 
according  to  the  requirements  of  Regulation  (EC)  No  641/2004  and  following  its  operational 
procedures.  The  internal  in-house  experimental  evaluation  of  the  method  was  carried  out  in 
May 2006.  

Following  the  evaluation  of  the  data  and  the  results  of  the  in-house  laboratory  tests,  the 
international collaborative study was organised (step 4) and took place in June-July 2006. 

A method for DNA extraction from A2704-12 seeds, submitted by the applicant, was evaluated 
by  the  CRL-GMFF;  laboratory  testing  of  the  method  was  carried  out  between  April  2006  and 
February  2007  in  order  to  confirm  its  performance  characteristics.  The  protocol  for  DNA 
extraction and a report on method testing is available at http://gmo-crl.jrc.it/. 

The operational procedure of the collaborative study included the following module: 

(cid:57)  Quantitative real-time PCR (Polymerase Chain Reaction). The methodology is an event-
specific  real-time  quantitative  TaqMan®  PCR  procedure  for  the  determination  of  the 
relative  content  of  event  A2704-12  DNA  to  total  soybean  DNA.  The  procedure  is  a 
simplex  system,  in  which  a  soybean  lectin  (Le1)  endogenous  assay  (reference  gene) 
and the target assay (A2704-12) are performed in separate wells.  

The  international  collaborative  study  was  carried  out  in  accordance  with  the  following 
internationally accepted guidelines:  

(cid:57)  ISO 5725 (1994).  
(cid:57)  The  IUPAC  “Protocol  for  the  design,  conduct  and  interpretation  of  method-

performance studies” (Horwitz, 1995). 

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2. 

List of participating laboratories  

As  part  of  the  international  collaborative  study  the  method  was  tested  in  thirteen  ENGL   
laboratories  to  determine  its  performance.  Clear  guidance  was  given  to  the  laboratories  with 
regards to the standard operational procedures to follow for the execution of the protocol. The 
participating laboratories are listed in alphabetical order in Table 1.  

Table  1.  Laboratories  participating  in  the  validation  of  the  detection  method  for  soybean  line 
A2704-12. 

Laboratory 

 Agricultural Biotechnology Center 

 Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (Bavarian    
Health and Food Safety Authority) 

 Centro Nacional de Alimentación - Agencia Española de Seguridad Alimentaria 

 CRA-W,  Dépt Qualité des productions agricoles 

 Ente Nazionale Sementi Elette (central office in Milano) / Laboratorio Analisi 
Sementi 
 Finnish Customs Laboratory 

 General Chemical State Laboratory, Food Division 

 Istituto Superiore di Sanità, ISS, National Center for Food Quality and Safety  
- GMO and Mycotoxins Unit 
 Laboratoire national de Santé 

 Landesuntersuchungsanstalt für das Gesundheits- und Veterinärwesen  
Sachsen Amtliche Lebensmittelüberwachung 
 LSGV Saarland (Landesamt für Soziales, Gesundheit und Verbraucherschutz) 

 National Veterinary Institute 

 The Food and Consumer Product Safety Authority (VWA) 

Country 

Hungary 

Germany 

Spain 

Belgium 

Italy  

Finland 

Greece  

Italy  

Luxembourg

Germany 

Germany 

Norway 

Netherlands 

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3.  Materials 

For the validation of the quantitative event-specific method, control samples consisting of: 
i) 

a  DNA  stock  solution  homozygous  for  the  GM-event  A2704-12  (Lot  Number 
32RRMM0020)  

and  
ii) 

non-GM  DNA  stock  solution  (Lot  Number  32RRMM0029)  extracted  from  a  genetically 
similar wild-type line  

were  provided  by  the  applicant  in  accordance  to  the  provisions  of  Regulation  (EC)  No 
1829/2003,  Art  2.11  [“control  sample  defined  as  the  GMO  or  its  genetic  material  (positive 
sample) and the parental organism or its genetic material that has been used for the purpose 
of the genetic modification (negative sample)].  

Samples  containing  mixtures  of  100%  A2704-12  and  non-GM  soybean  genomic  DNA  at 
different  GMO  concentrations  were  prepared  by  the  CRL-GMFF,  using  the  control  samples 
provided, in a constant amount of total soybean DNA.  

Participants received the following materials: 

(cid:57)  Five  calibration  samples  (200  µl  of  DNA  solution  each)  for  the  preparation  of  the 

standard curve, labelled from S1 to S5.  

(cid:57)  Twenty unknown DNA samples (100 µl of DNA solution each), labelled from U1 to U20.  
(cid:57)  Amplification reagent control for use on each PCR plate. 
(cid:57)  Reaction reagents:  

(cid:137)  Universal PCR Master Mix 2X, 2 vials:   5 ml each  
(cid:137)  Sterile distilled water:    

4 ml 

(cid:57)  Primers  and  probes  (1  tube  each)  for  the Le1  reference  gene  and  for  the  A2704-12 

specific systems as follows: 

Le1 system 
(cid:137)  KVM164 primer  (10 µM):  
(cid:137)  KVM165 primer (10 µM):  
(cid:137)  TM021 TaqMan® probe (10 µM):  

A2704-12 soybean system 
(cid:137)  KVM175 primer  (10 µM):  
(cid:137)  SMO001 primer (10 µM):  
(cid:137)  TM031 TaqMan® probe (10 µM):  

160 µl 
160 µl  
160 µl    

320 µl  
320 µl 
160 µl 

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4. 

Experimental design 

Twenty unknown samples (labelled from U1 to U20), representing five GM levels, were used 
in the validation study (Table 2). On each PCR plate, samples were analysed in parallel with 
both the A2704-12 and Le1 specific systems. In total, two plates were run per participating 
laboratory,  with  two  replicates  for  each  GM  level  analysed  on  each  run.  In  total,  four 
replicates  for  each  GM  level  were  analysed.  PCR  analysis  was  performed  in  triplicate  for  all 
samples. Participating laboratories carried out the determination of the GM% according to the 
instructions  provided  in  the  protocol  and  using  the  electronic  tool  provided  (Excel 
spreadsheet). 

Table 2.  A2704-12 GM contents 

A2704-12 GM % 

(GM copy number/soybean genome copy number *100) 

0.10 

0.40 

0.90 

2.00 

3.30 

5.  Method 

Description of operational steps followed 

For  specific  detection  of  event  A2704-12  genomic  DNA,  two  specific  primers  amplify  a  64-bp 
fragment of an internal sequence of the A2704-12 event that consists of the unique junction of 
rearranged  sequences  of  the  original  transforming  plasmid.  The  amplicon  is  located  on  a 
junction  fragment  containing  one  copy  of  the  3’  bla  sequence  and  one  copy  of  the  5’  bla 
sequence,  integrated  in  an  inverted  orientation,  as  compared  to  the  transforming  plasmid 
configuration.  

PCR  products  are  measured  during  each  cycle  (real-time)  by  means  of  a  target-specific 
oligonucleotide  probe  labelled  with  two fluorescent  dyes:  FAM as a  reporter  dye  at  its  5′  end 
and TAMRA as a quencher dye at its 3′ end. 

For  the  relative  quantification  of  event  A2704-12  DNA,  a  soybean-specific  reference  system 
amplifies  a  105-bp  fragment  of  the  soybean  endogenous  gene Le1  (lectin, accession  number 
K00821), using two Le1 gene-specific primers and a Le1 gene-specific probe labelled with FAM 
and TAMRA.  
For relative quantification of event A2704-12 DNA in a test sample, the normalised ∆Ct values 
of calibration samples are used to calculate, by linear regression, a standard curve (plotting ∆Ct 
values  against  the  logarithm  of  the  amount  of  event  A2704-12  DNA).  The  normalised  ∆Ct 
values  of  the  unknown  samples  are  measured  and,  by  means  of  the  regression  formula,  the 
relative amount of event A2704-12 DNA is estimated. 

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Calibration  samples  denominated  from  S1  to  S5  were  prepared  by  mixing  the  appropriate 
amount  of  A2704-12  DNA  from  the  stock  solution  with  non-GM  cotton  DNA  to  obtain  the 
following  relative  contents  of  A2704-12:  3.6%,  1.8%,  0.9%,  0.45%  and  0.09%.  Total  DNA 
amount per reaction was 200 ng, when 5 µl of a DNA solution at the concentration of 40 ng/µl 
were loaded.  

The GM contents of the calibration samples and total DNA quantity used in PCR are provided in 
Table 3 (%GM calculated considering the 1C value for soybean genome as 1.13 pg) (3). 

Table 3. % GM values of the standard curve samples. 

Sample code 

S1 

S2 

S3 

S4 

S5 

Total amount of DNA in 
reaction (ng/5 µl) 

200 

200 

200 

200 

200 

% GM (DNA/DNA) 

3.60 

1.80 

0.90 

0.45 

0.09 

6.  Deviations reported 

No deviations from the protocol were reported. 

7. 

Summary of results 

PCR efficiency and linearity 

The  values  of  the  slopes  of  the  standard  curve  [from  which  the  PCR  efficiency  is  calculated 
using  the  formula  ((10^(-1/slope))-1)*100]  and  of  the  R2  (expressing  the  linearity  of  the 
regression)  reported  by  participating  laboratories  for  both  runs  (plates  A  and  B)  are 
summarised in Table 4.  

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Table 4. Values of reference curve slope, PCR efficiency and linearity (R2) 

LAB  PLATE Slope 

PCR 
Efficiency 
(%) 

Linearity
(R2) 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

-3.23 

-3.32 

-3.31 

-3.41 

-3.39 

-3.36 

-3.54 

-3.67 

-3.49 

-3.49 

-3.55 

-3.41 

-3.60 

-3.41 

-3.45 

-3.49 

-4.23 

-3.55 

-3.50 

-3.44 

-3.59 

-3.49 

-3.52 

-3.29 

-3.60 

-3.57 

95.85 

99.73 

99.58 

96.27 

97.37 

98.37 

91.60 

87.12 

93.53 

93.57 

91.22 

96.37 

89.58 

96.60 

94.90 

93.47 

72.37 

91.29 

93.25 

95.29 

89.95 

93.57 

92.18 

98.46 

89.59 

90.75 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

0.99 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

0.97 

0.98 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

  Mean -3.50 

93.15 

1.00 

The mean PCR efficiency was above 93%. The linearity of the method was on average above 
0.99. Therefore, the data reported confirm the appropriate performance characteristics of the 
method tested. 

GMO quantification 

Table  5  shows  the  mean  values  of  the  four  replicates  for  each  GM  level  as  calculated  and 
provided by all laboratories. Each mean value is the average of three PCR repetitions. 

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Table 5. GM% mean values determined by laboratories for unknown samples. 

(GM% = GM copy number/soybean genome copy number *100) 

Sample GMO content  

LAB 

0.1 

0.4 

0.9 

2.0 

3.3 

  REP 1  REP 2  REP 3  REP 4 REP 1  REP 2  REP 3  REP 4 REP 1 REP 2 REP 3 REP 4 REP 1 REP 2 REP 3 REP 4  REP 1  REP 2  REP 3 REP 4

1 

2 

3 

4 

5 

6 

7 

8 

9 

0.09  0.09  0.11  0.10 0.41  0.39  0.42  0.48 0.89 0.81 1.03 0.91

2.21 2.13 2.40 2.32  3.78  4.07  3.51 3.66

0.10  0.13  0.11  0.13 0.42  0.40  0.46  0.44 0.87 0.90 1.04 1.21

2.23 2.42 2.18 1.98  4.33  4.09  3.45 3.96

0.11  0.12  0.12  0.11 0.42  0.42  0.43  0.48 0.92 1.09 1.06 1.17

2.03 1.99 2.16 2.34  3.77  4.00  4.06 3.85

0.11  0.11  0.14  0.14 0.41  0.37  0.47  0.41 0.85 0.83 1.09 1.12

2.01 2.33 2.09 2.06  3.31  3.69  3.20 3.52

0.10  0.11  0.11  0.10 0.40  0.37  0.41  0.41 0.89 0.94 0.86 0.84

2.08 2.02 2.21 1.96  3.27  2.99  3.35 3.21

0.10  0.10  0.10  0.09 0.43  0.39  0.39  0.38 0.85 0.78 0.78 0.82

2.13 1.96 2.13 1.93  3.04  3.18  3.51 3.41

0.15  0.17  0.12  0.11 0.41  0.40  0.44  0.30 0.92 0.89 0.82 1.01

1.76 1.88 1.57 1.56  2.78  3.55  2.34 3.02

0.13  0.13  0.12  0.12 0.41  0.42  0.44  0.34 0.88 0.92 0.86 0.82

1.90 1.84 1.82 1.68  3.01  3.03  2.81 2.80

0.07  0.11  0.12  0.13 0.23  0.23  0.59  0.49 0.37 0.61 0.69 0.76

1.02 1.01 2.02 2.97  1.21  1.63  4.75 3.92

10 

0.12  0.11  0.15  0.13 0.47  0.43  0.53  0.47 0.92 0.95 1.21 1.08

2.21 2.32 2.41 2.28  3.46  3.91  4.07 4.29

11 

0.14  0.14  0.11  0.11 0.44  0.47  0.45  0.40 1.07 0.99 0.91 1.06

2.16 2.34 2.20 2.08  4.09  4.00  3.46 3.72

12 

0.14  0.17  0.13  0.13 0.46  0.40  0.45  0.43 0.96 1.12 1.06 0.99

1.74 2.10 2.09 2.11  3.35  4.11  3.10 3.01

13 

0.11  0.12  0.12  0.12 0.44  0.43  0.42  0.41 0.88 0.86 0.85 0.91

2.03 1.90 2.01 1.93  2.96  3.25  3.30 2.94

In  Figure  1  the  relative  deviation  from  the  true  value  for  each  GM  level  tested  is  shown  for 
each  laboratory.  The  coloured  bars  represent  the  relative  GM  quantification  obtained  by  the 
participating laboratories as well as the mean value. 

Figure 1. Relative deviation (%) from the true value of A2704-12 for all laboratories 

Laboratories

1

2

3

4

5

6

7

8

9

10

11

12

13 Mean

0.1 GM%

0.4 GM%

0.9 GM%

2.0 GM%

3.3 GM%

50.00

40.00

30.00

20.00

10.00

0.00

-10.00

-20.00

-30.00

-40.00

%
e
u

l
a
V
e
u
r
T
m
o
r
f

n
o

i
t
a
i
v
e
d

e
v
i
t
a
l
e
R

As observed in Figure 1, a general trend of overestimation is visible for most of the laboratories 
at the lowest GM levels 0.1% and 0.4%. The highest relative deviation from the true value of 
A2704-12 is observed at 0.1% GM level, although the mean value is well within the limit of the 
trueness acceptance level (bias < 20%). The mean overestimation of higher GM levels 0.9%, 

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2.0%  and  3.3%  is  very  limited.  One  laboratory  underestimated  the  GM  level  0.9%  by  more 
than 30%. Overall, the average relative deviation from the true value was acceptable at all GM 
levels tested, indicating a satisfactory accuracy of the method. 

8.  Method performance requirements 

Among  the  performance  criteria  established  by  ENGL  and  adopted  by  the  CRL-GMFF 
(http://gmo-crl.jrc.it/guidancedocs.htm, see also Annex 1), repeatability and reproducibility are 
assessed  through an  international  collaborative  trial,  carried  out  with  the  support  of  ENGL 
laboratories (see Table 1). Table 6 illustrates the estimation of repeatability and reproducibility 
at various GM levels, according to the range of GM percentages tested during the collaborative 
trial.  

The  relative reproducibility standard deviation (RSDR),  that  describes  the  inter-laboratory 
variation,  should  be  below  33%  at  the  target  concentration  and  over  the  majority  of  the 
dynamic range, while it should be below 50% at the lower end of the dynamic range.  

As  it  can  be  observed  in  Table  6,  the  method  fully  satisfies  this  requirement  at  all  GM  levels 
tested. In fact, the highest value of RSDR (%) is 16% at the 0.1% and 0.9% levels, thus within 
the acceptance criterion. 

Table 6.  A2704-12: summary of validation results. 

Unknown sample GM% 

 Laboratories having returned results 

 Samples per laboratory 

 Number of outliers 

 Reason for exclusion 

 Mean value 

Expected value (GMO %) 

0.1 

13 

4 

0 

- 

0.4 

13 

4 

1 

1 C. test 

0.9 

13 

4 

0 

- 

2.0 

13 

4 

1 

3.3 

13 

4 

1 

1 C. test 

1 C. test 

0.12 

0.42 

0.92 

2.07 

3.47 

 Relative repeatability standard deviation, RSDr (%)  

13 

8 

11 

6 

9 

 Repeatability standard deviation  

0.02 

0.03 

0.10 

0.13 

0.30 

 Relative reproducibility standard deviation, RSDR (%)  

 Reproducibility standard deviation  

 Bias (absolute value) 

 Bias (%) 

16 

0.02 

0.02 

18 

9 

0.04 

0.02 

6 

16 

0.15 

0.02 

2 

10 

0.21 

0.07 

3 

14 

0.47 

0.17 

5 

C  =  Cochran’s  test;  G=  Grubbs’  test;  identification  and  removal  of  outliers  through  Cochran  and  Grubbs  tests, 

according to ISO 5725-2. 

Bias is estimated according to ISO 5725 data analysis protocol. 

Table 6 further documents the relative repeatability standard deviation (RSDr), as estimated for 
each GM level. In order to accept methods for collaborative study evaluation, the CRL requires 

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that  RSDr  values  be  below  25%,  as  indicated  by  ENGL  (Definition  of  Minimum  Performance 
Requirements for Analytical Methods of GMO Testing” (http://gmo-crl.jrc.it/guidancedocs.htm).  

As  it  can  be  observed  from  the  values  reported  in  Table  6,  the  method  satisfies  this 
requirement throughout the whole dynamic range tested.  

The trueness of the method is estimated using the measures of the method bias for each GM 
level. According to ENGL method performance requirements, trueness should be ± 25% across 
the entire dynamic range. In this case the method satisfies this requirement across the entire 
dynamic range tested; in fact, the highest value of bias is 18% at the 0.1% level, well within 
the acceptance criterion. 

9.  Conclusions 

The  overall  method  performance  has  been  evaluated  with  respect  to  the  method  acceptance 
criteria  and  method  performance  requirements  recommended  by  the  ENGL  (as  detailed  at 
http://gmo-crl.jrc.it/guidancedocs.htm).  The  method  acceptance  criteria  were  reported  by  the 
applicant  and  used  to  evaluate  the  method  prior  to  the  international  collaborative  study  (see 
Annex 1 for a summary of method acceptance criteria and method performance requirements). 

The  results  obtained  during  the  collaborative  study  indicate  that  the  analytical  module  of  the 
method  submitted  by  the  applicant  complies  with  ENGL  performance  criteria.  The  method  is 
therefore  applicable  to  the  control  samples  provided  (see  paragraph  3  “Materials”),  in 
accordance  with  the  requirements of  Annex  I-2.C.2  to  Commission  Regulation  (EC)  No 
641/2004. 

10.  Quality assurance 

The CRL-GMFF carries out all operations according to ISO 9001:2000 (certificate number: CH-
32232)  and  ISO  17025:2005  (certificate  number:  DAC-PL-0459-06-00)  [DNA  extraction, 
qualitative  and  quantitative  PCR  in  the  area  of  Biology  (DNA  extraction  and  PCR  method 
validation for the detection and identification of GMOs in food and feed materials)] 

11.  References 

1.  Horwitz,  W.  (1995)  Protocol  for  the  design,  conduct  and  interpretation  of  method 

performance studies, Pure and Appl. Chem, 67, 331-343.  

2.  International  Standard  (ISO)  5725.  1994.  Accuracy  (trueness  and  precision)  of 
measurement  methods  and  results.  International  Organization  for  Standardization, 
Genève, Swizerland.  

3.  Arumuganathan  K,  Earle  ED.  1991.  Nuclear  DNA  content  of  some  important  plant 

species. Plant Molecular Biology Reporter 9: 208-218 

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12.  Annex  1:  method  acceptance  criteria  and  method 
performance requirements as set by the European Network 
of GMO Laboratories (ENGL) 

Method  Acceptance  Criteria  should  be  fulfilled  at  the  moment  of  submission  of  a  method  (Phase  1: 

acceptance for the collaborative study).  

Method  Performance  Requirements  should  be  fulfilled  in  a  collaborative  study  in  order  to  consider  the 

method as fit for its purpose (Phase 2:  evaluation of the collaborative study results).  

 Method Acceptance Criteria 

Applicability  

Definition: The description of analytes, matrices, and concentrations to which a method can be applied.  

Acceptance Criterion: The applicability statement should provide information on the scope of the method 

and include data for the indices listed below for the product/s for which the application is submitted. The 

description  should  also  include  warnings  to  known  interferences  by  other  analytes,  or  inapplicability  to 

certain matrices and situations.   

Practicability  

Definition: The ease of operations, the feasibility and efficiency of implementation, the associated unitary 

costs (e.g. Euro/sample) of the method.   

Acceptance Criterion: The practicability statement should provide indication on the required equipment for 

the  application  of  the  method  with  regards  to  the  analysis  per se  and  the  sample  preparation.  An 

indication of costs, timing, practical difficulties and any other factor that could be of importance for  the 

operators should be indicated. 

Specificity 

Definition: Property of a method to respond exclusively to the characteristic or analyte of interest. 

Acceptance  Criterion:  The  method  should  be  event-specific  and  be  functional  only  with  the  GMO  or  GM 

based product for which it was developed. This should be demonstrated by empirical results from testing 

the  method  with  non-target  transgenic  events  and  non-transgenic  material.  This  testing  should  include 

closely related events and cases where the limit of the detection is tested. 

Dynamic Range  

Definition:  The  range  of  concentrations  over  which  the  method  performs  in  a  linear  manner  with  an 

acceptable level of accuracy and precision. 

Acceptance Criterion: The dynamic range of the method should include the 1/10 and at least 5 times the 

target  concentration.  Target  concentration  is  intended  as  the  threshold  relevant  for  legislative 

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requirements.  The  acceptable  level  of  accuracy  and  precision  are  described  below.  The  range  of  the 

standard  curve(s)  should  allow  testing  of  blind  samples  throughout  the  entire  dynamic  range,  including 

the lower (10%) and upper (500%) end. 

Accuracy  

Definition: The closeness of agreement between a test result and the accepted reference value.  

Acceptance  Criterion:  The  accuracy  should  be  within  ±  25%  of  the  accepted  reference  value  over  the 

whole dynamic range.  

Amplification Efficency 

i

Definition: The rate of amplification that leads to a theoretical slope of –3.32 with an efficiency of 100% 
in each cycle. The efficiency of the reaction can be calculated by the following equation: Efficiency = [10(-
1/slope)] – 1 

Acceptance Criterion: The average value of the slope of the standard curve should be in the range of (- 

3.1 ≥ slope ≥ - 3.6) 

R2 Coefficient 

Definition:  The  R2  coefficient  is  the  correlation  coefficient  of  a  standard  curve  obtained  by  linear 

regression analysis. 

Acceptance Criterion: The average value of R2 should be ≥ 0.98. 

Repeatability Standard Deviation (RSDr) 

Definition:  The  standard  deviation  of  test  results  obtained  under  repeatability  conditions.  Repeatability 

conditions are conditions where test results are obtained with the same method, on identical test items, in 

the same laboratory, by the same operator, using the same equipment within short intervals of time.  

Acceptance Criterion: The relative repeatability standard deviation should be below 25% over the whole 

dynamic range of the method.  

Note: Estimates of repeatability submitted by the applicant should be obtained on a sufficient number of 

test results, at least 15, as indicated in ISO 5725-3 (1994). 

Limit of Quantitation (LOQ) 

Definition: The limit of quantitation is the lowest amount or concentration of analyte in a sample that can 

be reliably quantified with an acceptable level of precision and accuracy.  

Acceptance  Criterion:  LOQ  should  be  less  than  1/10th  of  the  value  of  the  target  concentration  with  an 

RSDr  ≤  25%.  Target  concentration  should  be  intended  as  the  threshold  relevant  for  legislative 

requirements. The acceptable level of accuracy and precision are described below. 

Limit of Detection (LOD) 

Definition: The limit of detection is the lowest amount or concentration of analyte in a sample, which can 

be reliably detected, but not necessarily quantified, as demonstrated by single laboratory validation.  

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Acceptance  Criterion:  LOD  should  be  less  than  1/20th  of  the  target  concentration.  Experimentally, 

quantitative  methods  should  detect  the  presence  of  the  analyte  at  least  95%  of  the  time  at  the  LOD, 

ensuring ≤ 5% false negative results. Target concentration should be intended as the threshold relevant 

for legislative requirements.  

Robustness  

Definition:  The  robustness  of  a  method  is  a  measure  of  its  capacity  to  remain  unaffected  by  small,  but 

deliberate deviations from the experimental conditions described in the procedure. 

Acceptance Criterion:  The response of an assay with respect to these small variations should not deviate 

more  than  ±  30%.  Examples  of  factors  that  a  robustness  test  could  address  are:    use  of  different 

instrument type, operator, brand of reagents, concentration of reagents, and temperature of reaction.  

Method Performance Requirements 

Dynamic Range  

Definition:  In  the  collaborative  trial  the  dynamic  range  is  the  range  of  concentrations  over  which  the 

reproducibility and the trueness of the method are evaluated with respect to the requirements specified 

below.  

Acceptance Criterion: The dynamic range of the method should include the 1/10 and at least five times 

the target concentration. Target concentration should be intended as the threshold relevant for legislative 

requirements.  

Reproducibility Standard Deviation (RSDR) 

Definition: The standard deviation of test results obtained under reproducibility conditions. Reproducibility 

conditions are conditions where test results are obtained with the same method, on identical test items, in 

different  laboratories,  with  different  operators,  using  different  equipment.  Reproducibility  standard 

deviation describes the inter-laboratory variation. 

Acceptance  Criterion:  The  relative  reproducibility  standard  deviation  should  be  below  35%  at  the  target 

concentration and over the entire dynamic range. An RSDR < 50 % is acceptable for concentrations below 

0.2%. 

Trueness 

Definition:  The  closeness  of  agreement  between  the  average  value  obtained  from  a  large  series  of  test 

results and an accepted reference value. The measure of trueness is usually expressed in terms of bias.  

Acceptance  Criterion:  The  trueness  should  be  within  ±  25%  of  the  accepted  reference  value  over  the 

whole dynamic range. 

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Event-specific Method for the Quantification  
of Soybean Line A2704-12  
Using Real-time PCR 

Protocol 

14 May 2007 

Directorate General-Joint Research Centre 
Institute for Health and Consumer Protection  
Biotechnology & GMOs Unit 

Method development: 

Bayer CropScience GmbH 

Collaborative trial: 

Community Reference Laboratory for GM Food and Feed (CRL-GMFF) 
Biotechnology & GMOs Unit 

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Content 

1. GENERAL INFORMATION AND SUMMARY OF THE METHODOLOGY........................ 4 

2. VALIDATION STATUS AND PERFORMANCE CHARACTERISTICS............................. 4 

2.1 GENERAL ...................................................................................................................4 
2.2 COLLABORATIVE TRIAL ...................................................................................................5 
2.3 LIMIT OF DETECTION (LOD) ...........................................................................................5 
2.4 LIMIT OF QUANTIFICATION (LOQ) ....................................................................................5 
2.5 MOLECULAR SPECIFICITY ................................................................................................5 

3. PROCEDURE............................................................................................................. 6 

3.1 GENERAL INSTRUCTIONS AND PRECAUTIONS.........................................................................6 
3.2 REAL-TIME PCR FOR QUANTITATIVE ANALYSIS OF A2704-12 ...................................................6 
3.2.1 General.............................................................................................................6 
3.2.2 Calibration.........................................................................................................7 
3.2.3 Real-time PCR set-up.........................................................................................7 
3.3 DATA ANALYSIS ...........................................................................................................9 
3.4 CALCULATION OF RESULTS ..............................................................................................9 

4.  MATERIALS .......................................................................................................... 9 

4.1 EQUIPMENT ................................................................................................................9 
4.2 REAGENTS................................................................................................................ 10 
4.3 PRIMERS AND PROBES ................................................................................................. 10 

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1. General information and summary of the methodology 

This protocol describes an event-specific real-time quantitative TaqMan® PCR procedure for the 
determination of the relative content of event A2704-12 DNA to total soybean DNA in a sample.  

The PCR assay was optimised for use in real-time PCR instruments for plastic reaction vessels. 
Glass capillaries are not recommended for the buffer composition described in this method. 

Template  DNA  extracted  by  means  of  suitable  methods  should  be  tested  for  quality  and 
quantity prior to use in PCR assay. Tests for the presence of PCR inhibitors (e.g. monitor run of 
diluted series, use of DNA spikes) are recommended. 

For  specific  detection  of  event  A2704-12  genomic  DNA,  a  64-bp  fragment  of  the  unique 
junction  of  rearranged  sequences  of  the  original  transforming  plasmid  is  amplified  using  two 
specific  primers.  PCR  products  are  measured  during  each  cycle  (real-time)  by  means  of  a 
target-specific oligonucleotide probe labelled with two fluorescent dyes: FAM as a reporter dye 
at its 5’ end and TAMRA as a quencher dye at its 3’ end. 

For  relative  quantification  of  event  A2704-12  DNA,  a  soybean-specific  reference  system 
amplifies a 105-bp fragment of the lectin gene (Le1), a soybean endogenous gene, using two 
specific  primers  and  a  Le1  gene-specific  probe  labelled  with  FAM  and  TAMRA  as  described 
above. 

The  measured  fluorescence  signal  passes  a  threshold  value  after  a  certain  number  of  cycles. 
This threshold cycle is called the “Ct” value. For quantification of the amount of event A2704-12 
DNA  in  a  test  sample,  the  normalised  ∆Ct  values  of  the  calibration  samples  are  used  to 
calculate by linear regression a reference curve ∆Ct-formula. The normalised ∆Ct values of the 
unknown  samples  are  measured  and,  by  means  of  the  regression,  the  relative  amount  of 
A2704-12 event DNA is estimated. 

2. Validation status and performance characteristics  

2.1 General 

The  method  was  optimised  for  suitable  DNA  extracted  from  soybean  leaves,  grains  or  seeds, 
containing mixtures of genetically modified and conventional soybean. 

The  reproducibility  and  trueness  of  the  method  was  tested  through  collaborative  trial  using 
samples at different GMO contents. 

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2.2 Collaborative trial 

The  method  was  validated  in  a  collaborative  study  by  the  Joint  Research  Centre  (JRC)  of  the 
European  Commission.  The  study  was  undertaken  with  thirteen  participating  laboratories  in 
June-July 2006.  

Each participant received twenty blind samples containing A2704-12 soybean genomic DNA at 
five GM contents, ranging from 0.1 % to 3.3 %.  

Each test sample was analysed by PCR in three repetitions. The study was designed as a blind 
quadruplicate  collaborative  trial;  each  laboratory  received  each  level  of  GM  A2704-12  in  four 
unknown samples. Two replicates of each GM level were analysed on the same PCR plate.  

A detailed validation report can be found under http://gmo-crl.jrc.it/statusofdoss.htm

2.3 Limit of detection (LOD) 

According to the method developer, the relative LOD of the method is at least 0.023% in 200 
ng of total soybean DNA. The relative LOD was not assessed in a collaborative study.  

2.4 Limit of quantification (LOQ) 

According to the method developer, the relative LOQ of the method is at least 0.045% in 200 
ng  of  total  soybean  DNA.  The  lowest  relative  GM  content  of  the  target  sequence  included  in 
collaborative trial was 0.1 %. 

2.5 Molecular specificity 

The  method  exploits  a  unique  DNA  sequence  of  the  junction  of  rearranged  sequences  of  the 
original  transforming  plasmid.  The  sequence  is  specific  to  A2704-12  event  and  thus  imparts 
event-specificity to the method.  

The  specificity  of  the  event-specific  assay  was  experimentally  tested  against  DNA  extracted 
from plant materials containing the specific targets of maize T25, MON 810, Bt11, Bt176, GA21, 
NK603,  CBH351,  Roundup  Ready  Soybean,  OSR  Ms1,  Ms8,  Rf1,  Rf2,  Rf3,  Topas19/2,  T45, 
LLRICE62 and LLCotton25.  None of the materials yielded detectable amplification in replicate 
experiments. 

The specificity of the soybean endogenous reference system Le1 was experimentally tested by 
the applicant on soybean related species (50 ng of genomic DNA in the PCR reaction and PCR 
reaction  of  40  thermo  cycles).  The Le1  endogenous  reference  system  produced  amplification 
signals for glycine max and glycine soj DNA samples. Out of the eight soybean related species, 
two did not react with the soybean endogenous system (i.e. lentil Castelluccio lentil and Vicia
faba Boccli) while weak amplification signals (i.e. above 37 cycles for a maximum of 7 replicate 

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out of 12 replicates) were obtained for six species (Lentil lentil 1, Vicia faba WJK-PRC-47, Vicia 
faba Madfredini, Arachis hypogaea US1175, Arachis rasteiro and Peas). 

3. Procedure 

3.1  General instructions and precautions 

•  The procedures require experience of working under sterile conditions. 

• 

Laboratory organisation,  e.g.  “forward  flow direction”  during  PCR-setup,  should  follow 
the  guidelines  given  by  relevant  authorities,  e.g.  ISO,  CEN,  Codex  Alimentarius 
Commission. 

•  PCR-reagents should be stored and handled in a separate room where no nucleic acids 
(with  exception  of  PCR  primers  or  probes)  or  DNA  degrading  or  modifying  enzymes 
have  been  handled  previously.  All  handling  of  PCR  reagents  and  controls  requires 
dedicated equipment, especially pipettes.  

•  All the equipment used should be sterilised prior to use and any residue of DNA has to 
be removed. All material used (e.g. vials, containers, pipette tips, etc.) must be suitable 
for PCR and molecular biology applications. They must be DNase-free, DNA-free, sterile 
and unable to adsorb protein or DNA. 

• 

In order to avoid contamination, filter pipette tips protected against aerosol should be 
used. 

•  Powder-free gloves should be used and changed frequently.  

• 

Laboratory  benches  and  equipment  should  be  cleaned  periodically  with  10%  sodium 
hypochloride solution (bleach).  

•  Pipettes should be checked regularly for precision and calibrated, if necessary. 

•  All handling steps - unless specified otherwise - should be carried out at 0 - 4°C. 

• 

In order to avoid repeated freeze/thaw cycles aliquots should be prepared. 

3.2  Real-time PCR for quantitative analysis of A2704-12 

3.2.1 General 

The PCR set-up for the taxon specific target sequence (Le1) and for the GMO (A2704-12) target 
sequence  should  be  carried  out  in  separate  vials.  Multiplex  PCR  (using  differential  fluorescent 
labels for the probes) has not been tested or validated.  

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The use of maximum 200 ng of template DNA per reaction well is recommended.  

The method is developed for a total volume of 25 µl per reaction mixture with the reagents as 
listed in Table 1 and Table 2. 

3.2.2 Calibration  

The calibration curve consists of five samples containing different amounts of A2704-12 DNA in 
a total amount of 200 ng soybean DNA. The GM content of the standard samples ranges from 
3.6% to 0.09%. 

A  calibration  curve  is  produced  by  plotting  the  ∆Ct-values  of  calibration  samples  against  the 
logarithm of the respective GM % contents; the slope (a) and the intercept (b) of the standard 
curve  (y  =  ax  +  b)  are  then  used  to  calculate  the  mean  %  GM  content  of  the  blind  samples 
based on their normalised ∆Ct values. 

3.2.3 Real-time PCR set-up 

1.  Thaw, mix gently and centrifuge the required amount of components needed for the run. 

Keep thawed reagents on ice.  

2.  In two reaction tubes (one for A2704-12 system and one for the Le1 system) on ice, add 
the following components (Tables 1 and 2) in the order mentioned below (except DNA) to 
prepare the master mixes. 

Table  1.  Amplification  reaction  mixture  in  the  final  volume/concentration  per  reaction  well  for 
the soybean Le1 reference system.  

Component 

Final concentration

µl/reaction 

TaqMan® Universal PCR Master Mix (2x) 

KVM164 primer (10 µM) 

KVM165 primer (10 µM) 

TM021 probe (10 µM) 

Nuclease free water 

Template DNA (max 200 ng) 

Total reaction volume: 

1x 

200 nM 

200 nM 

200 nM 

# 

# 

12.5  

0.5 

0.5 

0.5 

6 

5  

25  

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Table  2.  Amplification  reaction  mixture  in  the  final  volume/concentration  per  reaction  well  for 
the A2704-12 specific system. 

Component 

Final concentration

µl/reaction 

TaqMan® Universal PCR Master Mix (2x) 

KVM175 primer forward (10 µM) 

SMO001 primer reverse (10 µM) 

TM031 Probe (10 µM) 

Nuclease free water 

Template DNA (max 200 ng) 
Total reaction volume: 

3.  Mix gently and centrifuge briefly.  

1x 

400 nM 

400 nM 

200 nM 

# 

# 

12.5 

1 

1 

0.5 

5 

5 
25 

4.  Prepare two reaction tubes (one for the A2704-12 and one for the Le1 master mixes) for 
each  DNA  sample  to  be  tested  (standard  curve  samples,  unknown  samples  and  control 
samples).  

5.  Add to each reaction tube the correct amount of master mix (e.g. 20 x 3 = 60 µl master 
mix for three PCR repetitions). Add to each tube the correct amount of DNA (e.g. 5 x 3 = 
15  µl  DNA  for  three  PCR  repetitions).  Vortex  each  tubes  for  approx.  10  sec.  This  step  is 
mandatory to reduce to a minimum the variability among the repetitions of each sample. 

6.  Spin down the tubes in a microcentrifuge. Aliquot 25 µl in each well. Seal the reaction plate 
with optical cover or optical caps. Centrifuge the plate at low speed (e.g. approximately 250 
x g for 1 minute at 4 °C to room temperature) to spin down the reaction mixture. 

7.  Place the plate into the instrument.  

8.  Run the PCR with cycling conditions described in Table 3: 

Table 3. Cycling program for A2704-12/Le1 systems 

Step 

Stage 

T°C 

Time 
(sec) 

Acquisition 

Cycles 

1 

2 

3 

UNG 

50 °C 

120 

Initial denaturation 

95 °C 

600 

Denaturation 

95 °C 

15 

No 

No 

No 

Amplification 

Annealing & 
Extension 

60 °C 

60 

Yes 

1 

1 

45 

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3.3  Data analysis 

Subsequent to the real-time PCR, analyse the run following the procedure below: 

a)  Set  the  threshold:  display  the  amplification  curves  of  one  system  (e.g.  A2704-12)  in 
logarithmic  mode.  Locate  the  threshold  line  in  the  area  where  the  amplification  profiles  are 
parallel (exponential phase of PCR) and where there is no “fork effect” between repetitions of 
the same sample. Press the “update” button to ensure changes affect Ct values. Switch to the 
linear  view  mode  by  clicking  on  the  Y  axis  of  the  amplification  plot,  and  check  that  the 
threshold previously set falls within the geometric phase of the curves. 

b)  Set  the  baseline:  determine  the  cycle  number  at  which  the  threshold  line  crosses  the  first 
amplification curve and set the baseline three cycles before that value (e.g. earliest Ct = 25, set 
the baseline crossing at Ct = 25 – 3 = 22). 

c) Save the settings. 

d) Repeat the procedure described in a) and b) on the amplification plots of the other system 
(e.g. Le1 system).     

e) Save the settings and export all the data into an Excel file for further calculations. 

3.4  Calculation of results 

After  having  defined  a  threshold  value  within  the  logarithmic  phase  of  amplification  as 
described above, the instrument’s software calculates the Ct-values for each reaction. 

The  reference  ∆Ct-curve  is  generated  by  plotting  the  ∆Ct-values  measured  for  the  calibration 
points  against  the  logarithm  of  the  GM%  content,  and  by  fitting  a  linear  regression  line  into 
these data.  

Thereafter, the reference curve ∆Ct-curve formula is used to estimate the relative amount (%) 
of A2704-12 event in the unknown samples of DNA.  

4. Materials 

4.1 

Equipment  

•  Real-time  PCR  instrument  for  plastic  reaction  vessels  (glass  capillaries  are  not 

recommended for the described buffer composition) 

•  Plastic  reaction  vessels  suitable  for  real-time  PCR  instrument  (enabling  undisturbed 

fluorescence detection) 

•  Software  for  run  analysis  (mostly  integrated  in  the  software  of  the  real-time  PCR 

instrument) 

CRL-GMFF: Protocol A2704-12 soybean   

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•  Microcentrifuge 
•  Micropipettes 
•  Vortex  
•  Rack for reaction tubes 
•  1.5/2.0 ml reaction tubes 

4.2  Reagents  

•  TaqMan® Universal PCR Master Mix (2X). Applied Biosystems Part No 4304437 

4.3 

Primers and Probes 

Name 

Oligonucleotide DNA Sequence (5’ to 3’) 

KVM175 

SMO001 

TM031 (Probe) 

KVM164 

KVM165 

TM021 (Probe) 

A2704-12 target sequence 

5’- GCA AAA AAG CGG TTA GCT CCT -3’ 

5’- ATT CAG GCT GCG CAA CTG TT -3’  

FAM-5’- CGG TCC TCC GAT CGC CCT TCC -3’-TAMRA 

Reference gene Le1 target sequence 

5’- CAC CTT TCT CGC ACC AAT TGA CA -3’ 

5’- TCA AAC TCA ACA GCG ACG AC -3’  

FAM-5’- CCA CAA ACA CAT GCA GGT TAT CTT GG - 3’-TAMRA 

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Soybean Seeds Sampling and DNA Extraction 

 Report on the Validation of a DNA Extraction 
Method from Soybean Seeds 

14 May 2007 

Directorate General-Joint Research Centre 
Institute for Health and Consumer Protection  
Biotechnology & GMOs Unit 

Method development and single laboratory validation: 

Bayer CropScience GmbH 

Method testing and confirmation: 

Community Reference Laboratory for GM Food and Feed (CRL-GMFF) 
Biotechnology & GMOs Unit 

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Content 

1. INTRODUCTION....................................................................................................... 4 

2. MATERIALS (EQUIPMENT/CHEMICALS/PLASTICWARE)........................................ 4 

3. DESCRIPTION OF THE METHODS ............................................................................ 6 

4. TESTING OF THE DNA EXTRACTION METHOD BY THE METHOD DEVELOPER......... 8 

5. EXPERIMENTAL TESTING OF THE DNA EXTRACTION METHOD BY THE 
COMMUNITY REFERENCE LABORATORY FOR GM FOOD AND FEED.......................... 12 

6. CONCLUSION......................................................................................................... 17 

7. QUALITY ASSURANCE............................................................................................ 17 

8. REFERENCES.......................................................................................................... 17 

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1. Introduction 

This  report  describes  a  plant  DNA  extraction  protocol  derived  from  the  publicly  available 
“Dellaporta”  method  (1).  This  protocol  can  be  used  for  the  extraction  of  DNA  from  soybean 
seeds  and  grains  ground  to  powder  with  a  Waring™  blender  or  with  any  other  appropriate 
seeds/grains crushing device. 

These  protocols  are  recommended  to be  executed  only  by  skilled  laboratory  personnel  as the 
procedures  comprise  the  use  of  hazardous  chemicals  and  materials.  It  is  strongly  advised  to 
take particular notice of products safety recommendations and guidelines. 

2. Materials (Equipment/Chemicals/Plasticware) 

2.1. Equipment 

The  following  equipment  is  used  in  the  DNA  extraction  procedure  described  (equivalents  may 
be used): 

1.  Waring blender, model 7010S/7010G/7010HS/7010HG or equivalent 
2.  70 mm Blender Base (Eberbach Corp. – Cat.No. 8495) for Waring blender or equivalent 
3.  Micro centrifuge with 18,000 x g for microcentrifuge tubes 
4.  Table centrifuge (swinging buckets) with 3,000 x g for Falcon tubes 
5.  Water bath adjustable to 65°C ± 1°C 
6.  Fluostar Galaxy type 0403 from BMG LabTechnologies 
7.  PC with Fluostar software (Fluo32) 

2.2. Chemicals 

The  following  reagents  are  used  in  the  DNA  extraction  procedure  described  (equivalents  may 
be used): 

1.  Na2-EDTA; Titriplex III (Merck Cat. No. 1.08418.1000) 
2.  Tris-HCl; Tris(hydroxymethyl)aminomethane hydrochloride (USB Cat. No. 22676) 
3.  NaCl; sodium chloride (Duchefa Cat No. S0520) 
4.  KAc; potassium acetate (Merck Cat. No. 1.04820.1000) 
5.  NaAc; sodium acetate (Merck Cat. No. 1.06268.1000) 
6.  SDS; sodium dodecyl sulphate (BDH Cat. No. 442444H) 
7.  2-mercaptoethanol (Sigma Cat. No. M6250) 
8.  RNase A (Roche Cat. No. 0109-142) 
9.  Ethanol p.a. (Merck Cat. No. 1.00983.1000) 
10.  Isopropanol p.a. (Merck Cat. No. 1.09634.2500) 
11.  Phenol:chloroform:isoamylalcohol  (25:24:1)  equilibrated  at  pH  8.0  (Sigma  Cat.  No.  P-

2069) 

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12.  Chloroform p.a. (Merck Cat. No. 1.02445.2500) 
13.  Polyethylene Glycol (MW 8000) (Sigma Cat. No. P2139) 

2.3. Solutions 

The following buffers and solutions are used in the DNA extraction procedure described: 

1.  Extraction Buffer pH 8.0 
•  100 mM Tris HCl pH 8.0 
•  50 mM EDTA  
•  500 mM NaCl 
•  10 mM 2-mercaptoethanol 

2.  Tris-EDTA buffer (TE) pH 8.0 
•  10 mM Tris HCl pH 8.0 
•  1 mM EDTA 

3.  Tris-EDTA buffer (TE0.1) pH 8.0 
•  10 mM Tris HCl pH 8.0 
•  0.1 mM EDTA 

4.  RNase A (10 mg/ml) 

5.  SDS 20% 

6.  Ethanol 70% 

7.  5M KAc 

8.  3M NaAc 

2.4. Plasticware 

1.  50 ml conical tubes 
2.  15 ml conical tubes 
3.  2 ml microcentrifuge tubes 
4. 

filter tips 

Note: All plasticware has to be sterile and free of DNases, RNases and nucleic acids. 

2.5. Precautions 

•  Phenol,  chloroform,  isoamyl  alcohol,  and  isopropanol  are  hazardous  chemicals; 
therefore, all manipulations have to be performed following safety guidelines and under 

CRL-GMFF: Soybean Seeds Sampling and DNA Extraction 

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• 

fume hood. 
It  is  recommended  to  use  clean  containers  for  Waring  blenders  for  grinding  the  seed 
bulk samples. 

•  All tubes and pipette tips have to be discarded as biological hazardous material 

2.6 Abbreviations: 
EDTA 
PCR 
RNase A  
TE 
Tris 

ethylenediaminetetraacetic acid 
polymerase chain reaction 
ribonuclease A 
Tris EDTA 
Tris(hydroxymethyl)aminomethane 

3. Description of the methods 

Sampling: 
Sampling approaches for seeds and grains are referred to in technical guidance documents and 
protocols described in: 

• 

• 

International  Organization  for  Standardizations,  Switzerland:  ISO  standard  6644,  ISO 
standard 13690, ISO standard 5725; 
International  Rules  for  Seed  Testing  (2004)  International  Seed  Testing  Association 
(ISTA), Switzerland. ISBN 3-906549-38-0; 
(2001)  Sampling  grains 

the  detection  of  Biotech  grains 

for 

•  USDA-GIPSA 

http://www.usda.gov/gipsa/. 

Scope and applicability: 
The “Dellaporta-derived” method for DNA extraction described below is suitable for the isolation 
of  genomic  DNA  from  ground  soybean  seeds  and  grains.  Application  of  the  method  to  other 
matrices may require adaptation and possible further specific validation. 

Principle: 
The  basic  principle  of  the  DNA  extraction  consists  of  first  releasing  the  DNA  present  in  the 
matrix  into  aqueous  solution  and  further  purifying  the  DNA  from  PCR  inhibitors.  The 
“Dellaporta-derived” method starts with a lysis step (thermal lysis in the presence of Tris HCl, 
EDTA,  NaCl  and  2-mercaptoethanol)  followed  by  isopropanol  precipitation  and  removal  of 
extraction  with 
contaminants 
phenol:chloroform:isoamylalcohol.  
A further DNA precipitate is then generated by using isopropanol, followed by RNAse treatment, 
phenol:chloroform:isoamylalcohol  purification  and final  isopropanol  precipitation.  The  resulting 
pellet is dissolved in TE buffer.  

lipophilic  molecules 

proteins 

such 

and 

by 

as 

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Seed crushing procedure: 

•  The seeds/grains are crushed using a Waring blender 
•  Grind  in  intervals  of  10  seconds  for  4  times  at  maximum  speed.  Shake  between 

intervals until all powder is loose to improve crushing-procedure 

•  All seeds/grains should be crushed until a fine powder is obtained. Thorough grinding 

will also produce a homogenous powder 

•  Prevent cross-contamination by dust particles between the samples 

Soybean seed DNA extraction protocol 

1.  Transfer 1 g powder into a 50 ml Falcon tube 
2.  Add 30 ml Extraction Buffer 
3.  Add 2.1 ml 20% SDS, mix well by inversion  
4.  Incubate at 65°C for 30 minutes 

Note: Mix samples every 10 minutes by inversion 

5.  Centrifuge for 20 minutes at 3,000 x g 
6.  Transfer 20 ml supernatant to a new 50 ml Falcon tube using a 25 ml pipette 
7.  Add 6 ml 5M KAc, shake vigorously for 1 minute 
8.  Incubate on ice for 30 minutes 

Note: Mix samples every 10 minutes by inversion 

9.  Centrifuge for 20 minutes at 3,000 x g 
10.  Transfer 20 ml supernatant to a new 50 ml Falcon tube using a 25 ml pipette 
11.  Add equal volume of isopropanol, mix gently for 1 minute 
12.  Incubate on ice for 5 minutes  
13.  Centrifuge for 20 minutes at 3,000 x g 
14.  Remove  supernatant  and  air-dry  the  pellet  at  37°C  until  all  isopropanol  residue  is 

evaporated 

15.  Dissolve the pellet in 10 ml TE 
16.  Shake the samples for 1 hour. Make sure the pellet is completely dissolved 
17.  Add 12 ml phenol:chloroform:isoamylalcohol (25:24:1) 
18.  Mix well for 1 minute 
19.  Centrifuge for 20 minutes at 3,000 x g 
20.  Transfer  the  upper  aqueous  phase  (8  ml)  to  a  new  50  ml  Falcon  tube  using  a  10  ml 

pipette. Do not disturb the interphase  

21.  Add 900 µl 3M NaAc 
22.  Add 6 ml isopropanol 
23.  Mix gently by inversion for 1 minute 
24.  Place on ice for 5 minutes 
25.  Centrifuge for 20 minutes at 3,000 x g to pellet the DNA 
26.  Remove  supernatant  and  air-dry  the  pellet  at  37°C  until  all  isopropanol  residue  is 

evaporated 

27.  Dissolve the pellet in 1 ml TE 

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28.  Shake the samples for 1 hour. Make sure the pellet is completely dissolved 
29.  Transfer the DNA solution to a new 2.0 ml microcentrifuge tube 
30.  Add 10 µl RNase A (10 mg/ml), mix gently and incubate for 20 min at 37°C 
31.  Add 800 µl phenol:chloroform:isoamylalcohol (25:24:1) 
32.  Mix well for 1 minute 
33.  Centrifuge for 10 minutes in a microcentrifuge at maximum speed 
34.  Transfer the upper aqueous phase (about 900 µl) to a new 2 ml microcentrifuge tube  
35.  Add 800 µl chloroform 
36.  Mix well for 1 minute 
37.  Centrifuge for 10 minutes in a micro centrifuge at maximum speed 
38.  Transfer the upper aqueous phase (about 800 µl) to a new 2 ml microcentrifuge tube 

containing 90 µl 3M NaAc  

39.  Add 600 µl isopropanol 
40.  Mix gently by inversion for 1 minute 
41.  Place on ice for 5 minutes 
42.  Centrifuge for 1 minute in a micro centrifuge at maximum speed to pellet the DNA 
43.  Remove all supernatant  
44.  Add 1 ml 70% ethanol to wash the DNA pellet. Make sure the DNA pellet is not stuck to 

the bottom. Shake the samples for 1 hour 

45.  Centrifuge for 5 minutes in a micro centrifuge at maximum speed 
46.  Remove  supernatant  and  air-dry  the  pellet  at  37°C  until  all  ethanol  residue  is 

evaporated 

47.  Add 200 µl TE 0.1 to the DNA pellet 
48.  Allow pellet to dissolve for about 10 hours at 4°C 
49.  Shake samples for minimal 3 hours at 4°C 
50.  Centrifuge for 1 minute in a micro centrifuge at maximum speed 
51.  Transfer the supernatant to a new microcentrifuge tube 

4.  Testing  of  the  DNA  extraction  method  by  the  method 
developer  

4.1 Performance characteristics 

A  soybean  seed  bulk  sample  was  ground  to  fine  powder.  Six  replicated  independent  DNA 
extractions were performed on 1 gram flour sub-samples. This experiment was repeated twice 
under repeatability conditions (within short intervals of time [i.e. days] by the same operator, 
using the same equipment) resulting in 18 DNA samples. 

4.2 DNA concentration, yield and repeatability standard deviation (RSDr) 

The  concentration  of  the  extracted  DNA  was  determined  by  fluorescence  detection  using  the 
PicoGreen  dsDNA  Quantitation  Kit.  1:40  dilutions  of  each  DNA  sample  were  prepared  in 
duplicate  and  mixed  with  the  PicoGreen  reagent.  The  DNA  concentration  was  determined  on 

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CRLVL13/05XP   

the basis of a nine-point standard curve ranging from 10 ng/ml to 200 ng/ml, using a Fluostar 
for fluorescence detection. Each concentration was obtained as an average of two readings per 
sample (Table 1). 

Table  1.  Results  of  the  18  (6  X  3)  DNA  extractions:  DNA  concentration  (ng/µl)  and  DNA 
extraction efficiency 

DNA extraction 

Sample mass 
(gram) 

[DNA] 
(ng / µl) 

Yield 
(µg DNA / gram sample) 

A1 

A2 

A3 

A4 

A5 

A6 

B1 

B2 

B3 

B4 

B5 

B6 

C1 

C2 

C3 

C4 

C5 

C6 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

1.0 

423.6 

480.5 

484.8 

450.8 

455.3 

506.1 

399.3 

426.6 

418.5 

484.2 

442.4 

435.5 

436.0 

502.5 

450.5 

463.0 

482.7 

483.8 

84.7 

96.1 

97.0 

90.2 

91.1 

101.2 

79.9 

85.3 

83.7 

96.8 

88.5 

87.1 

87.2 

100.5 

90.1 

92.6 

96.5 

96.8 

Table 1 lists the DNA concentrations and DNA yield of all 18 DNA extractions. The average DNA 
concentration was 457 ± 31 ng / µl. 
The  average  DNA  extraction  efficiency  was  91.4  ±  6.1  µg  DNA  /  g  of  sample,  the  relative 
Repeatability Standard Deviation (RSDr) was 6.7%. 

Therefore,  the  soybean  DNA  extraction  procedure  is  sufficiently  efficient  and  yields  adequate 
amounts of genomic DNA with a sufficient level of repeatability. 

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4.3 Analysis of DNA fragmentation 

Analysis  of  DNA  fragmentation  was  performed  by  ethidium  bromide-stained  agarose  gel 
electrophoresis. One microliter of each undiluted DNA was analyzed on a 1% agarose gel (TBE 
buffer), including a molecular weight marker (Figure 1). 

Figure 1. Agarose gel electrophoresis of eighteen genomic DNA samples extracted from soybean 
seeds (1 µl undiluted DNA). 

              1     2     3     4    5     6     7     8    9    10   11    12   13   14   15   16    17  18               

2072 bp 
1500 bp 

The  18  genomic  DNA  samples  extracted  as  described  above  appeared  as  distinct  fluorescent 
banding patterns migrating through the gel corresponding to high molecular weight DNA. None 
of the 18 genomic DNA samples showed indications of significant degradation. 

4.4 Evidence of the absence of PCR inhibitory compounds 

The absence of PCR inhibitory compounds in the DNA preparations was demonstrated by Real-
time  PCR  using  the  oligonucleotides  directed  to  the  endogenous  control  gene lectin on  serial 
dilutions of the DNA preparations. 

All  DNA  extracts  were  adjusted  to  a  concentration  of  40  ng/µl.  From  this  sample  (named 
“undiluted sample”), a fourfold serial dilution series was prepared with water (1:4, 1:16, 1:64, 
1:256).  
To assess the presence of inhibitors, the Ct values (y axis) of the dilution series were plotted 
against the logarithm of the DNA amount (x axis). By linear regression, a trend line (y = ax + 
b) was calculated, as well as a correlation coefficient, r2, as a measure of linearity (Table 2). 
The ideal slope value, ‘a’, (optimal PCR efficiency) than becomes a = -3.32 (typically ‘a’ values 
between -3.1 and -3.6 indicate excellent PCR efficiencies).  

Correlation  coefficients  of  r2  >0.98  indicate  an  excellent  linear  relationship,  and  thus,  equally 
efficient PCR amplification over the measured dynamic range. 
The Ct value for the “undiluted sample” (40 ng/µl, 200 ng/reaction) was extrapolated from the 
equation  calculated  by  linear  regression.  Subsequently  the  extrapolated  Ct  for  the  undiluted 

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CRLVL13/05XP   

sample was compared with the measured Ct. Differences between measured and extrapolated 
Ct of < 0.5 are indicative of low level or absence of inhibition. 

The results reported in Table 2 show no evidence of the presence of PCR inhibitors; in fact all 
18 ∆Ct values are < 0.5, suggesting absence of PCR inhibitors. 

Table 2. Results of the analysis for PCR inhibitory compounds; comparison of extrapolated Ct 
values versus measured Ct values. 

DNA  Slope, a 

Intercept, b  Linearity, R2 Ctmeasured Ctextrapolated

A1 

A2 

A3 

A4 

A5 

A6 

B1 

B2 

B3 

B4 

B5 

B6 

C1 

C2 

C3 

C4 

C5 

C6 

-3.4477 

30.1770 

-3.5102 

30.3226 

-3.5644 

30.2912 

-3.4918 

30.2546 

-3.4705 

30.2414 

-3.5217 

30.4561 

-3.3889 

30.2363 

-3.4336 

30.5378 

-3.4494 

30.6269 

-3.5080 

30.7840 

-3.3633 

30.5680 

-3.3576 

30.5944 

-3.4920 

30.7847 

-3.5200 

30.7951 

-3.4440 

30.7391 

-3.4819 

30.8506 

-3.4039 

30.6721 

-3.4822 

30.7781 

0.9997 

0.9999 

0.9995 

0.9999 

0.9977 

0.9999 

0.9999 

0.9992 

0.9999 

0.9996 

0.9999 

0.9995 

0.9999 

0.9996 

0.9997 

0.9999 

0.9999 

0.9999 

22.16 

22.35 

22.38 

22.46 

22.34 

22.55 

22.55 

22.79 

22.71 

23.06 

22.93 

22.80 

22.79 

22.93 

22.84 

23.08 

22.98 

22.86 

22.24 

22.25 

22.09 

22.22 

22.26 

22.35 

22.44 

22.64 

22.69 

22.71 

22.83 

22.87 

22.75 

22.70 

22.81 

22.84 

22.84 

22.77 

∆Ct  
(Ctmeasured-
Ctextrapolated)

-0.08 

0.10 

0.29 

0.24 

0.09 

0.20 

0.11 

0.15 

0.02 

0.35 

0.10 

-0.07 

0.04 

0.24 

0.03 

0.24 

0.15 

0.10 

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5.  Experimental  testing  of  the  DNA  extraction  method  by 
the Community Reference Laboratory for GM Food and Feed 

The aim of the experimental testing was to verify that the DNA extraction method provides DNA 
of suitable quantity and quality for the intended purpose.  

The  DNA  extraction  method  should  allow  preparation  of  the  analyte  in  quality  and  quantity 
appropriate  for  the  analytical  method  used  to  quantify  the  event-specific  analyte  versus  the 
reference analyte. 

The CRL-GMFF tested the “Dellaporta-derived” method proposed by the applicant on samples of 
food and feed consisting of ground soybean seeds provided by the applicant. 

To assess the suitability of the DNA extraction method for real-time PCR analysis, the extracted 
DNA was tested using a qualitative PCR run on the real-time PCR equipment.  

5.1 Preparation of samples 

About  200  g  of  soybean  seed  material  were  ground  using  a  GRINDOMIX  GM  200  (Retsch 
GmbH) mixer. 

5.2 DNA extraction 

DNA  was  extracted  following  the  “Dellaporta-derived”  method  described  above;  the  DNA 
extraction  was  carried  out  on  6  test  portions  (replicates)  and  repeated  over  three  different 
days, giving a total of 18 DNA extractions. 

5.3 DNA concentration, yield and repeatability 

Concentration  of  the  DNA  extracted  was  determined  by  fluorescence  detection  using  the 
PicoGreen dsDNA Quantitation Kit (Molecular Probes). 

Each DNA extract was measured twice, and the two values were averaged. DNA concentration 
was determined on the basis of a five point standard curve ranging from 1 to 500 ng/µl using a 
Biorad VersaFluor fluorometer.  

The DNA concentration for all samples is reported in the Table 3. 

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CRLVL13/05XP   

Table 3. DNA concentration (ng/µl) of eighteen samples extracted in three days: yellow boxes 
for samples extracted on day 1, green boxes for samples extracted on day 2 and blue boxes for 
samples extracted on day 3. 

Sample 

Concentration 
(ng/µl) 

1 

2 

3 

4 

5 

6 

1 

2 

3 

4 

5 

6 

1 

2 

3 

4 

5 

6 

197 

188 

182 

186 

190 

150 

172 

147 

125 

99 

91 

91 

187 

159 

164 

163 

177 

177 

(cid:57)  DNA concentration (ng/µl) 

Overall average of all samples:    
Standard deviation of all samples  

Coefficient of variation   

158 ng/µl 
34.7 ng/µl 
21.9 % 

(cid:190)  Yield (total volume of DNA solution: 200 µl) 

Overall average of all samples:    
Standard deviation 

Coefficient of variation   

31.6 µg 
6.9 µg 
21.9 % 

5.4 Fragmentation state of DNA 

The size of the extracted DNA was evaluated by agarose gel electrophoresis; 8 µl of the DNA 
solution were analysed on a 1.0% agarose gel (Figure 1).    

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Figure  1.  Agarose  gel  electrophoresis  of  eighteen  genomic  DNA  samples  extracted  from 
soybean seeds. Lanes 2-7: samples extracted on day 1; lanes 8-13 samples extracted on day 2; 
lanes 14-19 samples extracted on day 3; lanes 1 and 20: 1kb DNA Marker. 

  1    2    3    4    5     6    7    8    9    10  11  12  13   14   15  16  17   18   19   20

10 kb 

1 kb 

The  eighteen  genomic  DNA  samples  extracted  as  described  above  appeared  as  distinct 
fluorescent banding patterns migrating through the gel corresponding to high molecular weight 
DNA. None of the DNA samples showed indications of significant degradation (‘smearing’). 

5.4 Purity / Absence of PCR inhibitors 

In order to assess the purity and to confirm the absence of PCR inhibitors, the extracted DNA 
solutions  were  adjusted  to  a  concentration  of  40  ng/µl  (hereafter  referred  as  “undiluted” 
samples).  

Subsequently fourfold serial dilutions of each extract were prepared with pure water (1:4, 1:16, 
1:64, 1:256) and analysed using a real-time PCR system detecting the target sequence of the 
endogenous control gene lectin, Le1.  

The Ct values obtained for “undiluted” and diluted DNA samples are reported in the Table 4.  

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Table 4. Ct values of undiluted and fourfold serially diluted DNA extracts after amplification of 
soybean  lectin  gene,  Le1 Yellow  boxes  for  samples  extracted  on  day  1,  green  boxes  for 
samples extracted on day 2 and blue boxes for samples extracted on day 3. 

.

Undiluted 

Diluted 

DNA extract 

(40 ng/µl) 

1:4 

1 

2 

3 

4 

5 

6 

1 

2 

3 

4 

5 

6 

1 

2 

3 

4 

5 

6 

20.77 

22.53 

21.07 

20.94 

21.07 

20.94 

22.56 

20.84 

22.68 

21.24 

21.15 

21.15 

22.68 

22.65 

22.78 

22.75 

22.12 

22.53 

22.80 

24.63 

22.97 

22.75 

22.99 

22.77 

24.58 

22.99 

24.86 

23.29 

23.13 

23.20 

24.70 

24.88 

24.91 

24.79 

24.15 

24.66 

1:16 

24.90 

26.43 

25.14 

24.78 

24.95 

24.79 

26.86 

24.91 

26.82 

25.33 

25.32 

25.39 

26.78 

26.82 

26.98 

26.78 

26.21 

26.84 

1:64 

26.79 

28.79 

27.03 

27.22 

26.90 

27.02 

28.99 

27.07 

28.87 

27.57 

27.36 

27.53 

28.92 

28.89 

29.22 

29.05 

28.37 

29.06 

1:256 

29.39 

31.11 

29.26 

29.73 

29.38 

28.75 

31.14 

29.31 

31.08 

29.56 

29.67 

29.70 

31.20 

31.38 

31.44 

31.22 

30.65 

31.33 

Table 5 below reports the comparison of extrapolated Ct values versus measured Ct values for 
all samples and the values of linearity (R2) and slope of all measurements. 

To  measure  inhibition,  the  Ct  values  of  the  four  diluted  samples  were  plotted  against  the 
logarithm  of  the  dilution  and  the  Ct  value  for  the  “undiluted”  sample  (40  ng/µl)  was 
extrapolated from the equation calculated by linear regression.  

Subsequently the extrapolated Ct for the “undiluted” sample was compared with the measured 
Ct. The evaluation is carried out considering that PCR inhibitors are present if the measured Ct 
value for the “undiluted” sample is suppressed by > 0.5 cycles from the calculated Ct value. In 
addition, the slope of the curve should be between -3.6 and -3.1. 

CRL-GMFF: Soybean Seeds Sampling and DNA Extraction 

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CRLVL13/05XP   

Table  5.  Comparison  of  extrapolated  Ct  values  versus  measured  Ct  values  (amplification  of 
soybean lectin gene, Le1) 

DNA 
extraction 

1 

2 

3 

4 

5 

6 

1 

2 

3 

4 

5 

6 

1 

2 

3 

4 

5 

6 

R2

0.994 

0.996 

0.997 

0.987 

0.995 

0.993 

0.998 

0.997 

0.999 

0.995 

0.999 

0.996 

0.999 

0.996 

1.000 

0.999 

0.999 

1.000 

Slope* 

Ct extrapolated 

mean Ct 
measured 

∆Ct** 

-3.6 

-3.6 

-3.4 

-3.9 

-3.5 

-3.3 

-3.6 

-3.5 

-3.4 

-3.5 

-3.6 

-3.6 

-3.6 

-3.6 

-3.6 

-3.6 

-3.6 

-3.7 

20.56 

22.28 

20.91 

20.27 

20.77 

20.79 

22.44 

20.79 

22.72 

21.18 

20.96 

21.04 

22.49 

22.60 

22.68 

22.57 

21.93 

22.41 

20.77 

22.53 

21.07 

20.94 

21.07 

20.94 

22.56 

20.84 

22.68 

21.24 

21.15 

21.15 

22.68 

22.65 

22.78 

22.75 

22.12 

22.53 

0.21 

0.24 

0.16 

0.67 

0.30 

0.15 

0.12 

0.05 

0.04 

0.06 

0.19 

0.11 

0.20 

0.05 

0.10 

0.19 

0.19 

0.12 

Note: In yellow boxes samples from 1 to 6 extracted on day 1; in green boxes samples from 1-6 extracted 

on day 2; in blue boxes samples from 1-6 extracted on day 3. 

*The expected slope for a PCR with 100% efficiency is -3.32 

**delta Ct = abs (Ct extrapolated - Ct measured) 

All  ∆Ct  values  of  extrapolated  versus  measured  Ct  are  <  0.5,  except  the  sample  number  4 
extracted on day 1, with a value of 0.67. 

R2 of linear regression is > 0.99 for all DNA samples except one slightly below (0.987). 

Curve slopes are all between -3.1 and -3.6, with two exceptions: sample 4 extracted on day 1, 
with  a  value of  -3.9  (same  sample  showing  a ∆Ct of  0.67) and  sample  6  extracted on  day  3, 
with a value of -3.7. 

CRL-GMFF: Soybean Seeds Sampling and DNA Extraction 

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CRLVL13/05XP   

6. Conclusion 

The  data  reported  confirm  that  the  extraction method,  applied  to soybean seeds  provided  by 
the  applicant,  produces  DNA  of  suitable  quantity  and  quality  for  subsequent  PCR  based 
detection  applications.  The  method  is  consequently  applicable  to  samples  of  soybean  seeds 
provided as samples of food and feed in accordance with the requirements of Annex I-2.C.2 to 
Commission Regulation (EC) No 641/2004. 

7. Quality assurance 

The CRL-GMFF carries out all operations according to ISO 9001:2000 (certificate number: CH-
32232)  and  ISO  17025:2005  (certificate  number:  DAC-PL-0459-06-00)  [DNA  extraction, 
qualitative  and  quantitative  PCR  in  the  area  of  Biology  (DNA  extraction  and  PCR  method 
validation for the detection and identification of GMOs in food and feed materials)] 

8. References 

1. Dellaporta et al. [1983] Plant Molecular Biology Reporter 1, nr 4, pp. 19-21. 

2. Sambrook J. and D. W. Russell. Molecular Cloning. A laboratory manual. Third edition. 2001. 
Cold Spring Harbor Laboratory Press. 

CRL-GMFF: Soybean Seeds Sampling and DNA Extraction 

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European Commission 

EUR 22910 EN – DG Joint Research Centre, Institute for Health and Consumer Protection 
Title: Event-specific Method for the Quantification of Soybean Line A2704-12 Using Real-time PCR – 
Validation Report and Protocol - Soybean Seeds Sampling and DNA Extraction  
Authors: M. Mazzara, C. Delobel, E. Grazioli, S. Larcher, C. Savini and G. Van den Eede 
Luxembourg: Office for Official Publications of the European Communities 
2007 – 47pp. – 21 x 29,7 cm  
EUR - Scientific and Technical Research series; ISSN 1018-5593 
ISBN 978-92-79-06928-4 

Abstract 

The  JRC  as  Community  Reference  Laboratory  for  GM  Food  and  Feed  (CRL-GMFF),  established  by 
Regulation  (EC)  No  1829/2003,  in  collaboration  with  the  European  Network  of  GMO  Laboratories 
(ENGL), has carried out a collaborative study to assess the performance of a quantitative event-specific 
method  to  detect  and  quantify  the  A2704-12  transformation  event  in  soybean  DNA  (unique  identifier 
ACS-GMØØ5-3). The collaborative trial was conducted according to internationally accepted guidelines 
(1, 2). 

In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food 
and feed and Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of 
Regulation  (EC)  No  1829/2003,  Bayer  CropScience  provided  the  detection  method  and  the  samples 
(genomic  DNA  extracted  from  wild-type  and  100%  soybean  A2704-12  event).  The  JRC  prepared  the 
validation  samples  (calibration  samples  and  blind  samples  at  unknown  GM  percentage  [DNA/DNA]). 
The international collaborative trial involved thirteen laboratories from ten European countries. 

The  results  of  the  international  collaborative  trial  met  the  ENGL  performance  requirements  and  the 
scientific  understanding  about  satisfactory  method  performance.  Therefore,  the  CRL-GMFF  considers 
the method validated as fit for the purpose of regulatory compliance. 

The results of the collaborative study are made publicly available at http://gmo-crl.jrc.it/. 

.

 
 
 
 
 
 
 
 
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 science and technology for 
the  Union.  Close  to  the  policy-making  process,  it  serves  the  common  interest  of  the  Member 
States, while being independent of special interests, whether private or national. 

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