Event-specific method for the quantification of oilseed rape line Ms8 using real-time PCR validation report.
Date: 2007-12-18
Institutions: Institute for Health and Consumer Protection
Subjects: biotechnology, consumer protection, evaluation method, genetically modified organism, oil seed rape, oleaginous plant, research method

Event-specific Method for the Quantification of 
Oilseed Rape Line Ms8 Using Real-time PCR 

Validation Report and Protocol 
Seeds Sampling and DNA Extraction of Oilseed Rape 

M. Mazzara, A. Bogni, C. Savini and G. Van den Eede 

11 January 2007

Method development: 
Bayer CropScience 

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

Institute for Health and Consumer Protection 
2007 

EUR 22917 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
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EUR 22917 EN 
ISBN 978-92-79-06934-5 
ISSN 1018-5593 
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© European Communities, 2007 

Reproduction is authorised provided the source is acknowledged 

Printed in Italy 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CRLVL06/04VR 

Event-specific Method for the Quantification 
of Oilseed Rape Line Ms8 Using 
Real-time PCR 

Validation Report 

Biotechnology & GMOs Unit 
Institute for Health and Consumer Protection 
DG Joint Research Centre 

11 January 2007 

Executive Summary 

The  JRC  as  Community  Reference  Laboratory  for  GM  Food  and  Feed  (CRL-GMFF)  (see 
Regulation  EC  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 Ms8 transformation event in oilseed rape DNA 
(unique  identifier  ACS-BN∅∅5-8).  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 with 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% oilseed 
rape  Ms8  event).  The  JRC  prepared  the  validation  samples  (calibration  samples  and  blind 
samples  at  unknown  GM  percentage  [DNA/DNA]).  The  collaborative  trial  involved  thirteen 
laboratories from eleven European Countries. 

The results of the 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  under  http://gmo-crl.jrc.it/. 
The method will also be submitted to ISO 21570 for consideration as an international standard. 

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Report on Steps 1-3 of the Validation Process 

CRLVL06/04VR 

Bayer  CropScience  submitted  the  detection  method  and  control  samples  for  oilseed  rape 
event  Ms8  (unique  identifier  ACS-BN∅∅5-8)  under  Article  8  and  20  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  with  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  assessed 
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,  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 Ms8 was positively concluded in October 2005.  

In  March-June  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%-3.6%  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.  

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

available on request. 

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CRLVL06/04VR 

Contents 

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 ......................................................................9 
GMO QUANTIFICATION.............................................................................. 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 oilseed rape event 
Ms8 (unique identifier ACS-BN∅∅5-8) in accordance to Articles 8 and 20 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  1829/2003)  organised  the  international  collaborative  ring 
trial for the event-specific method for the detection and quantification of Ms8 oilseed rape. The 
study  involved  thirteen  laboratories,  each  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)  641/2004  and  following  its  operational 
procedures. 
The  internal  in-house  experimental  evaluation  of  the  method  was  carried  out  in  March-June 
2006. 
Following  the  evaluation  of  the  data  and  the  results  of  the  in-house  laboratory  tests,  the 
international ring trial was organised (step 4) and took place in July 2006. 

A  method  for  DNA  extraction  from  oilseed  rape  seeds,  submitted  by  the  applicant,  was 
evaluated  by  the  CRL-GMFF;  laboratory  testing  of  the  method  was  carried  out  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  Ms8  DNA  to  total  oilseed  rape  DNA.  The  procedure  is  a 
simplex system, in which an oilseed rape (OSR) CruA (Cruciferin A) endogenous assay 
(reference gene) and the target assay (Ms8) are performed in separate wells.  

The  international  collaborative  ring-trial  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  ring  trial  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  common  execution  of  the 
protocol. The participating laboratories are listed in alphabetical order in Table 1.  

Table 1. ENGL laboratories participating in the validation study of oilseed rape Ms8. 

Laboratory 

 Behoerde fuer Wissenschaft und Gesundheit 

 Bundesinstitut fuer Risikobewertung (BfR) 

Country 

Germany 

Germany 

 Danish Plant Directorate - Laboratory for diagnostics in Plants, Seed and Feed 

Denmark 

 Dr E Wessling Chemical Laboratory 

 Ente Nazionale Sementi Elette/ Laboratorio Analisi Sementi 

 Finnish Customs Laboratory 

 Institute for Agricultural & Fisheries Research (ILVO) 
 Scientific Institute of the Flemish Community 

 National Food Administration 

 National Institute of Biology 

 National Institute of  Food Hygiene and Nutrition GMO lab 

 Scottish Agricultural Science Agency 

Hungary 

Italy 

Finland 

Belgium 

Sweden 

Slovenia 

Hungary 
United 
Kingdom 

 Swiss Federal Research Station for Animal Production and Dairy Products 

Switzerland 

 Umweltbundesamt GmbH 

Austria 

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3.  Materials 

For the validation of the quantitative event-specific method, control samples consisting of i) a 
DNA  stock  solution  hemizygous  for  the  GM-event  Ms8  (Lot  Number  32RRMM0008-3)  and ii) 
non-GM  DNA  stock  solution  (Lot  Number  32RRMM0100)  extracted  from  a  genetically  similar 
wild-type  line  provided  by  the  applicant  in  accordance  to  the  provisions  of  Regulation  (EC) 
No1829/2003,  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% Ms8 and non-GM oilseed rape genomic DNA at different 
GMO concentrations were prepared by the CRL-GMFF, using the control samples provided, in a 
constant amount of total oilseed rape DNA.  

Participants received the following materials: 

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

standard curve, denominated from S1 to S5.  

(cid:57)  Twenty unknown DNA samples (100 µl of DNA solution each), denominated from U1 to 

U20.  

(cid:57)  Amplification reagent control for use on each PCR plate. 
(cid:57)  Reaction  reagents,  primers  and  probes  for  the CruA  reference  gene  and  for  the  Ms8 

specific systems as follows:  

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

4 ml 

(cid:57)  Primers and probes (1 tube each) as follows: 

CruA reference system 
(cid:137)  MDB510 primer (10 µM):  
(cid:137)  MDB511 primer (10 µM):  
(cid:137)  TM003 TaqMan® probe (10 µM):  

Ms8 oilseed rape system 
(cid:137)  KVM085 primer  (10 µM):  
(cid:137)  HCA048 primer (10 µM):  
(cid:137)  TM011 TaqMan® probe (10 µM):  

320 µl 
320 µl  
160 µl    

160 µl  
160 µl 
160 µl 

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Table  2  shows  the  GM  contents  of  the  unknown  samples  (U1-U20)  distributed  to  ring  trial 
participants.  

Table 2.  Ms8 GM contents 

Ms8 GM % 
(GM copy number/OSR genome copy number *100) 
0.1 
0.4 
0.9 
1.8 
3.6 

4. 

Experimental design 

Twenty unknown samples (U1-U20), representing five GM levels, were used in the validation 
study.  On  each  PCR  plate,  samples  were  analysed  in  parallel  with  both  the  Ms8  and CruA 
specific system. 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). 

5.  Method 

Description of operational steps followed 

For  specific  detection  of  event  Ms8  genomic  DNA,  a  129-bp  fragment  of  the  recombination 
region of parts of the construct inserted into the plant genome 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 is used as the reporter 
dye at its 5′ end and TAMRA as a quencher dye at its 3′ end. 

For relative quantification of event Ms8 DNA, an OSR-specific reference system amplifies a 101-
bp fragment of CruA (Cruciferin A) oilseed rape endogenous gene (GenBank X14555), using a 
pair  of  CruA  gene-specific  primers  and  a  CruA  gene-specific  probe  labelled  with  VIC  and 
TAMRA.  

For  relative  quantification  of  event  Ms8  DNA  in  a  test  sample,  the  normalised  ΔCt  values  of 
calibration samples are used to calculate, by linear regression, a standard curve (plotting ΔCt 
values against the logarithm of the amount of Ms8 event DNA). The normalised ΔCt values of 
the  unknown  samples  are  measured  and,  by  means  of  the  regression  formula,  the  relative 
amount of Ms8 event DNA is estimated. 

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Calibration  samples  from  S1  to  S5  were  prepared  by  mixing  the  appropriate  amount  of  Ms8 
DNA from the stock solution in control non-GM oilseed rape DNA to obtain the following relative 
contents of Ms8: 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 oilseed rape genomes as 1.15 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 

Ten laboratories reported no deviations from the protocol. 
One  laboratory  used  the  Hex  instead  of  the  VIC  detection  channel  to  detect  the  specific 
fluorescence from the VIC probe. However, the laboratory declared that HEX wavelengths both 
for excitation and emission spectra are similar to the VIC and the filter used is the same.  
One laboratory programmed as unknowns the standards amplified with the reference gene. 
One  laboratory  ran  the  samples  in  twenty  microliters,  as  only  a  384-well  plate  machine  was 
available; final concentrations of the supplied primers/probe, buffer and the DNA amount were 
maintained according to the original protocol. 

7. 

Summary of results 

PCR efficiency and linearity 

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

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

CRLVL06/04VR 

LAB  PLATE

Slope 

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 

-3.26 
-3.33 
-3.18 
-2.84 
-3.64 
-3.83 
-3.26 
-3.72 
-3.34 
-3.13 
-3.91 
-3.22 
-3.56 
-3.33 
-3.48 
-3.48 
-3.16 
-3.38 
-3.55 
-2.76 
-3.35 
-3.35 
-3.43 
-3.41 
-3.26 
-3.40 

PCR 
Efficiency 
(%) 
97.3 
99.7 
93.8 
75.1 
88.2 
82.4 
97.5 
85.6 
99.3 
91.3 
80.1 
95.4 
90.8 
99.7 
93.8 
93.6 
92.6 
97.6 
91.3 
69.9 
98.9 
98.7 
95.8 
96.3 
97.2 
97.0 

Mean

-3.37 

92.3 

Linearity
(R2) 

1.00 
1.00 
0.98 
0.99 
0.99 
0.99 
0.98 
0.99 
1.00 
1.00 
0.99 
1.00 
1.00 
1.00 
1.00 
1.00 
1.00 
0.99 
1.00 
0.95 
1.00 
1.00 
0.98 
0.98 
0.99 
1.00 

0.99 

The mean PCR efficiency was 92.3% and the linearity of the method was on average 0.99. 
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  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 all unknown samples (U1-U20). 

Sample GMO content  
(GM% = GM copy number/OSR genome copy number *100) 

LAB 

0.10 

0.40 

0.90 

1.80 

3.60 

  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 

10 

11 

12 

13 

0.09  0.09  0.10

0.11  0.39  0.36

0.41  0.39

0.77  0.70  0.79

0.92  1.60  1.65

1.61  1.63  2.94

3.30  3.17  3.14

0.05  0.07  0.04

0.14  0.41  0.30

0.21  0.11

0.49  0.70  0.59

1.52  1.31  1.80

2.97  1.17  1.79

5.56  3.56  7.37

0.11  0.09  0.13

0.11  0.39  0.44

0.49  0.42

0.96  0.81  0.79

0.99  2.15  1.48

1.60  1.39  2.98

2.56  2.63  2.30

0.09  0.10  0.07

0.14  0.36  0.34

0.37  0.35

0.70  0.69  0.95

0.93  2.22  1.46

2.02  1.60  2.58

2.98  3.17  2.18

0.11  0.17  0.10

0.13  0.37  0.42

0.35  0.36

0.90  0.87  0.85

0.93  1.96  2.28

2.10  1.86  3.52

5.13  3.99  3.49

0.12  0.12  0.10

0.08  0.35  0.48

0.29  0.38

0.93  0.97  0.98

0.78  1.65  2.29

1.52  1.82  3.88

3.65  3.55  4.20

0.12  0.13  0.09

0.10  0.40  0.38

0.34  0.42

0.91  0.95  0.85

0.95  1.77  1.86

1.54  1.70  3.37

3.26  3.31  3.06

0.11  0.12  0.13

0.12  0.33  0.36

0.41  0.36

0.89  0.78  0.70

0.89  1.61  1.74

1.74  1.92  3.03

2.97  3.27  3.23

0.09  0.09  0.11

0.12  0.37  0.36

0.40  0.43

0.95  0.74  0.90

0.91  1.72  1.70

1.77  1.86  3.46

3.43  3.36  3.38

0.10  0.12  0.10

0.08  0.48  0.46

0.41  0.36

1.17  0.95  0.86

1.14  1.89  1.81

3.09  2.71  3.82

7.48  7.19  5.74

0.10  0.11  0.09

0.11  0.38  0.40

0.39  0.39

0.85  0.84  0.83

0.94  1.68  1.76

1.66  1.76  3.35

3.33  3.07  3.22

0.07  0.06  0.84

0.05  0.42  0.20

0.07  0.45

0.84  0.61  0.47

0.20  1.19  0.85

1.17  0.81  3.01

0.41  0.61  2.91

0.11  0.12  0.11

0.18  0.36  0.37

0.70  0.53

1.19  0.73  0.80

1.26  1.78  1.84

1.98  2.69  3.78

3.26  4.07  4.70

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; green bars represent the overall mean.  

As  observed  in  Figure  1,  the  majority  of  laboratories  overestimated  the  true  value  at  0.1%, 
whereas several laboratories slightly underestimated the GM content at 3.6%. One laboratory 
heavily  underestimated  the  true  value  at  the  1.8%  level,  but  was  not  indicated  as  an  outlier 
according to Cochrane and Grubbs tests (ISO 5725-2). 

Overall, the average relative deviation (green bar) was definitely modest at all the GM levels, 

indicating a satisfactory accuracy of the method.  

Figure 1. Relative deviation (%) from the true value of Ms8 for all laboratories 

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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  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  around  23%  at  the  0.1%  and  1.8%  levels, 
well within the acceptance criterion. 

Table 6.  Oilseed rape Ms8: summary of validation results. 

Expected value (GMO %) 

Unknown sample GM% 

0.1 

0.4 

0.9 

1.8 

3.6 

 Laboratories having returned results 

 Samples per laboratory 

 Number of outliers 

 Reason for exclusion 

 Mean value 

 Relative repeatability standard deviation, RSDr (%)  

 Repeatability standard deviation  

 Relative reproducibility standard deviation, RSDR (%)  

 Reproducibility standard deviation  

13 

4 

1 

1 C 

0.11 

22 

0.02 

23 

0.02 

13 

4 

1 

1 C 

0.39 

18 

0.07 

21 

0.08 

13 

4 

2 

13 

4 

1 

1 C/1 G  

1 C 

0.89 

14 

0.12 

14 

0.13 

1.78 

17 

0.30 

23 

0.41 

13 

4 

3 

3 C  

3.33 

11 

0.37 

17 

0.58 

 Bias (absolute value) 

 Bias (%) 

0.007 

-0.014 

-0.009 

-0.019 

-0.269 

7.4 

-3.5 

-1.0 

-1.0 

-7.5 

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  ring  trial  evaluation,  the  CRL 
requires  that  RSDr  values  be  below  25%,  as  indicated  by  ENGL  (Definition  of  Minimum 
[http://gmo-
Performance  Requirements 

for  Analytical  Methods  of  GMO  Testing” 

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crl.jrc.it/guidancedocs.htm]).  As  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  7.5  at  the  3.6%  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 under 
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 ring trial (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 

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

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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 Efficiency 

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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CRLVL06/04VP 

Event-specific Method for the Quantification of  

Oilseed Rape Line Ms8 Using Real-time PCR 

Protocol 

Method development: 
  Bayer CropScience 

Collaborative trial: 
Joint Research Centre – European Commission 
Biotechnology & GMOs Unit  

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Contents 

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....................................................................................5 
2.4 LIMIT OF QUANTITATION ...............................................................................5 
2.5 MOLECULAR SPECIFICITY ...............................................................................5 

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

3.1  GENERAL INSTRUCTIONS AND PRECAUTIONS ...................................................6 
REAL-TIME PCR FOR QUANTITATIVE ANALYSIS OF MS8 OILSEED RAPE....................7 
3.2 
3.2.1 General.........................................................................................7 
3.2.2 Calibration....................................................................................7 
3.2.3 Real-time PCR set-up...................................................................7 
3.3  DATA ANALYSIS......................................................................................9 
CALCULATION OF RESULTS....................................................................... 10 
3.4 

4.  MATERIALS....................................................................................... 10 

4.1 
4.2 
4.3 

EQUIPMENT ........................................................................................ 10 
REAGENTS .......................................................................................... 11 
PRIMERS AND PROBES............................................................................ 11 

5.  REFERENCES..................................................................................... 11 

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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 Ms8 DNA to total oilseed rape (OSR) 
DNA in a sample.  

The PCR assay was optimised for the 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  the  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 Ms8 genomic DNA, a 129-bp fragment of the recombination 
region  between  the  insert  and  the  plant  genome  (located  at  the  3’  flanking  region)  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  quantitation  of  event  Ms8  DNA,  an  oilseed  rape-specific  reference  system 
amplifies a 101-bp fragment of the Cruciferin A gene (CruA), an oilseed rape endogenous 
gene, using a pair of specific primers and a CruA gene-specific probe labelled with VIC 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  quantitation  of  the  amount  of 
event Ms8 DNA in a test sample, the normalised ΔCt values of the calibration samples are 
used to calculate by linear regression a reference curve ΔCt-formula. The normalised ΔCt 
values  of  the  unknown  samples  are  measured  and,  by  means  of  the  reference  ΔCt-
formula, the relative amount of Ms8 event DNA is estimated. 

2. Validation status and performance characteristics  

2.1 General 

The  method  has  been  optimised  for  suitable  DNA  extracted  from  oilseed  rape  grains  or 
seeds. 

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The  reproducibility  and  trueness  of  the  method  were  tested  through  an  international 
collaborative ring trial using DNA samples at different GMO contents. 

2.2 Collaborative trial 

The method was validated in a collaborative trial by the Joint Research Centre (JRC) of the 
European  Commission.  The  study  was  undertaken  with  13  laboratories  in  July-August 
2006.  

Each participant received twenty unknown samples containing Ms8 oilseed rape genomic 
DNA at five GM contents, between 0.1 % and 3.6 %.  

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  Ms8  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 

According to the method developer, the relative LOD of the method is at least 0.045% in 
200  ng  of  total  oilseed  rape  DNA.  The  relative  LOD  was  not  assessed  in  a  collaborative 
trial. The lowest relative GM content of the target sequence included in collaborative trial 
was 0.1 %. 

2.4 Limit of quantitation 

According to the method developer, the relative LOQ of the method is at least 0.09% in 
200 ng of total oilseed rape DNA. The lowest relative GM content of the target sequence 
included in collaborative trial was 0.1 %. 

2.5 Molecular specificity 

The  method  utilises  a  unique  DNA  sequence  of  the  recombination  region  between  the 
insert  and  the  plant  genome.  The  sequence  is  specific  to  Ms8  event  and  thus  imparts 
event-specificity to the method. 

The  specificity  of  the  forward  and  reverse  oligonucleotide  primer  was  experimentally 
tested in duplicated end-point PCRs against DNA extracted from plant materials containing 
the specific targets of maize T25, MON810, Bt11, Bt176, GA21, NK603, CBH351, Roundup 

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Ready  Soybean,  soybean  A2704-12,  oilseed  rape  Ms1,  Ms8,  Rf1,  Rf2,  Rf3,  Topas19/2, 
T45, LLCotton25 and LLRice62.  
None  of  the  GM-lines,  except  the  positive  control  Ms8,  yielded  detectable  amplicons  in 
duplicate experiments. 

3. Procedure 

3.1  General instructions and precautions 

•  The procedures require experience of working under sterile conditions. 

•  Laboratory  organisation, e.g.  “flow  direction”  during PCR-setup, should follow the 
guidelines  given  by  relevant  authorities  like  e.g.  ISO,  CEN,  Codex  alimentarius 
commission. 

•  PCR-reagents should be stored and handled in a separate room and freezer and in 
equipment  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 must 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 shall not adsorb protein or DNA. 

• 

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

•  Use only powder-free gloves and change them frequently.  

•  Clean 

laboratory  benches  and  equipment  periodically  with  10%  sodium 

hypochloride solution (bleach).  

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

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

• 

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

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3.2  Real-time  PCR  for  quantitative  analysis  of  Ms8  oilseed 

rape 

3.2.1 General 

The  PCR  set-up  for  the  taxon  specific  target  sequence  (CruA)  and  for  the  GMO  (Ms8) 
target  sequence  should  be  carried  out  in  separate  vials.  Multiplex  PCR  (using  differential 
fluorescent labels for the probes) has not been tested or validated. 

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 fixed percentages of Ms8 DNA in 
a  total  amount  of  200  ng  oilseed  rape  DNA.  The  GM  content  of  the  standard  samples 
ranges from 3.6% to 0.09%. 

A  calibration  curve  is  produced  by  plotting  the  ΔCt-values  of  calibration  samples  against 
the  logarithm  of  the  respective  GM  %  contents;  the  slope  (a) and  the  intercept (b)  of 
the calibration 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 at 1-4°C on ice.  

2.  In two reaction tubes (one for Ms8 system and one for the CruA system) on ice, add 
the  following  components  (Tables  1  and  2)  in  the  order  mentioned  below  (except 
DNA) to prepare the master mixes. 

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

Component 

Final 
concentration 

µl/reaction 

TaqMan® Universal PCR Master Mix (2x) 

MBD510 For primer (10 μM) 

MBD511 Rev primer (10 μM) 

TM003 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  

Table 2. Amplification reaction mixture in the final volume/concentration per reaction well 
for the Ms8 specific system. 

Component 

Final 
concentration 

µl/reaction 

TaqMan® Universal PCR Master Mix (2x) 

KVM085 For primer (10 μM) 

HCA048 Rev primer (10 μM) 

TM011 Probe (10 μM) 

Nuclease free water 

Template DNA (max 200 ng) 
Total reaction volume: 

1x 

400 nM 

400 nM 

200 nM 

# 

# 

12.5 

1 

1 

0.5 

5 

5 

25 

3.  Mix gently and centrifuge briefly.  

4.  Prepare  two  reaction  tubes  (one  for  the  Ms8  and  one  for  the CruA  master  mix)  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 

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(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  the  variability  among  the  repetitions  of  each 
sample to a minimum. 

6.  Spin down the tubes in a micro-centrifuge. 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 oilseed rape Ms8/CruA systems 

Step 

Stage 

T°C 

Time 
(sec) 

Acquisition 

Cycles

1 

2 

3 

UNG 

50 °C 

120 

Initial denaturation 

95 °C 

600 

Amplification 

Denaturation 

95 oC 

Annealing & 
Extension 

60 oC 

15 

60 

No 

No 

No 

Yes 

1 

1 

45 

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.  Ms8)  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). 

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c) Save the settings 

d)  Repeat  the  procedure  described  in  a)  and  b)  on  the  amplification  plots  of  the  other 
system (e.g. CruA 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  ΔCt-curve  is  generated  by  plotting  the  ΔCt-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 ΔCt-curve formula is used to estimate the relative amount (%) 
of Ms8 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) 
•  Microcentrifuge 
•  Micropipettes 
•  Vortex  
•  Rack for reaction tubes 
•  1.5/2.0 ml tubes 

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4.2  Reagents  

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

4.3  Primers and Probes 

Oligonucleotide DNA Sequence (5’ to 3’) 

Name 
Ms8 target sequence 
KVM085 
HCA048 
TM011 (Probe) 
Reference gene CruA target sequence 
MDB510 
MDB511 
TM003 (Probe)  VIC 5’ – AGT CCT TAT GTG CTC CAC TTT CTG GTG CA -3’ TAMRA 

5’ – GTT AGA AAA AGT AAA CAA TTA ATA TAG CCG G -3’ 
5’ – GGA GGG TGT TTT TGG TTA TC -3’ 
FAM 5'- AAT ATA ATC GAC GGA TCC CCG GGA ATT C -3' TAMRA 

5’ – GGC CAG GGT TTC CGT GAT -3’ 
5’ – CCG TCG TTG TAG AAC CAT TGG -3’ 

5. References  

Arumuganathan, K., Earle, E.D. (1991). Nuclear content of some important plant species. Plant Mol. 

Biol. Reporter 9, 208-218. 

11/11

 
 
 
 
 
 
 
 
 
 
 
 
 
CRLVL07/04XP corrected version 1 

Seeds Sampling and DNA Extraction  
of Oilseed Rape 

 Report on the Validation of an Oilseed Rape 
DNA Extraction Method from Seeds 

Biotechnology & GMOs Unit 
Institute for Health and Consumer Protection 
DG Joint Research Centre 

Method development and single laboratory validation: 
  Bayer BioScience N.V. 

Method testing and confirmation: 
European Commission, Directorate General-Joint Research Centre  
Biotechnology & GMOs Unit  

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Content 

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

2. DESCRIPTION OF THE METHODS .....................................................................4 

3. EQUIPMENT/CHEMICALS/PLASTICWARE ........................................................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 ...................11 

6. CONCLUSION ..................................................................................................15 

7. QUALITY ASSURANCE .....................................................................................15 

8. REFERENCES ...................................................................................................15 

9. ABBREVIATIONS .............................................................................................15 

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1. Introduction 

A plant DNA extraction protocol is described here as derived from the publicly available “Dellaporta” 
method  (1).  The  modified  protocol  can  be  used  for  extraction  of  DNA  from  seeds  and  grains  of 
oilseed rape ground to powder using a mortar and pestle.  

These protocols are recommended to be executed by skilled laboratory personal as the procedures 
comprise working with hazardous chemicals and materials. It is strongly advised to take particular 
notice of all product safety recommendations and guidelines. 

2. 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; 

•  USDA-GIPSA 

(2001)  Sampling  grains 

for 

the  detection  of  Biotech  grains 

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

Scope and applicability: 
The  “Oilseed  rape  seed  DNA  extraction  protocol”  method  for  DNA  extraction  described  below  is 
suitable for the isolation of genomic DNA from a wide variety of oilseed rape tissues and derived 
matrices.  However,  validation  data  presented  here  are  restricted  to  ground  oilseed  rape  seeds. 
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,  concurrently  or  subsequently,  purifying  the  DNA  from  PCR 
inhibitors. The “Oilseed rape seed DNA extraction protocol” method starts with a lysis step (thermal 
lysis  in  the  presence  of  Tris  HCl,  EDTA,  SDS  and  β-mercaptoethanol)  followed  by  removal  of 
contaminants such as lipophilic molecules and proteins by extraction with phenol and chloroform.  

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A  DNA  precipitate  is  then  generated  by  using  isopropanol.  The  pellet  is  dissolved  in  TE-buffer. 
Remaining inhibitors are removed by an anion exchange chromatography step using the DNA Clean 
& Concentrator 25 kit (Zymo Research). 

Seed crushing procedure:  

(cid:131)  Collect 11 grams of seeds (+/- 3000 seeds) 
(cid:131)  Crushed the seeds in a 200 ml mortar using a pestle 
(cid:131)  Grind thoroughly till all seeds are pulverized and a fine powder is obtained 

Oil Seed Rape seed DNA extraction protocol 

1.  Transfer 11 grams of grounded seeds into a 250 ml centrifuge bucket 
2.  Add 150 ml Extraction Buffer 
3.  Add 10.5 ml 20% SDS, mix well by inversion 
4.  Incubate at 65oC for 40 minutes. Note: mix samples every 10 minutes by inversion 
5.  Centrifuge 10 minutes at 10.000 x g 
6.  Filter the supernatant over a Miracloth filtration membrane 
7.  Transfer 10 ml filtered supernatant to a new 15 ml Falcon tube 
8.  Add 3 ml 5M KAc, shake vigorously for 1 minute 
9.  Incubate on ice for 30 minutes. Note: mix samples every 10 minutes by inversion 
10.  Centrifuge for 30 minutes at 3.000 x g 
11.  Transfer 8 ml supernatant to a new 15 ml Falcon tube using a 10 ml pipette 
12.  Add 6 ml isopropanol, mix gently for one minute 
13.  Incubate on ice for 5 minutes 
14.  Centrifuge for 15 minutes in a table centrifuge at 3.000 x g 
15.  Remove  supernantant  and  air-dry  the  pellet  at  37oC  until  all  isopropanol  residue  is 

evaporated 

16.  Dissolve the pellet in 1 ml TE 
17.  Transfer the DNA solution to a new 2.0 ml Eppendorf tube 
18.  Add 10 μl RNase A (10 mg/ml), mix gently and incubate for 15 minutes at 37oC 
19.  Add 800 μl phenol:chloroform:isoamylalcohol (25:24:1) 
20.  Mix well for 1 minute 
21.  Centrifuge for 10 minutes in a microcentrifuge at maximum speed 
22.  Transfer the upper aqueous phase (900 μl) to a new 2 ml Eppendorf tube. Do not disturb 

the interphase 

23.  Add 800 μl chloroform 
24.  Mix well for 1 minute 
25.  Centrifuge for 10 minutes in a microcentrifuge at maximum speed 
26.   Transfer the upper aqueous phase (800 μl) to a new 2 ml Eppendorf tube containing 90 μl 

3M NaAc. Do not disturb the interphase 

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27.  Add 600 μl isopropanol 
28.  Mix gently by inversion for 1 minute 
29.  Centrifuge for 1 minute in a microcentrifuge at maximum speed to pellet the DNA 
30.  Remove all supernantant 
31.  Add 1 ml 70% ethanol to wash the DNA pellet. Make sure the pellet is not attached to the 

bottom. Shake the samples for 1 hour 

32.  Centrifuge for 5 minutes in a microcentrifuge at maximum speed 
33.  Remove supernatant and air-dry the pellet at 37 oC until all ethanol residue is evaporated  
34.  Add 100 μl TE 0.1X to the DNA pellet 
35.  Store samples over night at 4 oC 
36.  Shake samples for 1 hour 

37.  Centrifuge for 1 minute in a microcentrifuge at maximum speed 

38.  Purify  the  DNA  samples  using  the  DNA  Clean  &  Concentrator  25  kit  according  to  the 

manufacturer’s instruction 

39.  Elute the DNA from the column twice using two times 100μl TE 0.1X 

3. Equipment/Chemicals/Plasticware 

3.1. Equipment 

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

1.  200 ml mortar and pestle 
2.  Sorval  RC-5B  Superspeed  Centrifuge  with  SLA-1500  rotor  (or  equivalent)  for  250  ml 

centrifuge buckets 

3.  Miracloth filtration membrane (Calbiochem Cat. No. 475855) 
4.  Microcentrifuge with 18.000 x g for Eppendorf tubes 
5.  Table centrifuge (swinging buckets) with 3000 x g for 15 ml Falcon tubes 
6.  Water bath adjustable to 65 oC ± 1 oC 
7.  UV spectrophotometer for DNA quantification 

3.2. 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) 

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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.  β-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-isoamylalcholol (25/24/1) (Sigma Cat. No. P-3803) 
12.  Chloroform p.a. (Merck Cat. No. 1.02445.2500) 

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

1.  Extraction buffer (Dellaporta buffer) 
•  100 mM Tris HCl pH 8.0 
•  50 mM EDTA pH 8.0 
•  500 mM NaCl 
•  10 mM β-mercaptoethanol  

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

3.  Tris-EDTA buffer (TE 0.1X) 
•  1 mM Tris HCl pH 8.0 
•  0.1 mM EDTA pH 8.0 

4.  RNase A (10 mg/ml) 

5.  SDS 20 % 

6.  Ethanol 70% 

7.  5M KAc 

8.  3M NaAc pH 5.2 

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3.3. Plasticware 

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

Item 

15 ml conical tubes 

2 ml microcentrifuge tube 

filter tips 

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

Experimental  data  are  provided  from  an  in-house  validation  in  which  the  analytical  module  has 
been successfully applied to the relevant matrix in the context of the application for authorisation. 
Four  samples  of  11  grams  (approximately  3000  Oilseed  rape  seeds)  were  grounded  to  fine 
powders, and all flour was taken from each ground sample for DNA extraction.  

The procedure was performed three times under repeatability conditions (within short intervals of 
time (i.e. days) by the same operator, using the same equipment) resulting in 12 DNA samples. 

4.1. DNA concentration, yield, repeatability  

The  concentration  of  the  extracted  DNA  was  determined  spectrophotometrically  (Ultrospec  2000, 
Pharmacia  Biotech).  20  μl  of  extracted  DNA  was  diluted  1:10  in  MilliQ  water.  Absorption  was 
measured for both blank (TE 0.1X diluted in MQ water) and diluted DNA solutions at 260 nm.  

DNA  concentration  was  calculated  based  on  the  assumption  that  an  OD  of  1  corresponds  to  50 
μg/ml DNA (2).  

Results are summarised in the following Table: 

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CRLVL07/04XP corrected version 1 

DNA 
extraction 

Sample 
mass (gr) 

[DNA] 
(ng/ul) 

DNA extraction efficiency (ug 
DNA/11 gram meal) 

1A 

2A 

3A 

4A 

1B 

2B 

3B 

4B 

1C 

2C 

3C 

4C 

11 

11 

11 

11 

11 

11 

11 

11 

11 

11 

11 

11 

50 

65 

58 

63 

69 

55 

53 

59 

65 

67 

55 

64 

10.0 

13.0 

11.6 

12.6 

13.8 

11.0 

10.6 

11.8 

13.0 

13.4 

11.0 

12.8 

The average DNA extraction efficiency was 12.1 + 1.2 μg DNA / 11 g sample.  

4.2. Analysis of DNA fragmentation 

Analysis  of  DNA  fragmentation  was  performed  by  ethidium  bromide-stained  1%  agarose  gel 
electrophoresis  compared  to  a  series  of  standard  (non  restriction  enzyme  digested)  lambda  DNA 
references  of  different  molecular  weight.  The  12  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  12  genomic  DNA  samples  showed  indications  of 
significant degradation (‘smearing’). 

1

2

3

4

5

6

7

8

9

0
1

1
1

2
1

2
  1

5
2

5

.

5

.

7
3

5

.

2
6

0
5

A
N
D
-
λ
g
N

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4.3. Evidence of the absence of PCR-inhibitory compounds 

Presence of PCR-inhibitory compounds in the DNA preparations was tested by real-time PCR using 
the  oligonucleotides  directed  to  the  endogenous  control  gene  Cruciferine  gene  (cruA)  on  serial 
dilutions  of  the  DNA  preparations.  The  threshold  cycle  (Ct)  values  of  a  real-time  PCR  analysis 
between the Ct values corresponding to the dilutions should match the dilution factor applied, e.g. 
if DNA is diluted 10X then the ΔCt should be approx. 3.32, if the DNA is diluted 2X, the ΔCt should 
be 1, etc.  

Deviations  from  this  relationship  may  indicate  that  the  extracted  DNA  contains  PCR  inhibitors,  or 
that the DNA solution is not homogenous. This relationship was used to analyse the serial dilutions 
of  the  DNA  preparations  for  the  presence  of  PCR  inhibitory  compounds  by  plotting  the  mean  CT 
values against the logarithm of the DNA mass, and determining the slope (PCR efficiency) and the 
linearity of the correlation. 

A two fold serial dilution of the extracted DNA was prepared yielding 8 different amounts of DNA 
(1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128) of which the Ct values were determined in triplicate 
in a real-time PCR run with the cruA oligonucleotide primers and probe. In total, the analysis was 
executed three times with DNA samples extracted on different days. To analyse the data, the mean 
Ct  values  (y  axis)  were  plotted  against  the  logarithm  of  the  DNA  mass  (x  axis),  and  by  linear 
regression  a  trend  line  (y  =  ax  +  b)  was  calculated,  as  well  as  a  correlation  coefficient,  r2,  as  a 
measure  of  linearity.  The  ideal  slope  value  a  (optimal  PCR  efficiency)  then  becomes  a  =  -3.32 
(typically a values between -3.1 and -3.6 indicate optimal 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  results  of  the  three  real-time 
PCR  runs  are  shown  in  the  table  below  and  show  no  evidence  of  the  presence  of  PCR-inhibitory 
compounds.  The  PCR  efficiency  has  been  calculated  by  the  following  equation:  Efficiency  (%)  = 
100 * [10(-1/slope)]-1 

DNA 

Slope, a 

Efficiency (%) 

Intercept, b 

Linearity, r2

1A 

1B 

1C 

-3.4400 

-3.4741 

-3.4975 

95.3 

94.0 

93.2 

29.469 

29.588 

30.059 

0.9986 

0.9978 

0.9962 

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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 method of DNA extraction 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 “Oilseed rape seed DNA extraction protocol” proposed by the applicant 
on samples of food and feed consisting of ground oilseed rape seeds provided by the applicant. 

To assess the suitability of the 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  300  g  of  oilseed  rape  seed  material  were  ground  using  a  GRINDOMIX  GM  200  (Retsch 
GmbH) mixer.  

5.1 DNA extraction 

DNA  was  extracted  following  the  “Oil  Seed  Rape  seed  DNA  extraction  protocol”  described  above 
and in-house validated by the applicant. 
The  DNA  extraction  was  carried  out  on  6  test  portions  (replicates)  and  repeated  over  three 
different days, giving a total of 18 DNA extracts. 

5.2 DNA concentration, yield, repeatability 

DNA  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 (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) is reported in the 
table below. 

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Sample 

1 
2 
3 
4 
5 
6 
1 
2 
3 
4 
5 
6 
1 
2 
3 
4 
5 
6 

Concentration 
(ng/μl) 
25.4 
33.7 
64.4 
36.2 
83.6 
37.5 
35.4 
30.1 
21.0 
36.2 
25.5 
28.1 
37.2 
50.8 
31.6 
43.8 
56.1 
45.1 

DNA concentration (ng/μl) 

Overall average of all samples:    
Standard deviation of all samples  

Coefficient of variation   

40.1  ng/μl 
15.5 ng/μl 
38.7 % 

Yield (total volume of DNA solution: 3600 μl) 

Overall average of all samples:    
Standard deviation 

Coefficient of variation   

8.0 μg 
3.1 μg 
38.7 % 

5.3 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.  In  yellow  boxes  samples  extracted  on  day  1,  in 
green  boxes  samples  extracted  on  day  2,  and  in  blue  boxes  samples  extracted  on  day  3.  A  DNA 
ladder 1kb (M) was used. 

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M

1

2

3

4

5

6

1

2

3

4

5

6

1

2

3

4

5

  M
6

High molecular weight DNA was observed for all samples 

5.4 Purity / Absence of PCR inhibitors 

To  assess  the  PCR  quality  of  the  DNA  extracted,  the  experimental  approach  previously  described 
(see paragraph 4.3) was followed.  
The Ct values obtained for the undiluted and diluted samples are reported in the table below. 

Ct values of undiluted and fourfold serially diluted DNA extracts after amplification of 
rapeseed Cruciferine gene, cruA 

Undiluted 
(40 ng/µl) 

Diluted 

DNA extract 
1 
2 
3 
4 
5 
6 

1 
2 
3 
4 
5 
6 

1 
2 
3 
4 
5 
6 

1:1 

22.06 

22.01 

20.90 

21.92 

20.86 

21.49 

21.70 

21.69 

22.35 

21.45 

21.94 

21.73 

21.25 

20.89 

22.30 

21.10 

21.56 

21.03 

1:4 

24.09 

23.94 

22.63 

23.62 

22.80 

23.49 

23.54 

23.89 

24.44 

23.21 

23.74 

23.53 

23.22 

22.69 

24.51 

23.12 

23.88 

22.91 

1:16 

25.98. 

26.05 

24.80 

25.69 

24.73 

25.51 

25.64 

26.09 

26.64 

25.36 

25.79 

25.86 

25.19 

24.57 

26.31 

24.87 

25.79 

24.96 

1:64 

28.28 

28.05 

26.78 

28.08 

27.01 

27.78 

27.69 

28.16 

28.84 

27.35 

27.96 

27.74 

27.44 

26.65 

28.34 

27.13 

28.08 

26.90 

1:256 

30.33 

30.34 

29.04 

30.39 

29.33 

30.08 

29.93 

30.34 

30.88 

29.26 

30.10 

29.61 

29.59 

28.83 

30.70 

29.48 

30.27 

28.98 

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Note: only samples 3 and 5 from the first extraction (yellow boxes), and samples 2, 4, 5, 6 from 
the  third  extraction (blue boxes)  were diluted  to  the  concentration  of  40  ng/ul.  All  other  samples 
with a measured DNA concentration lower than 40 ng/ul, were tested undiluted. 

The table below summarises the comparison of extrapolated Ct values versus measured Ct values 
for all samples, as well as reporting the values of linearity (R2) and slope of all measurements. 

Comparison of extrapolated Ct values versus measured Ct values (amplification of 
rapeseed Cruciferine gene, cruA) 

DNA 
extraction 

1 

2 

3 

4 

5 

6 

1 

2 

3 

4 

5 

6 
1 

2 

3 

4 

5 

6 

R2
0.998 
0.998 
0.999 
0.997 
0.997 
0.998 
0.999 
0.998 
0.999 
0.998 
0.999 
0.994 
0.998 
0.996 
0.944 
0.994 
0.997 
0.998 

Slope 
-3.49 
-3.52 
-3.52 
-3.77 
-3.64 
-3.66 
-3.53 
-3.56 
-3.57 
-3.34 
-3.53 
-3.35 
-3.55 
-3.41 
-3.42 
-3.54 
-3.56 
-3.35 

Ct extrapolated 
21.91 
21.80 
20.51 
21.27 
20.49 
21.20 
21.39 
21.76 
22.33 
21.27 
21.59 
21.65 
21.02 
20.56 
22.32 
20.82 
21.64 
20.90 

mean Ct 
measured 
22.06 
22.01 
20.90 
21.92 
20.86 
21.49 
21.70 
21.69 
22.35 
21.45 
21.94 
21.73 
21.25 
20.89 
22.30 
21.10 
21.56 
21.03 

 ΔCt 
0.15 
0.21 
0.39 
0.65 
0.36 
0.28 
0.31 
0.08 
0.02 
0.18 
0.35 
0.08 
0.23 
0.33 
0.02 
0.29 
0.08 
0.13 

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 delta Ct values of extrapolated versus measured Ct are < 0.5, with one exception, the sample 
number 4 of the first extraction with a value of 0.65. 
R2 of linear regression is > 0.99 for all DNA samples, except one (0.944). 

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6. Conclusion 

The data reported confirm that the extraction method provides DNA of suitable quantity and quality 
for subsequent PCR based detection applications. The method is therefore applicable to the samples 
of  food  and  feed  provided  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  its  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  S,  Wood  J  and  Hicks  JB.  [1983]  Plant  DNA  mini  preparation.  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. 

9. Abbreviations 

EDTA 
PCR 
RNase A  
TE 
Tris 

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

Community Reference Laboratory for GM Food and Feed 

Seeds Sampling and DNA Extraction of Oilseed Rape  

15/15 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
European Commission 

EUR 22917EN – DG Joint Research Centre, Institute for Health and Consumer Protection 
Title: Event-specific Method for the Quantification of Oilseed Rape Line Ms8 Using Real-time PCR 
Validation Report and Protocol - Seeds Sampling and DNA Extraction of Oilseed Rape 
Authors: M. Mazzara, A. Bogni, C. Savini and G. Van den Eede 
Luxembourg: Office for Official Publications of the European Communities 
2007 – 46 pp. – 21 x 29,7 cm  
EUR - Scientific and Technical Research series; ISSN 1018-5593 
ISBN 978-92-79-06934-5 

Abstract 

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF) (see Regulation 
EC 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 Ms8 transformation event in oilseed rape DNA (unique identifier ACS-BN(cid:31)(cid:31)5-8). 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  with  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% oilseed rape Ms8 event). The JRC 
prepared the validation samples (calibration samples and blind samples at unknown GM percentage 
[DNA/DNA]). The collaborative trial involved thirteen laboratories from eleven European Countries. 

The  results  of  the  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  under  http://gmo-crl.jrc.it/.  The 
method  will  also  be  submitted  to  ISO  21570  for  consideration  as  an  international  standard..

 
 
 
 
 
 
 
 
 
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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