Verification of performance of Ms8 and Rf3 event-specific methods on the hybrid Ms8xRf3 using real-time PCR validation report.
Date: 2007-12-19
Institutions: Institute for Health and Consumer Protection
Subjects: biotechnology, consumer protection, evaluation method, genetically modified organism, oil seed rape, oleaginous plant, research method

Verification of Performance of Ms8 and Rf3 
Event-specific Methods on the Hybrid Ms8xRf3 
Using Real-time PCR 

Validation Report and Protocol 

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

15 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 22929 EN

 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The mission of the Institute for Health and Consumer Protection is to provide scientific support to the 
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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 22929 EN 
ISBN 978-92-79-06986-4 
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© European Communities, 2007 

Reproduction is authorised provided the source is acknowledged 

Printed in Italy 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CRLVL07/05VR 

Verification of Performance of Ms8 and Rf3 
Event-specific Methods on the Hybrid Ms8xRf3 
Using Real-time PCR 

Validation Report 

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

15 January 2007 

Executive Summary 

The  JRC  as  Community  Reference  Laboratory  for  GM  Food  and  Feed  (CRL-GMFF)  (see 
Regulation  EC  No  1829/2003),  has  carried  out  an  in-house  verification  study  to  assess  the 
performance  of  two  quantitative,  event-specific  methods  for  use  with  the  hybrid  oilseed  rape 
line  (unique  identifier  ACS-BN∅∅5-8xACS-BN∅∅3-6)  which  combines  both  the  Ms8  and  Rf3 
transformation  events.  Both  methods  have  been  previously  fully  validated  individually  on 
parental lines, to detect and quantify each insert on extracted DNA. The study 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  methods  and  the  samples  (genomic  DNA  extracted  from  the  wild-type  and  100% 
event  Ms8xRf3  hybrid  oilseed  rape).  The  JRC  prepared  the  in-house  validation  samples 
(calibration samples and blind samples at unknown GM percentage).  

The  results  of  the  in-house  verification  study  were evaluated  with  reference  to  ENGL  method 
performance  requirements  (http://gmo-crl.jrc.it/doc/Method%20requirements.pdf)  and  to  the 
validation results for the individual parental lines (http://gmo-crl.jrc.it/statusofdoss.htm). 

The  results  of  CRL-GMFF  in-house  verification  studies  are  publicly  available  at  http://gmo-
crl.jrc.it/.  

Community Reference Laboratory for GM Food and Feed 
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Report on Steps 1-3 of the Validation Process 

CRLVL07/05VR 

Bayer CropScience submitted the detection methods and control samples for the hybrid oilseed 
rape  containing  the  stacked  events  Ms8xRf3  (unique  identifier  ACS-BN∅∅5-8xACS-BN∅∅3-6) 
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 
http://gmo-
CRL-GMFF 
(“Description 
crl.jrc.it/guidancedocs.htm). 

Validation 

Process”, 

the 

of 

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/doc/Method%20requirements.pdf  )  (see  Annex  1  for  a  summary 
of  method  acceptance  criteria  and  method  performance  requirements).  During  step  2,  two 
scientific assessments were performed and a request of complementary information addressed 
to the applicant. Upon reception of complementary information, the scientific evaluation of the 
detection  method  for  the  hybrid  oilseed  rape  Ms8xRf3  was  positively  concluded  in  October 
2005.  

The  event-specific  detection  methods  for  the  two  oilseed  rape  lines  hosting  the  single  events 
Ms8  and  Rf3  were  validated  by  the  CRL-GMFF  following  the  conclusion  of  the  respective 
international  collaborative  ring  trials  and  the  publication  of  the  respective  validation  reports 
(http://gmo-crl.jrc.it/statusofdoss.htm).  Hence,  the  detection  methods  for  the  hybrid  oilseed 
rape Ms8xRf3 did not undergo a full validation process. The CRL-GMFF performed an in-house 
verification  of  the  detection  methods  to  verify  that  they  exhibit a  comparable performance on 
hybrid  samples  combining  both  traits  (as  provided  in  accordance  to  Annex  1.2.C.2  of 
Commission Regulation (EC) No 641/2004). 

In  July  2006,  the  CRL-GMFF  experimentally  verified  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  DNA  copy  number  basis.  The  experiments  were  performed  under 
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. 

Community Reference Laboratory for GM Food and Feed 
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CRLVL07/05VR 

Contents 

1. 

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

2.  MATERIALS........................................................................................................ 6 

3.   EXPERIMENTAL DESIGN.................................................................................... 7 

4.  METHOD............................................................................................................. 7 

DESCRIPTION OF THE OPERATIONAL STEPS.......................................................................... 7 

5.  DEVIATIONS REPORTED ................................................................................... 7 

6. 

SUMMARY OF RESULTS ..................................................................................... 8 

PCR EFFICIENCY AND LINEARITY ...................................................................................... 8 

7.  METHOD PERFORMANCE REQUIREMENTS........................................................ 9 

8. 

COMPARISON OF METHOD PERFORMANCE BETWEEN HYBRID AND PARENTAL 
LINES............................................................................................................... 10 

9. 

CONCLUSIONS................................................................................................. 11 

10.  QUALITY ASSURANCE ..................................................................................... 11 

11.   REFERENCES.................................................................................................... 11 

12.   ANNEX 1: METHOD ACCEPTANCE CRITERIA AND METHOD PERFORMANCE 

REQUIREMENTS AS SET BY THE EUROPEAN NETWORK OF GMO 
LABORATORIES (ENGL)................................................................................... 12 

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1.  Introduction 

Bayer CropScience submitted both detection methods (for Ms8 and Rf3) and control samples 
for the hybrid oilseed rape hosting the stacked events Ms8xRf3 (unique identifier ACS-BN∅∅5-
8xACS-BN∅∅3-6)  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  Directorate  General  Joint  Research  Centre  (JRC,  Biotechnology  and  GMOs  Unit  of  the 
Institute  of  Health  and  Consumer  Protection)  as  Community  Reference  Laboratory  for  GM 
Food  and  Feed  (see  Regulation  EC  1829/2003)  carried  out  an  in-house  verification  of  both 
event-specific  methods  for  the  detection  and  quantification  of  Ms8  and  Rf3  in  the  hybrid 
oilseed  rape  line  combining  the  two  traits  derived  through  traditional  breeding  techniques 
between progeny of the parental Ms8 and Rf3 oilseed rape lines. The single methods had been 
previously validated by international collaborative trial on the single parental lines (http://gmo-
crl.jrc.it/statusofdoss.htm).  

Upon reception of methods, samples and related data (step 1), the CRL-GMFF carried out the 
assessment of the documentation (step 2) and the in-house evaluation of the methods (step 
3),  according  to  the  requirements  of  Regulation  (EC)  641/2004  and  following  CRL-GMFF 
operational procedures. 
The CRL-GMFF method verification was performed 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 in-house verification included the following module: 

(cid:57)  Quantitative real-time PCR (Polymerase Chain Reaction). The methodology consists of two 
event-specific real-time quantitative TaqMan® PCR procedures for the determination of the 
relative content of event Ms8 and Rf3 DNA to total oilseed rape DNA in the hybrid line. The 
procedure  is  a  simplex  system,  in  which  an  oilseed  rape  (OSR) CruA  gene  (Cruciferin A) 
endogenous assay (reference gene) and the target assays (Ms8 or Rf3) are performed in 
separate wells.  

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

(cid:57)  ISO 5725:1994 (1).  
(cid:57)  The  IUPAC  “Protocol  for  the  design,  conduct  and  interpretation  of  method-performance 

studies” (2). 

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2.  Materials 

For the verification of the quantitative event-specific methods, control samples consisting of a 
DNA stock solution (Lot number 32RRMM0128) extracted from leaves of plants harbouring the 
Ms8XRf3  events  in  hemizygous  state,  and  genomic  DNA  (Lot  number  32RRMM0101)  from 
leaves of wild type plants genetically similar to the GM-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%  Ms8xRf3  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.  

The  protocols  (reagents,  concentrations,  primer/probe  sequences,  amplification  profile) 
followed  in  the  in-house  verification  are  as  those  already  published  as  validated  methods  for 
the individual Ms8 and Rf3 events.  

Table 1 shows the five levels of unknown samples used in the verification of the Ms8 and Rf3 
methods on extracted hybrid Ms8xRf3 DNA. 

Table 1.  Ms8 and Rf3 GM contents in Ms8xRf3 

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

Rf3 GM % 
(GM copy number/oilseed rape genome copy 
number *100) 
0.1 
0.4 
0.9 
1.8 
3.6 

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3.   Experimental design 

Eight  runs  for  each  event-specific  method  were  carried  out.  In  each  run,  samples  were 
analysed in parallel with both the GM-specific system and the CruA reference system. Five GM-
levels per run were examined and two replicates for each GM level were analysed. PCR analysis 
was  performed  in  triplicate  for  all  samples.  On  the  whole,  for  each  method  (Ms8  and  Rf3), 
quantification  of  the  five  GM  levels  was  performed  as  an  average  of  sixteen  replicate 
samples/GM-level. An Excel spreadsheet was used for determination of GM%. 

4.  Method 

Description of the operational steps 

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. Similarly, for specific detection of event Rf3 genomic DNA, a 139-bp fragment of the 
recombination region of parts of the construct inserted into the plant genome is amplified using 
two specific primers. Both 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 
reporter dye at its 5′ end and TAMRA as a quencher dye at its 3′ end. 

For relative quantification of event Ms8 and Rf3 DNA, an OSR-specific reference system which 
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, was used.  

For  relative  quantification  of  events  Ms8  and  Rf3  DNA  in  a  test  sample,  the  normalised  ∆Ct 
values  of  calibration  samples  (denominated  from  S1  to  S5)  are  used  to  calculate,  by  linear 
regression, a reference curve (plotting ∆Ct values against the logarithm of the amount of event 
DNA). The normalised ∆Ct values of the unknown samples are measured and, by means of the 
regression formula, the relative amount of Ms8 or Rf3 event DNA is estimated. 

Calibration  samples  for  the  preparation  of  the  standard  curve,  denominated  from  S1  to  S5, 
were  prepared  by  mixing  the  appropriate  amount  of  Ms8xRf3  DNA  from  the  stock  solution  in 
non-GM control oilseed rape DNA to obtain the following relative contents of Ms8xRf3: 3.60%, 
1.80%,  0.90%,  0.45%  and  0.09%.  The  total  DNA  amount  was  200  ng,  when  5  µl  per 
reaction/well was used (40 ng/µl).  

For detailed information on the preparation of standard curve calibration samples please refer 

to the protocols of validated methods at http://gmo-crl.jrc.it/statusofdoss.htm 

5.  Deviations reported 

No deviations from the protocols of the two previously validated methods were introduced. 

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CRLVL07/05VR 

6.  Summary of results 

PCR efficiency and linearity 

The values of the slope of the ∆Ct-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) in 

the eight runs for both GM events are summarised in Tables 2 and 3. 

Table 2. Values of slope, PCR efficiency and linearity (R2) of the ∆Ct standard curve for the 

Ms8 method on hybrid Ms8xRf3 

Run 

Slope 

Ms8 

PCR 
Efficiency 
(%) 

Linearity 
(R2) 

1 

2 

3 

4 

5 

6 

7 

8 

-3.35 

-3.43 

-3.10 

-3.28 

-3.15 

-3.07 

-3.18 

-3.35 

Mean 

-3.24 

98.6 

95.6 

89.9 

98.4 

92.3 

88.4 

93.7 

98.6 

94.5 

0.997 

0.989 

0.997 

0.991 

0.994 

0.999 

0.995 

0.993 

0.995 

Table 3. Values of slope, PCR efficiency and linearity (R2) of the (cid:31)Ct standard curve for the 

Rf3 method on hybrid Ms8xRf3 

Run 

Slope 

1 

2 

3 

4 

5 

 6 

7 

8 

-3.184 

-3.342 

-3.685 

-3.433 

-3.508 

-3.448 

-3.458 

-3.471 

Rf3 

PCR 
Efficiency 
(%) 

Linearity 
(R2) 

93.9 

99.2 

86.8 

95.6 

92.8 

95.0 

94.6 

94.1 

0.995 

0.997 

0.987 

0.986 

0.996 

0.997 

0.990 

0.994 

Mean 

-3.441 

94.0 

0.993 

The mean PCR efficiencies were higher than 90% and the linearity of both methods (R2 value) 
was above 0.99. 
Data  reported  in  Table  2  and  3  confirm  the  appropriate  performance  characteristics  of  both 
methods tested on the hybrid material. 

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7.  Method performance requirements 

The results of the in-house verification study for Ms8 and Rf3 against hybrid Ms8xRf3 oilseed 
rape material are reported in Tables 4 and 5, respectively. Both sets of results are evaluated 
with respect to the method acceptance criteria, as established by ENGL and adopted by CRL-
GMFF  (http://gmo-crl.jrc.it/guidancedocs.htm,  see  also  Annex  1).  Further,  Tables  4  and  5 
detail estimates of accuracy and precision for each GM level and for both methods. 

Table 4. Estimates of accuracy and precision for the Ms8 method on hybrid oilseed rape 

Ms8xRf3. 

Ms8 

Unknown 
sample GM% 

Mean 
SD 
RSDr (%) 
Bias% 

0.1 

0.09 
0.01 
13 
-9.4 

Expected value (GMO %) 

0.4 

0.36 
0.04 
11 
-9.6 

0.9 

0.91 
0.10 
11 
0.9 

1.8 

1.86 
0.18 
9.5 
3.5 

3.6 

3.56 
0.27 
7.7 
-1.1 

Table 5. Estimates of accuracy and precision for the Rf3 method on hybrid oilseed rape 

Ms8xRf3. 

Rf3 

Unknown 
sample GM% 

Mean 
SD 
RSDr (%) 
Bias% 

Expected value (GMO %) 

0.1 

0.10 
0.01 
8.9 
0.0 

0.4 

0.36 
0.04 
11 
-10.5 

0.9 

0.93 
0.07 
7.7 
3.5 

1.8 

1.75 
0.20 
12 
-2.8 

3.6 

3.50 
0.32 
9.1 
-2.6 

The trueness of the method is estimated using the measures of the method bias for each GM 
level.  According  to  the  ENGL  acceptance  criteria  and  method  performance  requirements,  the 
accuracy  of  the  quantification,  measured  as  bias  from  the  accepted  value,  should  be ±  25% 
across  the  entire  dynamic  range.  As  shown  in  Tables  4  and  5,  both  methods  satisfy  such  a 
requirement throughout their respective dynamic ranges. 

Tables  4  and  5  further  document  the  relative repeatability standard deviation (RSDr) as 
estimated for each GM level. In order to accept methods for collaborative ring trial evaluation, 
the  CRL-GMFF  requires  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]). 

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As  can  be  observed  from  the  values  reported  in  Tables  4  and  5,  both  methods  satisfy  this 

requirement across their respective dynamic ranges. 

CRLVL07/05VR 

8.  Comparison  of  method  performance  between  hybrid 
and parental lines 

A  synoptic  comparison  of  the  two  method  performances  on  the  hybrid  oilseed  rape  and 

parental lines respectively is shown in Tables 6 and 7. 

Table  6.  Comparison  of  accuracy  and  precision  of  Ms8  method  on  the  hybrid  and  parental 
line. 

Accuracy and precision of Ms8 

Accuracy and precision of Ms8 

quantification on hybrid Ms8xRf3 

quantification on parental line Ms8* 

GM% 

Bias (%) 

RSDr (%) 

GM% 

Bias (%) 

RSDr (%) 

0.1 

0.4 

0.9 

1.8 

3.6 

-9.4 

-9.5 

0.9 

3.4 

-1.1 

13 

11 

10 

9.5 

7.7 

0.1 

0.4 

0.9 

1.8 

3.6 

7.4 

-3.5 

-0.9 

-1.0 

-7.5 

22 

18 

14 

17 

11 

*method validated (http://gmo-crl.jrc.it/statusofdoss.htm) 

Table 7. Comparison of accuracy and precision of Rf3 method on the hybrid and parental line. 

Accuracy and precision of Rf3 

Accuracy and precision of Rf3 

quantification on hybrid Ms8xRf3 

quantification on parental line Rf3* 

GM% 

Bias (%) 

RSDr (%) 

GM% 

Bias (%) 

RSDr (%) 

0.1 

0.4 

0.9 

1.8 

3.6 

0.0 

-10 

3.5 

-2.8 

-2.6 

8.9 

11 

7.7 

12 

9.1 

0.1 

0.4 

0.9 

1.8 

3.6 

6.9 

4.4 

4.5 

-2.4 

-5.2 

13 

12 

14 

12 

13 

*method validated (http://gmo-crl.jrc.it/statusofdoss.htm) 

Overall, the individual Ms8 and Rf3 event-specific methods show comparable performances in 
terms of accuracy and precision of quantification when applied to the single parental lines or 
to the hybrid product. 

Therefore,  the  in-house  method  verification  has  demonstrated  that  the  Ms8  and  the  Rf3 
methods  can  be  equally  applied  in  the  quantification  of  the  respective  events  in  the  hybrid 
oilseed rape product. 

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9.  Conclusions 

The  overall  method  performance  of  the  two  event-specific  methods  for  the  quantitative 
detection of events Ms8 and Rf3 combined in the hybrid Ms8xRf3, have 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), and to 
the validation results for the individual parental lines (http://gmo-crl.jrc.it/statusofdoss.htm). 

The results obtained during the present verification study indicate that the analytical modules 
of  the  methods  submitted  by  the  applicant  comply  with  ENGL  performance  criteria.  The 
methods  are  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. 

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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. 

Community Reference Laboratory for GM Food and Feed 
Validation Report Oilseed Rape Ms8xRf3 

12/14 

 
 
 
CRLVL07/05VR 

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 

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. 

Community Reference Laboratory for GM Food and Feed 
Validation Report Oilseed Rape Ms8xRf3 

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CRLVL07/05VR 

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.  

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. 

Community Reference Laboratory for GM Food and Feed 
Validation Report Oilseed Rape Ms8xRf3 

14/14 

 
 
Protocol Ms8 – Community Reference Laboratory for GM Food and Feed  

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 
3.2 

GENERAL INSTRUCTIONS AND PRECAUTIONS .............................................................. 6 
REAL-TIME PCR FOR QUANTITATIVE ANALYSIS OF MS8 OILSEED RAPE........................ 7 
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 ∆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  reference  ∆Ct-
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 

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CRLVL06/04VP 

the specific targets of maize T25, MON810, Bt11, Bt176, GA21, NK603, CBH351, Roundup 
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  ∆Ct-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  ∆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 ∆Ct-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 

Name 

Oligonucleotide DNA Sequence (5’ to 3’) 

KVM085 

HCA048 

Ms8 target sequence 

5’ – GTT AGA AAA AGT AAA CAA TTA ATA TAG CCG G -3’ 

5’ – GGA GGG TGT TTT TGG TTA TC -3’ 

TM011 (Probe) 

FAM 5'- AAT ATA ATC GAC GGA TCC CCG GGA ATT C -3' TAMRA 

Reference gene CruA t rget sequence

a

MDB510 

MDB511 

5’ – GGC CAG GGT TTC CGT GAT -3’ 

5’ – CCG TCG TTG TAG AAC CAT TGG -3’ 

TM003 (Probe)  VIC 5’ – AGT CCT TAT GTG CTC CAC TTT CTG GTG CA -3’ TAMRA 

5. References  

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

Biol. Reporter 9, 208-218. 

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Protocol Rf3 – Community Reference Laboratory for GM Food and Feed  

CRLVL07/04VP 

Event-specific Method for the Quantification of  
Oilseed Rape Line Rf3 Using Real-time PCR 

Protocol 

Method development: 
  Bayer CropScience 

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

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Protocol Rf3 – Community Reference Laboratory for GM Food and Feed  

CRLVL07/04VP 

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 RF3 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 .......................................................................................... 10 
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 Rf3 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 Rf3 genomic DNA, a 139-bp fragment of the recombination 
region  between  the  insert  and  the  plant  genome  (located  at  the  5’  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  Rf3  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 Rf3 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  reference  ∆Ct-
formula, the relative amount of Rf3 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  collaborative  trial 
using DNA samples at different GMO contents. 

2.2 Collaborative trial 

The  method  was  validated  in  an  international  collaborative  trial  organized  by  the  Joint 
Research Centre (JRC) of the European Commission, Community Reference Laboratory for 
GM Food and Feed. The study was undertaken with 13 laboratories in July-August 2006.  

Each  participant  received  twenty  unknown  samples  containing  Rf3  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  Rf3  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  Rf3  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  Rf3,  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  Rf3  oilseed 

rape 

3.2.1 General 

The  PCR  set-up  for  the  taxon  specific  target  sequence  (CruA)  and  for  the  GMO  (Rf3) 
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 Rf3 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  ∆Ct-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 Rf3 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 Rf3 specific system. 

Component 

Final 
concentration 

µl/reaction 

TaqMan® Universal PCR Master Mix (2x) 

KVM084 For primer (10 µM) 

DPA165 Rev primer (10 µM) 

TM010 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 Rf3 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 
(e.g. 5 x 3 = 15 µl DNA for three PCR repetitions). Vortex each tubes for approx. 10 

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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 Rf3/CruA systems 

Step 

Stage 

1 

2 

3 

UNG 

Initial denaturation 

Amplification 

Denaturation 

Annealing & 
Extension 

T°C 

Time 
(sec) 

50 °C 

120 

95 °C 

600 

95 oC 

60 oC 

15 

60 

Acquisition  Cycles

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.  Rf3)  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 

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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  ∆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 ∆Ct-curve formula is used to estimate the relative amount (%) 
of Rf3 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 

4.2  Reagents  

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

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4.3  Primers and Probes 

Name 

Oligonucleotide DNA Sequence (5’ to 3’) 

KVM084 

DPA165 

5’ –AGC ATT TAG CAT GTA CCA TCA GAC A-3’ 

5’ – CAT AAA GGA AGA TGG AGA CTT GAG -3’ 

Rf3 target sequence 

TM010 (Probe) 

FAM 5'-  CGC ACG CTT ATC GAC CAT AAG CCC A -3' TAMRA 

Reference gene CruA t rget sequence

a

MDB510 

MDB511 

5’ – GGC CAG GGT TTC CGT GAT -3’ 

5’ – CCG TCG TTG TAG AAC CAT TGG -3’ 

TM003 (Probe)  VIC 5’ – AGT CCT TAT GTG CTC CAC TTT CTG GTG CA -3’ TAMRA 

5. References  

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

Biol Reporter 9, 208-218. 

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European Commission 

EUR 22929 EN – DG Joint Research Centre, Institute for Health and Consumer Protection 
Title: Verification of Performance of Ms8 and Rf3 
Event-specific Methods on the Hybrid Ms8xRf3 Using Real-time PCR - Validation Report and Protocol 
Authors: M. Mazzara, E. Grazioli, C. Savini and G. Van den Eede 
Luxembourg: Office for Official Publications of the European Communities 
2007 – 40 pp. – 21 x 29,7 cm  
EUR - Scientific and Technical Research series; ISSN 1018-5593 
ISBN 978-92-79-06986-4 

Abstract 

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF) (see Regulation 
EC  No  1829/2003),  has  carried  out  an  in-house  verification  study  to  assess  the  performance  of  two 
quantitative,  event-specific  methods  for  use  with  the  hybrid  oilseed  rape  line  (unique  identifier  ACS-
BN(cid:31)(cid:31)5-8xACS-BNøø3-6)  which  combines  both  the  Ms8  and  Rf3  transformation  events.  Both 
methods have been previously fully validated individually on parental lines, to detect and quantify each 
insert  on  extracted  DNA.  The  study  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 methods 
and the samples (genomic DNA extracted from the wild-type and 100% event Ms8xRf3 hybrid oilseed 
rape). The JRC prepared the in-house validation samples (calibration samples and blind samples at 
unknown GM percentage).  

The  results  of  the  in-house  verification  study  were  evaluated  with  reference  to  ENGL  method 
performance requirements (http://gmo-crl.jrc.it/doc/Method%20requirements.pdf) and to the validation 
results for the individual parental lines (http://gmo-crl.jrc.it/statusofdoss.htm). 

The  results  of  CRL-GMFF  in-house  verification  studies  are  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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