The certification report of the genetic identification of a Hippoglossus hippoglossus (Atlantic halibut) fish powder EURM®-020.
Date: 2020-02-18
Institutions: Joint Research Centre
Subjects: fish, genetics, organic certification, research method, sea fish, testing

CERTIFICATION REPORT 

 The certification of the genetic identification  
of a Hippoglossus hippoglossus (Atlantic halibut)  
fish powder: EURM®-020

EUR 30097 EN 

 
 
  
 
 
 
 
 
European Commission  
Joint Research Centre 
Directorate F – Health, Consumers and Reference Materials 

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This  publication  is  a  Reference  Materials  Report  by  the Joint Research  Centre,  the  European  Commission’s  in-house  science  service.  It  aims  to  provide 
evidence-based scientific support to the European policy-making process. The scientific output expressed does not imply a policy position of the European 
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All images © European Union 2020 

JRC119890 

EUR 30097 EN 

ISBN 978-92-76-16230-8 (PDF) 
ISSN 1831-9424 (online) 
doi:10.2760/170544 

Luxembourg: Publications Office of the European Union, 2020 
© European Union, 2020 

Reproduction is authorised provided the source is acknowledged. 

Abstract 

This  report  describes  the  production  of  EURM-020,  a  fish  tissue  material  certified  for  the  identification  of  the  fish  species  Hippoglossus  hippoglossus 
(Atlantic halibut). This material was produced following ISO 17034:2016 and is certified in accordance with ISO Guide 35:2017. 
The material was produced from one fillet originating from a single fish which was genetically identified as Hippoglossus hippoglossus (Atlantic halibut). In 
facilitating storage, handling and shipment, the fish fillet was cut into small pieces, slowly dried using a freeze drying process and cryogenically milled to 
obtain a  fine powder.  The  powder was bottled, and  the  bottles  were  closed under  an argon atmosphere to  ensure the conservation of the  material. In 
storing the material, the bottles were placed at 4 °C. 
The  fish  species  present  in  EURM-020  was  determined  to  be  Hippoglossus  hippoglossus  (Atlantic  halibut)  via  genetic  identification  based  upon  the 
sequencing results of two specific regions of mitochondrial DNA (also called DNA barcodes): the cytochrome b gene (cytb) and the cytochrome c oxidase 
subunit I gene (COI).  
Between-unit homogeneity was established by evaluating the quality and quantity of the extracted DNA from multiple bottles. In addition, the two DNA 
barcodes  were  amplified  by  polymerase  chain  reaction  (PCR)  and  analysed  by  bi-directional  Sanger  sequencing  for  each  bottle  sampled.  The  stability 
during  dispatch  and  storage  was assessed  through  quantification  of extracted  DNA  by UV  spectrophotometry,  and  gel-electrophoresis  of the extracted 
DNA and PCR products of the two DNA barcodes. The process was conducted following ISO Guide 35:2017. 
The material was characterised based on a comparison of measurements produced by two laboratories with demonstrated competencies and adhering to 
ISO/IEC 17025. Data analysis and species identification was completed at the JRC.  
The material is certified for its identity which was verified with two different mitochondrial DNA based methods. It is unlikely that there is an alternative 
species that shares both the sequences of the cytb and COI gene barcode regions with the species in EURM-020. Consequently, there is no doubt regarding 
the  certified  identity,  Hippoglossus  hippoglossus,    for  EURM-020  and  the  uncertainties  of  the  certified  value  relating  to  inhomogeneity,  instability  and 
characterisation are negligible.  
The  material  is  intended  for  the  quality  control  or  assessment  of  method  performance.  As  with  any  reference  material,  it  can  be  used  for  validation 
studies.  EURM-020  is  available  in  glass  vials  containing  at  least  200  mg  of  dried  Hippoglossus  hippoglossus  (Atlantic  halibut)  powder.  The  minimum 

amount of sample to be used is 7 mg. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CERTIFICATION REPORT 

The certification of the genetic identification  
of a Hippoglossus hippoglossus (Atlantic halibut)  
fish powder: EURM®-020 

A.M. Kortekaas, H. Leys, J. Seghers, S. Trapmann 

European Commission, Joint Research Centre 
Directorate F – Health, Consumers and Reference Materials 
Geel, Belgium 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Disclaimer 

Certain  commercial  equipment,  instruments,  and  materials  are  identified  in  this  paper  to  specify  adequately  the 
experimental procedure. In no case does  such identification imply recommendation or endorsement by the European 
Commission, nor does it imply that the material or equipment is necessarily the best available for the purpose.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Summary 

This  report  describes  the  production  of  EURM-020,  a  fish  tissue  material  certified  for  the 
identification  of  the  fish  species  Hippoglossus  hippoglossus  (Atlantic  halibut).  This  material  was 
produced following ISO 17034:2016 [1] and is certified in accordance with ISO Guide 35:2017 [2]. 

The  material  was  produced  from  one  fillet  originating  from  a  single  fish  which  was  genetically 
identified  as  Hippoglossus  hippoglossus  (Atlantic  halibut).  In  facilitating  storage,  handling  and 
shipment, the fish fillet was cut into small pieces,  slowly  dried using a freeze drying process and 
cryogenically milled to obtain a fine powder. The powder was bottled, and the bottles were closed 
under an argon atmosphere to ensure the conservation of the material. In storing the material, the 
bottles were placed at 4 °C. 

The  fish  species  present  in  EURM-020  was  determined  to  be  Hippoglossus  hippoglossus 
(Atlantic halibut) via genetic identification based upon the sequencing results of two specific regions 
of mitochondrial DNA (also called DNA barcodes): the cytochrome b gene (cytb) and the cytochrome 
c oxidase subunit I gene (COI).  

Between-unit homogeneity was established by evaluating the quality and quantity of the extracted 
DNA from multiple bottles. In addition, the two DNA barcodes were amplified by polymerase chain 
reaction  (PCR)  and  analysed  by  bi-directional  Sanger  sequencing  for  each  bottle  sampled.  The 
stability  during  dispatch  and  storage  was  assessed  through  quantification  of  extracted  DNA  by 
UV spectrophotometry,  and  gel-electrophoresis of the extracted DNA and PCR products of  the two 
DNA barcodes. The process was conducted following ISO Guide 35:2017 [2]. 

The  material  was  characterised  based  on  a  comparison  of  measurements  produced  by  two 
laboratories with demonstrated competencies and adhering to ISO/IEC 17025 [3]. Data analysis and 
species identification was completed at the JRC.  

The  material  is  certified  for  its  identity  which  was  verified  with  two  different  mitochondrial 
DNA based  methods.  It  is  unlikely  that  there  is  an  alternative  species  that  shares  both  the 
sequences of the cytb and COI gene barcode regions with the species in EURM-020. Consequently, 
there is no doubt regarding the certified identity, Hippoglossus hippoglossus, for EURM-020 and the 
uncertainties  of  the  certified  value  relating  to  inhomogeneity,  instability  and  characterisation  are 
negligible.  

The material is intended for the quality control or assessment of method performance. As with any 
reference  material,  it  can  be  used  for  validation  studies.  EURM-020  is  available  in  glass  vials 
containing  at  least  200  mg  of  dried  Hippoglossus  hippoglossus  (Atlantic  halibut)  powder.  The 
minimum amount of sample to be used is 7 mg. 

1 

 
The following value for the nominal property was assigned: 

Fish powder  

Certified value 2) 

Uncertainty 3) 

Taxon 1) 

Hippoglossus hippoglossus 4) 
(Atlantic halibut) 

Negligible 

1) As obtained by bi-directional Sanger sequencing of two fish DNA identification barcodes, the mitochondrial cytochrome b gene 
(cytb) and the mitochondrial cytochrome c oxidase subunit I gene (COI) and subsequent comparison with reference sequences from 
GenBank® (NCBI), FishTrace (EC) and the Bar Code of Life Database System (BOLD).  
2) Certified values are values that fulfil the highest standards of accuracy. The given value represents the consensus value of 
accepted  sets  of  data,  with  each  set  obtained  by  a  different  laboratory  or  with  a  different  method  of  determination.  The 
certified  value  is  traceable  to  reference  DNA  sequences  in  the  GenBank  (NCBI),  FishTrace  (EC)  and  BOLD  databases.  The 
identity at the species level, was defined as a minimum of 99 % similarity with the DNA sequences of the specimens (voucher 
samples, specimens with morphological identification) deposited in the GenBank (NCBI), FishTrace (EC) or BOLD databases.  
3) The identity was verified with two different (genetic) methods. It is unlikely that there is an alternative species that shares the 
sequences of these independent genes. Consequently, there is no doubt regarding the certified identity, Hippoglossus hippoglossus, 
for EURM-020 and its related uncertainty is negligible.  
4) Hippoglossus hippoglossus (Linnaeus, 1758) 

2 

 
 
 
 
 
 
 
Table of contents 

Summary............................................................................................................................... 1 
Table of contents ................................................................................................................. 3 
Glossary ................................................................................................................................ 5 
Introduction .............................................................................................................. 7 
1 
Background ............................................................................................................... 7 
1.1  
Choice of the material ................................................................................................ 8 
1.2 
Design of the project .................................................................................................. 8 
1.3 
Participants ............................................................................................................... 8 
2 
Project management and evaluation .......................................................................... 8 
2.1 
Provider of raw material and processing .................................................................... 8 
2.2 
Homogeneity study .................................................................................................... 8 
2.3 
Stability study ............................................................................................................ 8 
2.4 
Characterisation ......................................................................................................... 9 
2.5 
Material processing and process control .................................................................. 9 
3 
Origin/Purity of the starting material ........................................................................... 9 
3.1 
Processing ................................................................................................................. 9 
3.2 
Process control .........................................................................................................10 
3.3 
Homogeneity ............................................................................................................10 
4 
4.1 
Between-unit homogeneity........................................................................................11 
4.2  Within-unit homogeneity and minimum sample intake...............................................12 
Stability....................................................................................................................12 
5 
Short-term stability study ..........................................................................................13 
5.1 
Long-term stability study ...........................................................................................14 
5.2 
Characterisation ......................................................................................................14 
6 
Selection of participants ............................................................................................14 
6.1 
6.2 
Study setup ...............................................................................................................14 
6.3  Methods used ...........................................................................................................15 
6.4 
Evaluation of results .................................................................................................16 
6.4.1  Technical evaluation ....................................................................................................16 
6.4.2  Evaluation of the genetic identification for the cytb barcode ...........................................17 
6.4.3  Evaluation of the genetic identification for the COI barcode ............................................18 
Value Assignment ....................................................................................................21 
7 
Metrological traceability and commutability ..........................................................22 
8 
8.1  Metrological traceability ............................................................................................22 
Commutability ...........................................................................................................22 
8.2 
Instructions for use .................................................................................................23 
9 
Safety information .....................................................................................................23 
9.1 
Storage conditions ....................................................................................................23 
9.2 
9.3 
Preparation and use of the material/Reconstitution ...................................................23 
9.4  Minimum sample intake ............................................................................................23 
Use of the certified value ..........................................................................................23 
9.5 
Acknowledgements ..................................................................................................24 
10 
References ...............................................................................................................25 
11 
Annexes ...............................................................................................................................28 

3 

 
4 

 
 
 
Glossary 

BLAST 

BOLD 

CEN 

COI 

CRM 

cytb 

DNA 

EC 

EDTA 

ERM® 

EU 

EURM 

ILC 

ISO 

IU 

JRC 

Basic Local Alignment Search Tool 

Barcode of Life Data system 

European Committee for Standardization 

Mitochondrial cytochrome c oxidase 1 gene 

Certified reference material 

Mitochondrial cytochrome b gene 

Deoxyribonucleic acid 

European Commission 

Ethylenediaminetetraacetic acid 

Trademark of European Reference Materials 

European Union 

European Reference Materials 

Interlaboratory comparison 

International Organization for Standardization  

International units 

Joint Research Centre of the European Commission 

mtDNA 

Mitochondrial DNA 

n 

N 

n.a. 

NCBI 

PCR 

PSA 

QA 

QC 

RM 

RMP 

rpm 

RT 

Number of replicates per unit 

Number of samples (units) analysed 

Not applicable 

National Center for Biotechnology Information 

Polymerase chain reaction 

Particle size analysis 

Quality assurance 

Quality control 

Reference material 

Reference material producer 

Revolutions per minute 

Room temperature 

RM Unit 

Reference Materials Unit of Directorate F 

s 

sextracts 

SI 

smeas 

Standard deviation 

Standard deviation of the extracts 

International System of Units 

Standard deviation of the measurement data 

5 

 
 
 
SOP 

sx̄ bottle 

T 

TaqMan® 

TE 

TRIS 

UV 

VIM 

Standard Operating Procedure  

Standard deviation of the mean of bottles  

Temperature 

Thermus aquaticus (Taq) DNA polymerase-based technology for fluorescent 
signal generation during real-time PCR 

Buffer containing TRIS and EDTA 

Tris(hydroxymethyl)aminomethane 

Ultraviolet 

International Vocabulary of Metrology – Basic and General Concepts and 
Associated Terms [ISO/IEC Guide 99:2007] 

V-KFT  

Volumetric Karl Fischer titration 

6 

 
 
 
 
1 

Introduction   

1.1   Background 

To  improve  transparency  in  the  fishery  and  aquaculture  supply  chain,  Europe  has  implemented 
Regulation  No.  1379/2013  on  the  common  organisation  of  markets  for  fishery  and  aquaculture 
products.  Article  35  sets  out  the  rules  on  the  mandatory  marking  or  labelling  of  pre-packed  or 
non-prepacked  fishery  and  aquaculture products.  For  example  indicating  the commercial  name  of 
the species and its scientific name, the production method, the area where the product was caught 
or farmed, the category of fishing gear used in capturing fisheries, whether the product has been 
defrosted and date of minimum durability [4].   

Labelling  of  fish  species  may  be  inadvertently  wrong  during  identification;  however,  deliberate 
mislabelling for financial gain occurs when species of higher value are substituted with lower value 
species. Additionally, species from unsustainable or illegal fisheries may enter the market through 
substitution [5]. 

Inspections  of  the  quality  and  authenticity  of  fresh  or  prepared  fish  products  on  the  market  are 
conducted to discourage operators from falsely labelling catches or products.  These inspections are 
carried  out  following  European  regulations  such  as  Regulation  No. 1224/2009  which  sets  out  a 
control regime, comprising the monitoring and inspection of the whole chain of fishery production 
and  marketing [6]  and  Regulation No.  178/2002, which covers the labelling of fishery  products to 
avoid misleading consumers and the traceability of food products [7]. 

The current technique used for the genetic identification of fish products is based on the sequencing 
of  specific  genes/regions  of  mitochondrial  DNA.  Mitochondrial  DNA  (mtDNA)  has  a  high  mutation 
rate,  and  as  a  consequence,  significant  amounts  of  sequence  variation  can  be  observed  between 
closely  related  species  [8].  Specific  genes  or  parts  of  genes  on  the  mtDNA  that  are  reliable  for 
species  identification  are  called  DNA  barcodes.  In  addition,  mtDNA  is  present  in  cells  at  higher 
concentrations compared to nuclear DNA and is, therefore, the preferred template [9].  

The high mutation rate of mtDNA can also lead to sequence variations within one species, and the 
use of specific barcodes for species identification can only be reliable if the within-species variation 
is less than the between-species variation. The gap in the distribution of distances between species 
is named the “barcoding gap”. For the identification of fish species, the mitochondrial cytochrome b 
gene (cytb) and the mitochondrial cytochrome c oxidase subunit I gene (COI) have been identified as 
reliable  barcodes  [10,  11].  Although,  genetic  identification  using  a  single  barcode  might  lead  to 
misidentification because two different species share the sequence of the barcode, it is unlikely that 
there  are  two  different  distinctive  species  that  share  the  sequences  on  several  independent 
genes [12]. 

Publicly  available  species-specific  DNA  sequences,  used  as  reference  for  the  identification  of  fish 
samples are available in databases such as Bar Code for Life Database System (BOLD), GenBank® 
(i.e.  the  National  Center  for  Biotechnology  Information's  (NCBI)  genetic  sequence  database)  and 
FishTrace  (i.e.  a  genetic  and  taxonomic  reference  database  for  species  identification  of  European 
commercial  marine  fish  species  from  the  European  Commission  (EC)).  They  all  contain  data  from 
“voucher specimen”, species identified via morphological and genetic methods, which are curated by 
museums  in  reference  collections.  With  the  exception  of  FishTrace,  the  databases  also  contain 
DNA sequences  of  samples  not  connected  to  reviewed  publications  and  may not  all  have  a  direct 
link to morphological identification. 

In  developing  and  validating  DNA  related  identification  methods,  laboratories  can  request  tissue 
aliquots of voucher specimens from  museums, although not an easy procedure. In 2018, the JRC 
decided  to  produce  reference  materials  for  fish  identification  of  commercially  important  fish 
species, which can be used for the validation and quality control of genetic identification methods 

7 

 
for fish species. The reference material EURM-020 is a fish powder of Hippoglossus hippoglossus 
(Atlantic halibut) and is characterised based on the genetic identification by two DNA barcodes, cytb 
and COI. After extraction of the DNA, parts of both genes have been amplified by polymerase chain 
reaction  (PCR)  and  sequenced  using  bi-directional  Sanger  sequencing.  The  EURM-020  material  is 
produced as a fish powder which can be stored at 4 °C and dispatched under cooled conditions. 

1.2  Choice of the material 

EURM-020  is  a  fish  powder  prepared  from  a  single  fillet  of  Hippoglossus  hippoglossus 
(Atlantic halibut).  It  is  a  dried  powder  to  facilitate  storage,  handling  and  shipment.  After 
reconstitution, the material closely resembles samples of fresh fish fillet.  

1.3  Design of the project 

The  certification  of  the  fish  species  present  in  EURM-020  was  based  upon  genetic  analysis 
performed  by  two  independent  laboratories,  selected  for  their  expertise  in  DNA  sequencing.  Both 
laboratories  analysed  two  DNA  barcodes,  known  to  be  reliable  for  fish  identification,  by 
PCR amplification  and  subsequent  bi-directional  Sanger  sequencing.  The  results  of  the  two 
independent laboratories were used for the certification of the genetic identification of the species 
which  is  a  result  of  the  unambiguous  data  of  the  accepted  sets  of  data,  each  set  obtained  in  a 
different  laboratory  or  with  a  different  method  of  determination.  The  traceability  chain  of  the 
genetic  identification  was  linked  to  reference  sequences  from  both  voucher  specimens  and  peer 
reviewed publications in the BOLD, GenBank (NCBI) and FishTrace (EC) databases. 

The material is certified for its identity. The material originates from a single fish and the identity 
was verified by two different genetic methods, therefore, there is no doubt regarding the identity. 
As a consequence, the uncertainties of the certified value relating to inhomogeneity, instability and 
characterisation are negligible. 

2 

Participants 

2.1  Project management and evaluation 

European  Commission,  Joint  Research  Centre,  Directorate  F  –  Health,  Consumers  and  Reference 
Materials, Geel, BE 
(accredited to ISO 17034:2016 for production of certified reference materials, BELAC No. 268-RM) 

2.2  Provider of raw material and processing 

European  Commission,  Joint  Research  Centre,  Directorate  F  –  Health,  Consumers  and  Reference 
Materials, Geel, BE  
(accredited to ISO 17034:2016 for production of certified reference materials, BELAC No. 268-RM) 

2.3  Homogeneity study 

European Commission, Joint Research Centre, Directorate F – Health, Consumers and Reference 
Materials, Geel, BE 

Eurofins Genomics Europe Applied Genomics GmbH, Ebersberg, DE 
(cytb measurements under scope of ISO/IEC 17025:2005 accreditation, DAkkS, D-PL-13372-01-00) 

2.4  Stability study 

European  Commission,  Joint  Research  Centre,  Directorate  F  –  Health,  Consumers  and  Reference 
Materials, Geel, BE 

8 

 
 
 
 
 
2.5  Characterisation 

European  Commission,  Joint  Research  Centre,  Directorate  F  –  Health,  Consumers  and  Reference 
Materials, Geel, BE 

Eurofins Genomics Europe Applied Genomics GmbH, Ebersberg, DE 
(Cytb measurements under scope of ISO/IEC 17025:2005 accreditation, DAkkS, D-PL-13372-01-00) 

SYNLAB Analytics & Services United Kingdom Ltd, Cramlington, UK 

3 

Material processing and process control 

3.1  Origin/Purity of the starting material 

The JRC purchased one fish of the species, Hippoglossus hippoglossus (Atlantic halibut) from a local 
fishmonger. The fish was filleted, packed, labelled and stored at -20 °C until use. Confirmation of 
identity and an assessment of purity were performed during the characterisation and homogeneity 
studies.  Genetic  identification  by  bi-directional  Sanger  sequencing  using  two  DNA  barcodes,  cytb 
and  COI,  confirmed 
this  EURM  was 
Hippoglossus   hippoglossus.  Since  only  a  single  fish  was  used  the  fish powder  produced  could be 
considered  pure.  This  was  confirmed  by  good  quality  electropherograms  generated  during  the 
characterisation and homogeneity studies.     

the  species  used 

identity  of 

that 

the 

for 

3.2  Processing 

During processing, contamination of the final EURM-020 material with foreign DNA was avoided by 
treating  all  contact  surfaces  with  DNA  degrading  solution  (DNA-Erase™,  MP  Biomedicals,  Irvine, 
CA, USA)  prior  to  use  and  by  using  clean  laboratory  clothing.  An  in-house  validation  study  had 
previously proven that the solution degraded DNA effectively under the conditions used. 

The Hippoglossus hippoglossus (Atlantic halibut) single fillet was thawed and the skin removed. The 
fillet was then cut into pieces of approximately 1 cm3 and about 1.5 kg of fillet cubes were freeze-
dried on trays using a program that started at -40 °C and increased gradually to 25 °C. The total 
freeze-drying process lasted 55 h (Epsilon 1-6D, Martin Christ, Osterode, DE). The dried cubes were 
then stored in stainless steel drums over liquid nitrogen prior to being cryogenically milled using a 
Palla  VM-KT  cryogenic  vibrating  mill  (KHD,  Köln,  DE).  The  resultant  powder  was  transferred  in  a 
plastic bag, placed in an airtight container and temporarily stored at 4 °C. The water content of the 
powder was further reduced by drying for 20 h under vacuum in the freeze-dryer at 20 °C, with an 
additional  20  h  under  vacuum  at  25  °C.  The  powder  was  then  manually  sieved  over  a  1000  μm 
stainless steel mesh and the remaining powder mixed in a DynaMIX CM200 (WAB, Muttenz, CH) for 
1 h  to homogenise.  The  final  powder,  about  354  g, was  packed  in  a plastic  bag  and  stored  in  an 
airtight container at 4 °C.  

The residual water mass fraction of the fish powder was 18.0 ± 1.6 g/kg, measured by volumetric 
Karl  Fischer  titration  (V-KFT,  841  KFD  Titrando,  Metrohm,  Herisau,  CH),  (N  =  1,  n =  3),  with  the 
expanded uncertainty calculated using a coverage factor k = 2.  

The  particle  size  distribution  of  the  powder  was  measured  based  on  the  deconvoluted  laser 
diffraction patterns (PSA, Sympatec, Clausthal-Zellerfeld, DE). The cumulative volume distribution of 
the particles derived from laser diffraction data was based on the equivalent spherical diameter, i.e. 
the  diameter  of  the  particles  derived  from  the  volume  occupied  upon  their  rotation.  The  mean 
particle diameter of the powder material was 94.7 μm ± 6.6 μm (s). 

A  feeder,  FD  –  SPAc  4A  (MCPI,  Meythet,  France),  was  used  to  fill  5  mL  amber  glass  bottles  with 
approximately 200 mg of the fish powder. Lyophilisation inserts were placed in the bottle-necks, the 
bottles were placed in a freeze-dryer to provide an argon atmosphere, (Epsilon 1-6D, Martin Christ, 
Osterode, DE)  and  were  closed inside  the  freeze-dryer.  Capping  was executed  using  an HV  100  B 

9 

 
 
semi-automatic  capping  machine  (Bausch  &  Ströbel,  Ilshofen,  DE)  while  labelling  was  completed 
manually. Each of the bottles was identified by a numbered label indicating the EURM code and the 
unit number according to filling order. Following an inventory and the selection of bottles for future 
analysis, according a random stratified sampling scheme, the remaining bottles were stored in the 
dark at 4 ± 3 °C. 

3.3  Process control  

To determine the residual mass fraction of water in EURM-020, 30 bottles were randomly selected 
for the V-KFT measurement. To reach the sample intake for V-KFT measurements, the content from 
3  individual  subsequent bottles  from  the  random  scheme,  was  used  for  one  V-KFT  measurement 
(N =  10,  n  =  1).  The  water  mass  fraction  of  EURM-020  was  33.4  ±  1.9  g/kg  with  the  expanded 
uncertainty calculated using a coverage factor k = 2. The associated expanded uncertainty (U) has 
been estimated during validation of the V-KFT method on fish tissue.  

The  particle  size  distribution  in  EURM-020  was  determined  based  on  the  deconvoluted  laser 
diffraction pattern. Five randomly selected bottles covering the whole EURM-020 production batch 
were  analysed  in  triplicate  (N  =  5,  n  =  3)  and  no  particles  larger  than  860  μm  were  detected 
(Figure 1).  The  mean  particle  diameters  and  standard  deviation  of  the  mean,  measured  by  laser 
diffraction, was 104.3 μm (sx̄ = 8.8 μm).  

]

%

l

[
n
o
i
t
c
a
r
f
e
m
u
o
v
e
v
i
t
a
u
m
u
C

l

100

90

80

70

60

50

40

30

20

10

0

1

10

100

1000

Equivalent sphere diameter [μm]

Figure  1:  Volume-based  cumulative  distribution  of  equivalent  sphere  diameters  in  EURM-020 
analysed  by  laser  diffraction  (N  =  5,  n  =  3).  The  total  particle  volume  for  the  material  is  set  as 
100 %. 

4 

Homogeneity 

A key requirement for any reference material aliquoted into units is the equivalence between those 
units. The complete batch of the EURM-020 was produced from a single fish and it can therefore be 
concluded that the certified species identity is valid for all the units. The RM is intended to be used 
as  a  control  material  for  genetic  identification  techniques  for  fish  species.  The  suitability  of 
EURM-020  depends  on  the  quality  of  the  DNA  that  can  be  extracted.  A  homogeneity  study  was 
carried out to confirm that DNA of sufficient quality can be extracted from all the units. 

10 

 
 
 
 
 
4.1  Between-unit homogeneity 

The number of bottles selected corresponds to approximately the cube root of the total number of 
bottles  produced.  Consequently,  10  bottles  were  selected  using  a  random  stratified  sampling 
scheme covering the whole batch for the between-unit homogeneity test. For this study, the batch 
was divided into 10 groups (with a similar number of bottles) and one bottle was selected randomly 
from each group.  

The  DNA  extraction  and  the  DNA  quality  checks  were  carried  out  at  the  JRC.  Two  independent 
replicates of between 7 to 8 mg were weighed into 2 mL low-DNA-binding microcentrifuge tubes. 
The replicates were divided over two extraction series using a randomised order. The material was 
reconstituted in 160 μL nuclease-free water (Promega) and vortexed for 10 seconds. Immediately, 
the DNA  was  extracted  following the  Animal  Tissue Protocol  from  the  DNeasy  Blood  &  Tissue  Kit 
(QIAGEN  Benelux  BV,  Antwerpen,  BE).  The  incubation  time  was  30  minutes,  which  was  found  to 
produce  sufficient  extracted  DNA  during  the  extraction  method  validation.  At  the  end  of  the 
extraction the DNA was eluted in a single eluate of 100 μL of buffer AE. In checking the integrity of 
the DNA, gel-electrophoresis was conducted using a Gel-red pre-stained 1 % Agarose gel (PowerPac 
3000 Electrophoresis Power Supply, Biorad). Some degree of DNA fragmentation was observed for 
all  the  samples,  but  the  quality  of  the  DNA  was  sufficient  for  PCR  amplification  and  Sanger 
sequencing of the targeted DNA barcodes of 413 bp and 652 bp (expected length of the nucleotides 
between the primers respectively for the targets of the cytb and COI genes). The average extracted 
DNA  concentration  measured  by  UV  spectrophotometry  (ND-1000  Spectrophotometer,  NanoDrop 
Technologies Inc., Wilmington, USA) was 128.0 ng/μL (sextracts = 25.7 ng/μL, sx̄bottles = 17.5 ng/μL) and 
was sufficient  to  provide template material for  PCR amplification and  sequencing. The  results  per 
bottle are summarised in Figure 1 in Annex B1. 

The samples were diluted in TE low buffer (pH 8,1 mmol/L Tris and 0.01 mmol/L EDTA) to the target 
DNA concentration (20 ng/μL) and shipped on dry ice to the laboratory (secton 2.3) that performed 
the  bi-directional  Sanger  sequencing  of  the  mitochondrial  cytb  and  COI  genes  after  the  PCR 
amplification and purification.  

The  primers  used  for  PCR  amplification  and  Sanger  sequencing  of  the  cytb  region  were  taken 
directly from the publication by Wolf, Burgerer et al. [10]. The primers used for the COI amplification 
and sequencing were provided by the standard operating procedure for the genetic identification of 
fish species using DNA barcoding (mitochondrial cytochrome c oxidase subunit I sequencing) from 
the Labelfish Consortium [13, 14, 15]. For PCR amplification of the COI barcode, a cocktail of two 
forward  primers  or two reverse  primers was used in  the respective master-mix  compositions.  The 
M13-tail  primers  were  used  for  the  Sanger  sequencing  of  the  COI  barcode. (Primers  for  cytb  and 
COI are described Annex A1). The measurements were performed under repeatability conditions and 
in a randomised manner.  

All  sequencing  data  were  analysed  at  the  JRC  and  sequencing  results  for  the  two  barcodes  (cytb 
and COI) were analysed separately. Initially, the electropherograms were analysed for their quality 
using  the  SeqManPro  application  within  Lasergene  15.1  software  (DNASTAR,  Madison,  US)  [37]. 
Primer sequences and regions of low quality at either end of the electropherogram were removed 
and  ambiguous  bases  and  no  base  calls  were  checked  against  the  sense  or  anti-sense  sequence 
and manually corrected where possible. Forward and reverse sequences were aligned per extraction 
to obtain a consensus sequence.  

The  20  cytb  consensus  sequences  were  compared  to  a  cytb  barcode  reference  sequence  for 
Hippoglossus hippoglossus (EU513792.1 in GenBank (NCBI) [16]) using the SeqManPro application 
within  the  Lasergene  15.1  software  (DNASTAR,  Madison,  US)  [37].  For  cytb,  15  of  20  sequences 
covered the complete length (413 bp) of the barcode sequence for Hippoglossus hippoglossus. The 
subsequent five sequences were somewhat shorter due to poorer quality base calls at the extremes 
of the barcodes (384, 395, 397 (2) and 405 bp). No sequence differences were observed between 

11 

 
the samples, however, one mismatch was observed between  all  the 20 consensus  sequences  and 
the reference sequence.  

The 20 COI consensus sequences were compared with the COI reference sequence for Hippoglossus 
hippoglossus (GBMTG1322.16 in BOLD (mined from NC_009709.1 in NCBI GenBank [17, 36])). The 
COI  barcode  sequence  has  a  length  of  652 bp.  None  of  the  20  sequences  obtained  covered  the 
complete length of the COI barcode, however, all sequences produced ranged between 618  bp  to 
645  bp.  No  sequence  differences  were  observed  between  the  sample  sequences,  however,  one 
mismatch was observed between the 20 consensus sequences and the reference sequence. 

The results of the homogeneity study showed that DNA of sufficient quality could be extracted from 
samples  contained  within  each  of  the  selected  bottles.  The  sequencing  results  produced  were  of 
good  quality,  indicating  no  significant  contamination  with  foreign  DNA  within  each  of  the  bottles 
sampled. The sequences of the extraction replicates and the consensus sequences per bottle have 
been  identified  as  Hippoglossus  hippoglossus  at  both  barcode  positions  sequenced,  cytb  and  COI. 
The  between-bottle  homogeneity  and  within-unit  homogeneity  is  confirmed  by  the  identical 
consensus  sequences  and  the  sequence  identity  results  of  above  99  %  with  the  Hippoglossus 
hippoglossus  reference  sequences  in  GenBank  (NCBI)  for  the  cytb  barcode  using  Basic  Local 
Alignment  Search  Tool  BLAST®  software,  (version  BLASTN  2.9.0+,  NCBI,  National  Library  of 
Medicine, Bethesda, US) [18, 19] and above 99 % similarity results with the reference sequences for 
the COI barcode using the Barcode of Life Data Identification Systems v4 (BOLD) [20].  

The results of the BLAST and BOLD searches are summarised in Table 1 to 2 in Annex B.  

4.2  Within-unit homogeneity and minimum sample intake 

The EURM-020 material is a pure powder that was produced from a fillet originating from a single 
fish and is therefore homogenous. A meaningful minimum sample intake at which the within-unit 
inhomogeneity  no  longer  contributes  to  analytical  variation  can  therefore  not  be  established. 
Homogeneity,  characterisation  and  stability  measurements  were  performed  with  a  sample  intake 
between 7 and 8 mg and were found to produce DNA concentrations fit for PCR amplification and 
Sanger sequencing, therefore, the minimum sample intake is 7 mg. 

5 

Stability 

Time,  temperature,  light  (including  ultraviolet  radiation)  and  water  content  were  regarded  as  the 
most  relevant  influences  on  the  stability  of  the  reference  materials.  The  impact  of  ultraviolet  or 
visible light was minimised by storing the material in brown glass bottles that reduce light exposure. 
In addition, materials are stored in the dark and dispatched in boxes, thus removing any possibility 
of  degradation  by  light.  The  water  content  was  adjusted  to  an  optimum  during  processing. 
Consequently, only the influences of time and temperature had to be investigated. 

Stability testing is necessary to establish the conditions for storage (long-term stability) as well as 
the  conditions  for  dispatching  the  materials  to  customers  (short-term  stability).  During  transport, 
especially  in  the  summer, temperatures  of up  to 60  °C  can be  reached,  and  stability  under  these 
conditions  must  be  demonstrated  if  the  samples  are  to  be  transported  without  any  additional 
cooling. 

The  stability studies were carried out using an isochronous design [34].  In this approach, samples 
were  stored  for  a  given  length  of  time  at  different  temperature  conditions.  At  the  specified  time 
points,  the  samples  were  moved  to  conditions  where  further  degradation  can  be  assumed  to  be 
negligible (reference conditions). At the end of the isochronous storage, the samples were analysed 
simultaneously  under  repeatability  conditions.  Analysis  of  the  material  (after  various  exposure 
times  and  temperatures)  under  repeatability  conditions  greatly  improves  the  sensitivity  of  the 
stability tests.  

12 

 
EURM-020 is certified for its genetic identity. As a result, stability behaviour cannot be investigated 
using  a  quantitative  method  and  consequently,  the  inability  to  estimate  stability  uncertainties. 
However, an indication of stability was obtained through a smell-test of the powder upon opening 
the  bottle  and  analysing  the  concentration  of  the  extracted  DNA  and  using  gel-electrophoresis  to 
examine the quality of the DNA in the extracts. Additionally, the quality of the extracted DNA was 
tested  by  performing  PCR  using  the  cytb  and  COI  PCR  methods  and  subsequently  comparing  the 
length  of  the  obtained  PCR  products  for  each  method  with the  expected  PCR  product  lengths.  All 
stability testing was conducted at the JRC and methods for DNA extraction and PCR were validated 
at the JRC. 

5.1  Short-term stability study 

 For the short-term stability study, samples were stored at 4 °C, 18 °C and 60 °C for 0, 1, 2 and 4 
weeks (at each temperature). The reference temperature was set at -70 °C. Two units per storage 
time and temperature, were selected using a random stratified sampling scheme. When the stability 
sample bottles  were  opened,  the  smell  of  the  fish powder  was  described  and  compared  with  the 
smell  of  the  samples  stored  at  the  reference  temperature.  From  each  bottle,  two  replicates  of 
between  7  to  8  mg  were  weighed  into  2  mL  low-DNA-binding  microcentrifuge  tubes  and  were 
reconstituted in 160 μL nuclease-free water (Promega) and vortexed for  10  seconds. Immediately 
following  reconstitution,  the  DNA  was  extracted  by  following  the  Animal  Tissue  Protocol  of  the 
DNeasy Blood & Tissue Kit (QIAGEN Benelux BV, Antwerpen, BE). The  incubating  time  was kept at 
20   minutes,  incubation  times  between  20  and  30  minutes  were  found  to  produce  sufficient 
extracted DNA  during the extraction method validation. At the end of the extraction  the DNA  was 
eluted  in  a  single  eluate  of  100 μL of  buffer  AE.  The  extracted  DNA  concentration  was measured 
Inc., 
using  UV-spectrophotometry 
Wilmington, USA). The DNA integrity was assessed by gel-electrophoresis on a Gel-red pre-stained 
1   %  Agarose  gel  (PowerPac  3000  Electrophoresis  Power  Supply,  Biorad).  PCR  product  size  was 
assessed through, cytb and COI specific PCR (C1000 Touch Thermal Cycler, Bio-Rad, NL), using the 
methods  described  in  Annex  A.2  and  A.3.  PCR  product  sizes  were  estimated  by  comparison  to  a 
100   bp  DNA  ladder  (Invitrogen)  using  gel-electrophoresis.  The  measurements  were  performed 
under  repeatability  conditions,  the  sample  replicates  were  extracted  on  separate  occasions,  and  a 
randomised sample sequence was used.  

(ND-1000  Spectrophotometer,  NanoDrop  Technologies 

The  data  were  evaluated  individually  for  each  temperature.  The  short-term  stability  study  results 
for  samples  stored  at  4  °C  and  18  °C  showed  that  these  samples  were  stable  at  these 
temperatures.  The  results  of  the  smell  test  on  the  powder  after  the  opening  of  the  bottle,  the 
concentration  of  extracted  DNA  measured  by  UV  spectrophotometry,  gel-electrophoresis 
assessment  of  extracted  DNA  and  the  cytb  and  COI  PCR  products  did  not  indicate  matrix  or 
DNA degradation. The  samples  stored at 4 ˚C and 18 ˚C showed similar results with the samples 
stored  at  the  reference  temperature  -70  °C.  However,  samples  stored  at  60  °C  were  found  to  be 
unstable at this temperature. These samples smelt different in comparison to samples stored at the 
reference temperature -70 °C, after opening, indicating decomposition of the fish powder at 60 °C. 
This was confirmed by the significant decrease in the concentration of extracted DNA from samples 
stored  at  60 °C  for  one  week  and  the  further  decline  in  the  samples  stored  for  four  weeks. 
Gel-electrophoresis  of 
reduced  DNA  concentrations. 
Gel-electrophoresis of the cytb and COI PCR products still showed bands at the expected sizes, the 
bands were weaker suggesting poor amplification of the degraded template material. 

the  DNA  extracts  also  showed 

Extracted DNA concentration results measured by UV spectrophotometer for each temperature are 
shown  in  Figures  1-3  of  Annex  C.  The  results  of  the  smell  test,  DNA  quality  tests  and  gel-
electrophoresis are not presented. 

The results of the short-term stability study showed that there was significant degradation of the 
material at 60 °C but no significant degradation was observed at 18 °C and below, therefore, cooled 
shipment is necessary to maintain sample integrity.  

13 

 
5.2 

Long-term stability study 

For  the  long-term  stability  study,  samples  were  stored  at  -20  °C  and  4  °C  for  0,  3,  6,  9  and 
12 months  (at  each  temperature)  and  the  isochronous  reference  temperature  was  -70  °C.  Two 
samples  per  storage  time  and  temperature  were  selected  using  a  random  stratified  sampling 
scheme. All the samples were assessed for the smell of the fish powder after opening. From each 
bottle  the  DNA  of  two  replicates  was  extracted  and  DNA  concentration  and  gel-electrophoresis 
assessment  of  extracted  DNA  and  cytb  and  COI  PCR  products  were  performed  as  described  in 
Section 5.1.  

The  long-term  stability  data  were  evaluated  individually  for  each  temperature.  The  results  of  the 
smell test, the extracted DNA concentration, gel-electrophoresis of the extracted DNA and the cytb 
and  COI  PCR  products,  showed  that  for  all  time  points,  the  tested  temperature  conditions 
(4 °C and  -20  °C)  showed  no  significant  difference  to  the  material  stored  at  the  reference 
temperature.  The higher variability  of  the  extracted  DNA  concentration  at  time  point  6  months  at 
4 °C was considered as an artefact. It can therefore be concluded that long-term storage at -20˚C 
or 4˚C has no detrimental impact on the material. 

The  results  of  extracted  DNA  concentration  measured  by  UV  spectrophotometer  for  each 
temperature of the 12-month stability study are shown in Figures 1 and 2 of Annex D. The results 
of the smell test, DNA quality tests and gel-electrophoresis are not presented. 

The  EURM-020  bottles  are  closed  under  an  argon  atmosphere,  and  the  long-term  stability  test 
showed  that  the  matrix  and  DNA  are  stable  when  stored  at  -20  °C  and  4  °C.  The  material  can, 
therefore, be stored at 4 °C. The stability of the material after opening has not been tested.  

After  the certification  study, the material will  be included in the  JRC's regular stability  monitoring 
programme, to further characterise the stability of the material. 

6 

Characterisation  

The  material  characterisation  is  the  process  of  determining  the  property  value  of  the  reference 
material. 

This was based on a comparison between two expert laboratories, i.e. the property of the material 
was  determined  in parallel  in  different  laboratories  to  demonstrate  the  absence  of  measurement 
bias. Due  to the  nature  of  the  analyte;  however,  both  participants  used  PCR  amplification  and  bi-
directional Sanger sequencing method for the measurements.  

6.1  Selection of participants  

Two  laboratories  were  selected  based  on  criteria  that  comprised  both  technical  competence  and 
quality management aspects. Each participant was required to operate a quality system. Having a 
formal  accreditation  was  not  mandatory,  but  meeting  the  requirements  of  ISO/IEC  17025  was 
obligatory.  Where  measurements  are  covered  by  the  scope  of  accreditation,  the  accreditation 
number is stated in the list of participants (Section 2). 

6.2  Study setup  

The sample preparations, DNA extraction and extracted DNA quality checks, were all performed at 
the  JRC.  DNA  extracts  were  sent  to  both  laboratories  that  independently  performed  cytb  and  COI 
PCR amplification and Sanger sequencing (bi-directional). The final result (assignation of taxon), is 
not influenced through the use of a common DNA extraction site, hence the datasets are considered 
independent.  

14 

 
For  the  characterisation  study,  both  laboratories  received  two  DNA  extracts  from  each  of  five 
bottles of EURM-020. They were requested to provide two independent results, two per bottle and 
thus  two  per  method  (cytb  and  COI)  per  bottle.  The  five  bottles  for  the  material  characterisation 
were selected using a random stratified sampling scheme and covered the whole batch of reference 
material. For one laboratory, the five bottles for the characterisation study were selected from the 
homogeneity  samples  as  these  had  been  selected  using  the  same  random  stratified  sampling 
scheme and therefore reduced the number of samples required and the overall costs of the project. 
The certified value for this EURM is the assigned identification which is a qualitative property. For 
such  cases,  it  is  not  necessary  to  follow  an  intermediate  precision  sample  scheme  (replicates 
measured  on  different  days).  Consequently,  the  sample  preparation  and  measurements  were 
completed under repeatability conditions, in a randomised order in a single day. 

6.3  Methods used 

DNA extraction and extracted DNA quality checks were all performed at the JRC. From each bottle 
two  replicates  of  between  7  to  8  mg  fish  powder  were  weighed  into  2  mL  low-DNA-binding 
microcentrifuge  tubes.  The  replicates  were  divided  over  two  extraction  series  using  a  randomised 
order.  The  material  was  reconstituted  in  160  μL  nuclease-free  water  (Promega)  and  vortexed  for 
10 seconds.  Immediately  after  reconstitution, the  DNA  was  extracted,  following  the  Animal  Tissue 
Protocol from the DNeasy Blood & Tissue Kit (QIAGEN Benelux BV, Antwerpen, BE). The incubating 
time  was  30  minutes.  During  in-house  validation  of  the  extraction  method  an  incubation  time 
between  20  and  30  minutes  proved  to  produce  sufficient  DNA  for  PCR  amplification  and 
sequencing.  The  DNA  was  eluted  in  a  single  eluate  of  100  μL  of  buffer  AE.  The  extracted 
DNA concentration  was  measured  by  UV-spectrophotometry 
(ND-1000  Spectrophotometer, 
NanoDrop  Technologies  Inc.,  Wilmington,  USA)  and  results  are  shown  in  Figure  1,  Annex  E2.  The 
average  DNA concentration  of  the  20  extracts  was  128.1  ng/μL  (sextracts  =  22.6  ng/μL, 
sx̄bottles = 15.5 ng/μL). The DNA integrity was checked by gel-electrophoresis on a Gel-red pre-stained 
1 % Agarose gel (PowerPac 3000 Electrophoresis Power Supply, Biorad). Although some degree of 
DNA  fragmentation  was  observed  for  all  samples,  the  quality  of  the  DNA  was  sufficient  for 
PCR  amplification and Sanger sequencing of the targeted DNA barcodes of 413 bp and 652 bp. The 
samples were diluted with TE low buffer (pH 8,1 mmol/L Tris and 0.01 mmol/L EDTA) to the target 
DNA concentration and shipped on dry ice conditions to the laboratories listed in Section 2.3. 

For the PCR amplification and sequencing both laboratories used the cytb primers as described in 
Table 1 in Annex A1 [10]. The laboratories used the COI primers as described in Table 2 in Annex A1 
[13, 14, 15], according to the Standard Operating Procedure (SOP) for the genetic identification of 
fish  species  using  DNA  barcoding  (mitochondrial  cytrochrome  1  sequencing)  prepared  by  the 
Labelfish Consortium in 2016. For the COI PCR amplification, one laboratory used a cocktail of the 
two  COI  forward  or  two  COI  reverse  primers  in  the  forward  and  reverse  PCR  master-mix 
compositions  respectively,  while  the  second  laboratory  prepared  four  different  PCR  master-mix 
compositions  using  single  COI  primers.  The  PCR  chemistry  used  and thermocycler  conditions  were 
slightly deviating from the SOP for both laboratories.  

Successful  amplification  of  the  cytb  and  COI  barcode  regions  were  confirmed  by  either  gel-
electrophoresis or capillary electrophoresis of the PCR products. The purified PCR products of cytb 
and  COI  were  bi-directionally  Sanger  sequenced  by  each  of  the  laboratories  using  their  own 
sequencing protocols. 

All  sequencing  data  were  analysed  at  the  JRC  using  the  SeqManPro  application  within  Lasergene 
15.1  software  (DNASTAR,  Madison,  US)  [37].  The  data  for  the  two  barcodes  (cytb  and  COI)  were 
analysed separately. 
The  resulting  consensus  sequences  were  uploaded  to  GenBank's  BLAST  [18,  19]  and  BOLD  [20] 
identification  tools  for  genetic  identification  of  the  fish  powder.  The  reference  sequences  for  the 
cytb barcode in the alignment hit table in the output from BLAST were checked for voucher status, 
related  publication  and  morphological  identification.  Reference  sequences  that  had  suspicious 
genetic  and  morphological  identification  were  no  longer  used  for  identification.  The  reference 

15 

 
sequences  in  the  hit  list  from  the  BOLD  identification  tool  for  the  COI  barcode  were  checked 
similarly.  

For  each  barcode,  cytb  and  COI,  the  consensus  sequences  per  extract  were  aligned  per  bottle  to 
obtain  one  sequence  per  bottle.  A  consensus  sequence  per  laboratory  was  aligned  from  the  five 
consensus  sequences  per  barcode.  A  final  consensus  sequence  for  the  cytb  and  the  COI  barcode 
was aligned with the sequences from both the laboratories and are given in FASTA format in Annex 
E1.   

6.4 

Evaluation of results 

The characterisation study resulted in two datasets for each of the cytb and COI barcode, thus four 
datasets in total. The taxon assignment results obtained by the JRC by analysing the data from the 
participants, grouped per laboratory and barcode method are displayed in Table 1 in Annex E1. 

6.4.1  Technical evaluation 

The  sequencing  data  obtained  from  each  laboratory  were  first  checked  for  compliance  with  the 
requested analysis protocol and for their validity based on technical reasons. The following criteria 
were considered during the evaluation: 

- Valid PCR-sequencing procedure 
- Compliance with the analysis protocol: analytical sequence according to the        
prepared scheme 
- PCR products showing a single band of about 464 and 738 bp in size for cytb and 
COI respectively as visualised on a gel 
- Quality of electropherograms; Bi‐directional sequences, check for ambiguous bases 
in  the  contig  and  sequence  length  (at  least  360  and  600  bp  for  cytb  and  COI 
respectively  to  obtain  optimum  length  for  differentiation  between  Hippoglossus 
hippoglossus and the sister species Hippoglossus stenopelis)  

The successful amplification of the DNA using the requested methods and the use of the specific 
primers  as  described 
in  Annex  A1  for  the  barcodes,  cytb  and  COI,  was  checked  by 
gel-electrophoresis  or  capillary  electrophoresis.  The  PCR  products  showed  a  single  band  of  the 
expected  length  of  approximately  464  bp  and  738  bp  for  cytb  and  COI  respectively.  The  PCR 
products were purified, and Sanger sequenced according to Standard Operating Procedures meeting 
the requirement of ISO/IEC 17025. The sequences obtained were clean and reached suitable lengths 
for species identification, > 384 bp for cytb and > 634 bp for COI. The two independent data sets 
per method (cytb and COI) gave identical sequence results. The identification resulted in the same 
taxon identification for all characterisation samples for both methods (cytb and COI) and from both 
laboratories.  The  individual  laboratories  have  species  identification  methods  under  their  scope  of 
accreditation. However, from the requested methods and the related primers, only the cytb method 
of one laboratory was covered under their scope of accreditation. For the other lab, this specific cytb 
method was new. Both laboratories used the primers mentioned in the SOP for the COI method, but 
the chemistry and PCR conditions were slightly changed; thermocycler temperatures were optimised 
and different PCR master mixes were used. This implies that for the cytb method for one laboratory 
and  for  the  COI  method  for  both  laboratories  the  PCR  amplification  was  not  fully  validated.  The 
quality  of  the  subsequent  results  were,  however,  controlled  and  checked  using  gel-  or  capillary 
electrophoresis  results  and  the  quality  of  the  electropherograms  produced  in  sequencing. 
Additionally,  the  similarity  of  all  the  sequencing  results  between  the  laboratories  gave  a  good 
indication  that  the  methods  used  were  valid  for  the  genetic  identification  of  the  species  in  this 
EURM. Based on the above mentioned, it was concluded that the datasets were technically valid.  

16 

 
 
 
 
6.4.2  Evaluation of the genetic identification for the cytb barcode 

The two data sets for the cytb barcode, five bottles per laboratory and two DNA extracts per bottle, 
were  analysed  separately.  Alignment  was  performed  using  the  SeqManPro  application  within 
Lasergene  15.1  software  (DNASTAR,  Madison,  US)  [37].  The  forward  and  reverse  sequences  were 
analysed and aligned to one consensus sequence per extraction replicate. Regions of low quality at 
either  end  of  the  electropherogram  were  removed  and,  where  possible,  ambiguous  bases  and  no 
base calls were checked against the sense or anti-sense sequence and manually corrected.  

The  10  consensus  extraction  sequences per laboratory  were  compared to the  reference  sequence 
for  Hippoglossus  hippoglossus  (EU513792.1  in  GenBank  [16]).  From  the  cytb  analysis  of  the  two 
sets  of  10  extraction  sequences,  17  of  the  obtained  consensus  sequences  covered  the  complete 
length  (413  bp)  of  the  cytb  barcode.  The  three  remaining  samples  were  shortened  due  to  poorer 
quality base calls at the extremes of the barcodes (384, 397 and 405 bp). No sequence differences 
were  observed  between  the  samples  however,  there  was  one  mismatching  base  between  the 
consensus  sequences  and  the  reference  sequence  for  Hippoglossus  hippoglossus  (EU513792.1  in 
GenBank  [16]).  The  mismatching  nucleotide  is  highlighted  in  the  cytb  barcode  sequence  given  in 
Annex E1. 

Additionally,  all  samples were  aligned  to  a  reference  sequence  of the  sister species  Hippoglossus 
stenopelis  (EU513796.1  in  NCBI  GenBank  [16]).  All  the  sequences  deviated  for  the  same  seven 
bases with this reference, including the above mentioned mismatching base.  

The two replicate sequences per bottle were aligned to obtain a consensus sequence per bottle and 
further  used  for  species  identification  by  using  Megablast  in  the  Standard  Nucleotide  Basic  Local 
(National  Center  for 
Alignment  Search  Tool  BLAST®  software, 
Biotechnology  Information  (NCBI),  U.S.  National  Library  of  Medicine,  Bethesda,  US)  [18,  19].  The 
BLASTN algorithm compares nucleotide sequences pairwise; searching for local regions of similarity 
in nucleotide sequences against the nucleotide database and calculates the statistical significance 
of matches [18]. Sequence similarity in BLAST refers to identity: similarity between two sequences; 
exactly  the  same  nucleotide  at  the  same  position  in  the  aligned  sequences  [38,  39,  40]  and  is 
expressed as % identity (Amount of identical nucleotides / length of the shortest of the compared 
sequences). 

(version  BLASTN  2.9.0+) 

The  length  of  these  consensus  sequences  for  all  bottles  covered  the  whole  length  of  the  cytb 
barcode (413 bp). Nucleotide BLAST produced an alignment hit table with 100 nucleotide sequence 
database entries of different species within the family Pleuronectidae (righteye flounders), of which, 
the  three  species  with  the  highest  similarity  are:  Hippoglossus  hippoglossus,  Hippoglossus 
stenolepis,  Reinhardtius  hippoglossoides.  The  12  Hippoglossus  hippoglossus  reference  sequences 
hits varied in overall length between 402 bp as a part of the cytb gene and the whole length of the 
mitochondrial DNA. 

All  consensus  sequences  of  the  bottles  used  for  the  characterisation  study  showed  for  the  cytb 
method a very high similarity with 11 Hippoglossus hippoglossus reference sequences ranging from 
99.8 % to 99.3 % identity (Table 1). For the sister species Hippoglossus stenolepis (Pacific halibut) it 
ranged  from  98.3  %  to  97.8  %  and  for  Reinhardtius  hippoglossoides  (Greenland  halibut),  from 
92.7  % to 91.0 %. One Hippoglossus hippoglossus reference sequence (Genbank MH031842.1) [21] 
showed  deviating  results  for  the  similarity  with  the  samples.  Detailed  information  in  the  related 
publication mentions that this Hippoglossus hippoglossus was revealed as Hippoglossus stenolepis 
and  according  to  the  authors  statement,  they  'could  not  make  any  strong  inference  about  the 
taxonomic status of this species of halibut because it lacks a voucher specimen' [21]. Therefore, this 
reference sequence was excluded for the identification  of the EURM-020 samples. The sequences 
of the characterisation samples deviated up to three nucleotides with the 11 reference sequences 
for  Hippoglossus  hippoglossus  in  the  hit-list.  Therefore,  they  could  not  reach  100 %  identity.  A 
summary  of  the  similarity  results  and  reference  sequences  used  is  given  in  Table  1.  All  the 
consensus  sequences  from  the  bottles  had  at  least  seven  different  bases  compared  to 
Hippoglossus stenolepis and at least 30 compared to Reinhardtius hippoglossoides.  

17 

 
The identification for the cytb barcode for the consensus sequences per bottle resulted in 99.8 % to 
99.3  %  identity  with  Hippoglossus  hippoglossus.  Due  to  mismatching  nucleotides  within  the 
sequences  of  the  individual  reference  sequences  used  for  the  cytb barcode  identification and  the 
different  lengths  of  the  sequences  compared,  outcomes  vary  between  reference  sequences.  They 
are  given  here  in  a  range  of  identity  percentage  instead  of  a  fixed  percentage.  The  identity 
percentage  never  exceeded  99.8  %  for  the  cytb  barcode  because  of  the  same  mismatching 
nucleotide  in  the  EURM-020  samples  compared  with  all  the  Hippoglossus  hippoglossus  reference 
sequences used.  

Table  1:  Average  sequence  alignment  similarity  results  of  the  characterisation  samples  of 
EURM-020,  expressed  as  %  identity,  compared  with  11  Hippoglossus  hippoglossus  reference 
sequences in GenBank for the cytb barcode using BLASTN 2.9.0+ (NCBI).  

GenBank accession 
code of cytb 
reference sequence 

Average identity [%] 
of EURM-020 with cytb 
reference sequence 

Taxon identity of 
cytb reference 
sequence 

EU513791.1 [16] 

AM749125.1 [17] 

AM749124.1 [17] 

AM749122.1 [17] 

AF413798.1 [24] 

EU492150.1 [22] 

EU492256.1 [23] 

EU513790.1 [16] 

AM749123.1 [17] 

HQ283277.1 [30] 

AF413799.1 [24] 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.5 

99.5 

99.5 

99.3 

Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 

Voucher ID 

Nucleotide 
mismatches of 
samples of 
EURM-020 with 
reference 
sequence 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

MNHN 2005-1582 

NRM 53139 

n.a. 

n.a. 

n.a. 

n.a. 

1 

2 

3 

6.4.3  Evaluation of the genetic identification for the COI barcode 

The two data sets for the COI barcode, five bottles per laboratory and two DNA extracts per bottle, 
were  analysed  separately.  From  each  extract,  the  forward  and  reverse  sequences  were  analysed 
and  aligned  to  one  consensus  sequence  per  sample  using  the  SeqManPro  application  within 
Lasergene 15.1 software (DNASTAR, Madison, US) [37]. Regions of low quality at either end of the 
electropherogram were removed and, ambiguous bases and no base calls were checked against the 
sense or anti-sense sequence and manually corrected where possible.  

The  10  consensus  extraction  sequences  per  laboratory  were  compared  to  the  Hippoglossus 
hippoglossus reference sequence (GBMTG1322.16 in BOLD/ mined from NC_009709.1 in GenBank 
[17, 36]). From the two sets of 10 sample sequences for the COI analysis from each lab, the length 
of the sequences was found to be between 626 bp and 652 bp. Of those 20 sequences, 17 were 
shorter  than  the  complete  COI  barcode  region  (652  bp)  due  to  poorer  quality  base  calls  at  the 
extremes.  No  sequence differences  were observed between the samples. However,  there was  one 
mismatching base between the consensus sequences and the reference sequence. The mismatching 
nucleotide is highlighted in the COI barcode sequence given in Annex E1. 

18 

 
 
The  sequences  were  also  aligned  to  a  reference  sequence  of  the  sister  species  Hippoglossus 
stenopelis.  All  extraction  sequences  showed  the  same  13  deviating  bases  compared  to  the 
reference  (GBMTG908_16  in  BOLD,  mined  from  NC_009710  in  NCBI  GenBank  [17])  of  the  sister 
species Hippoglossus stenolepis. 

The two sample sequences per bottle were aligned to obtain a consensus sequence per bottle and 
further used for species identification. For three of the  consensus sequences, the entire  region of 
the COI barcode (652 bp) was covered. For the remaining seven consensus sequences, the lengths 
varied  from  634  bp  to  651  bp.  Identification  for  the  COI  method  was  performed  using  the 
BOLD  Identification Systems v4 [20]. The search database for animal identification used, the Public 
Record Barcode Database, contains all published COI records from BOLD with a minimum sequence 
length of 500 bp and is a library with a collection of records from the published projects section of 
BOLD. In BOLD, nucleotide sequences are first translated into amino acids sequences, verified that 
they  originate  from  the  COI  protein  and,  after  passing  some  additional  quality  checks,  a  linear 
search of the reference library is followed [20]. Sequence similarity in BOLD refers to the ratio to 
which nucleotide or protein sequences are  related and takes substitutions into account;  expressed 
as similarity percentage [39, 40]. For sequences showing less than 1 % divergence to a reference 
sequence,  the  BOLD  identification  system  delivers  a  species  identification  [20].  The  BOLD 
identification  application  generated  a  hit  list  of  the  order  Pleuronectiformes  (flatfish)  and  family 
Pleuronectidae  of  99  individuals  of  species.  The  species  that  gave  the  highest  similarity  are: 
Hippoglossus  hippoglossus,  Hippoglossus  stenolepis  and  Reinhardtius  hippoglossoides.  The 
25   Hippoglossus  hippoglossus  reference  sequence  hits  varied  in  length  between  606  bp  and 
1563  bp for the COI gene region. 

All consensus sequences of the bottles used for the characterisation study for the COI barcode had 
comparable similarity ranges using the BOLD identification application. The sequence similarity for 
Hippoglossus hippoglossus ranged between 99.8 % and 99.4 % and the samples were all identified 
as  Hippoglossus  hippoglossus  (Table  2).    For  the  sister  species  Hippoglossus  stenolepis  similarity 
ranged from 98.3 % to 97.6 % and for Reinhardtius hippoglossoides from 93.1 % to 92.7 %. One of 
the 25 Hippoglosssus hippoglossus reference sequences in the hit list (GBMIN131002-17 in BOLD, 
KX164003  in  Genbank  [33])  showed  a  deviating  alignment  with  the  samples.  Upon  closer 
investigation, the neighbour-joining tree (Kimura 2-parameter distance model for COI-5P marker) in 
BOLD  [20,  31,  32]  showed  that  this  reference  sequence  is  clustered  within  the  Hippoglossus 
stenopelis sister species. Because of the lack of a publication and therefore proof for morphological 
taxonomy for this reference sequence it has been excluded from further analysis of the data.  

Using the SeqManPro application within the Lasergene 15.1 software [37] revealed that within the 
COI barcode region of the characterisation samples, there was one deviating nucleotide, compared 
to all 24 Hippoglossus hippoglossus reference sequences in the hit list from the BOLD application. 
The sequences of the characterisation samples deviated up to four nucleotides with the reference 
sequences in the hit list (Table 2). All the characterisation samples had at least 11 different bases 
compared to Hippoglossus stenolepis and at least 44 compared to Reinhardtius hippoglossoides.  

Also,  a  BLAST  search  for  the  COI  barcode  gave  comparable  results  for  the  sample  replicate 
sequences, from 99.8 % to 99.3 % identity with Hippoglossus hippoglossus.  

For the COI barcode, the identification of the consensus sequences per bottle resulted in 99.8 % to 
99.4  %  similarity  with  Hippoglossus  hippoglossus.  Due  to  mismatching  nucleotides  within  the 
sequences of the individual reference sequences used for the COI barcode, identification outcomes 
vary between reference sequences. They are given here in a range of similarity instead of a fixed 
percentage. The similarity for the COI barcode was never higher than 99.8 % because of the same 
mismatching nucleotide in the EURM-020 samples compared with all reference sequences.  

19 

 
Table 2: Average sequence alignment similarity results of characterisation samples of EURM-020, 
expressed  as  %  similarity,  compared  with  24  Hippoglossus  hippoglossus  reference  sequences  for 
the  COI  barcode  using  the  BOLD  system  v4  identification  tool  (Animal  Public  Record  Barcode 
Database)  

BOLD process 
ID of COI 
reference 
sequence 

GenBank accession 
code of COI 
reference sequence  

Average 
similarity 
[%] of 
EURM-020 
with COI 
reference 
sequence 

Taxon identity 
of COI 
reference 
sequence 

Nucleotide 
mismatches of 
samples EURM-
020 with 
reference 
sequence 

Voucher ID 

GBMNA14652-19 

AM749122.1 [17] 

GBGC3118-07 

AM749123.1 [17] 

SCFAC777-06 

SCFAC105-05 

KC015478.1 [25] 

KC015480.1 [25] 

SCAFB738-07 

KC015482.1 [25] 

GBGCA8489-15 

KJ128511.1 [26] 

GBMTG1322-16 

NC_009709.1 [17][36] 

GBGC3116-07 

AM749125.1 [17] 

FOAE640-06 [27] 

n.a. 

GLF055-14 [28] 

n.a. 

SCFAC836-06 

KC015479.1 [25] 

SCFAC657-06 

KC015481.1 [25] 

SCFAC110-05 

KC015483.1 [25] 

SCFAC648-06 

KC015484.1 [25] 

BNSF511-12 

KJ204922.1 [29] 

BNSF534-12 

KJ204923.1 [29] 

BNSF645-12 

KJ204924.1 [29] 

BNSFI074-12 

KJ204925.1 [29] 

GBGC3117-07 

AM749124.1 [17] 

SCAFB551-07 

KC015477.1 [25] 

GBGC7349-09 

EU513652.1 [16] 

GBGC7348-09 

EU513653.1 [16] 

GBGC7347-09 

EU513654.1 [16] 

FOAE641-06 [26] 

n.a. 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.7 

99.7 

99.7 

99.7 

99.7 

99.7 

99.7 

99.7 

99.7 

99.7 

99.7 

99.5 

99.5 

99.4 

99.4 

99.4 

99.4 

Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 

n.a. 

n.a. 

06-604 

1 

05-406-006d4 

07-126 

NRM:53139 

n.a. 

n.a. 

BW-A2852/ CSIRO H 
4303-12 

GLF055 

06-889 

06-739 

2 

05-399-006e3 

06-724 

MT02932 

MT02955 

MT03047 

MT04123 

n.a. 

06-786 

n.a. 

n.a. 

n.a. 

BW-A2853 

3 

4 

20 

 
 
 
7 

Value Assignment 

A certified value was assigned. 

Certified values are values that fulfil the highest standards of accuracy. Two datasets were used to 
assign  the  certified  value,  the  identity,  of  EURM-020  and  is  based  on  two  genetic  identification 
methods using two different mitochondrial DNA barcodes, cytb and COI measured by bi-directional 
Sanger  sequencing.  The  certified  value,  the  taxon  assignment  for  EURM-020,  refers  to  reference 
sequences in the GenBank (NCBI) and FishTrace (EC) databases for cytb and the BOLD database for 
COI.  

The length of the resultant sequence of EURM-020 for  the cytb barcode was 413  bp and for the 
COI  barcode 652 bp. The sequences of EURM-020 for both barcodes are given in FASTA format in 
Annex E1.  

The assigned taxon, for all characterisation samples from both datasets for the cytb barcode, was 
Hippoglossus  hippoglossus,  based  on  the  highest  scores  of  similarity,  99.8  %  identity,  with  the 
Hippoglossus  hippoglossus  reference  sequences  retrieved  by  the  BLAST  search  tool  (version 
BLASTN  2.9.0+NCBI) [18, 19]). The cytb sequences of the characterisation samples mismatched one 
nucleotide  with  the  reference  sequences  with  the  highest  similarity  scores.  The  assigned  taxon  is 
linked to the reference sequences in the GenBank (NCBI) and FishTrace (EC) databases (Table 3).  

Table 3: Hippoglossus hippoglossus reference sequences for the cytb barcode in GenBank used for 
taxon assignment of EURM-020.  

GenBank accession 
code of cytb 
reference sequence 

Average identity [%] 
of EURM-020 with cytb 
reference sequence 

Taxon identity of 
cytb reference 
sequence 

Voucher ID 

Nucleotide 
mismatches of 
samples of EURM-
020 with reference 
sequence 

EU513791.1 [16] 

AM749125.1 [17] 

AM749124.1 [17] 

AM749122.1 [17] 

AF413798.1 [24] 

EU492150.1 [22] 

EU492256.1 [23] 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

1 

MNHN 2005-1582 

NRM 53139 

The taxon assignment for all characterisation samples from both datasets for the COI barcode, was 
Hippoglossus  hippoglossus,  based  on  the  highest  scores,  99.8  %  similarity  with  Hippoglossus 
hippoglossus  reference  sequences  retrieved  by  the  Data  Identification  Systems  v4  of  BOLD  [20]. 
The COI sequences of the characterisation samples mismatched one nucleotide with the reference 
sequences  with  the  highest  similarity  scores.  The  assigned  taxon  is  linked  to  the  reference 
sequences in the BOLD Animal Public Record Barcode Database (Table 4). 

21 

 
 
Table 4: Hippoglossus hippoglossus reference sequences for the COI barcode in the BOLD Animal 
Public Record Barcode Database used for taxon assignment of EURM-020.  

BOLD process 
ID reference 
sequence  

GenBank accession 
code of COI 
reference 
sequence 

Average 
similarity [%] 
of EURM-020 
with COI 
reference 
sequence 

Taxon identity 
of COI 
reference 
sequence 

Nucleotide 
mismatches of 
samples EURM-
020 with 
reference 
sequence 

Voucher ID 

GBMNA14652-19 

AM749122.1 [17] 

GBGC3118-07 

AM749123.1 [17] 

SCFAC777-06 

SCFAC105-05 

KC015478.1 [25] 

KC015480.1 [25] 

SCAFB738-07 

KC015482.1 [25] 

GBGCA8489-15 

KJ128511.1 [26] 

GBMTG1322-16 

NC_009709.1 [17][36] 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 
Hippoglossus 
hippoglossus 

n.a. 

n.a. 

06-604 

1 

05-406-006d4 

07-126 

NRM:53139 

n.a. 

The species identity for EURM-020 was defined by a minimum 99 % sequence similarity with the 
DNA  sequences  of  the  specimens  (voucher  samples  and  specimens  with  morphological 
identification),  deposited  in  the  GenBank  (NCBI),  FishTrace  (EC)  and  BOLD  databases.  It  is unlikely 
that there is an alternative species that shares the sequences of the two independent genes used in 
this study, therefore, there is no doubt regarding the certified identity, Hippoglossus hippoglossus, for 
EURM-020. Consequently, the uncertainty is negligible. 

8 

Metrological traceability and commutability 

8.1  Metrological traceability  

Identity 

The genetic identity is a clearly defined molecular property. The genetic identity was confirmed by 
two different mtDNA barcode methods, cytb and COI, and is traceable to DNA reference sequences 
of  which  the  morphological  identity  of  the  related  species  was  executed,  in  the  GenBank  (NCBI), 
FishTrace (EC) and BOLD databases. 

8.2  Commutability 

The  commutability  of  a  CRM  defines  its  fitness  for  use  and  is,  therefore,  a  crucial  characteristic 
when  applying  different  measurement  methods.  When  the  commutability  of  a  CRM  is  not 
established,  the  results  from  routinely  used  methods  cannot  be  legitimately  compared  with  the 
certified value.  

The  CRM  is  prepared  from  fresh  fish  tissue  that  was  dried  and  milled  to  a  powder.  The  certified 
value established, was obtained by PCR amplification and bi-directional Sanger sequencing of two 
different DNA barcodes, cytb and COI. After reconstitution, the material closely resembles samples 
of  fresh  fillet.  The  analytical  behaviour  will,  therefore,  be  the  same  as  is  the  case  for  fresh  fish 
tissue samples tested with these DNA barcodes using PCR sequencing.  

22 

 
 
 
 
 
 
9 

Instructions for use 

9.1  Safety information 

The usual laboratory safety measures apply.  

The material is for in vitro use only. 

9.2  Storage conditions 

The materials should be stored at 4 ± 3 °C in the dark. Care should be taken to avoid any changes 
in  the  moisture  content  once  the  units  are  opened  since,  the  material  is  hygroscopic.  The  user 
should close any bottles immediately after taking a sample.  

Please  note  that  the  European  Commission  cannot  be  held  responsible  for  changes  that  occur 
during storage of the material at the customer's premises, especially for opened bottles. 

9.3  Preparation and use of the material/Reconstitution 

To prepare for use, the material must be reconstituted according to the following procedure: Weigh 
7 to 8 mg of the fish powder into a 2 mL low-DNA-binding microcentrifuge tube and add 160 μL 
nuclease-free  water,  vortex  for  10  seconds  and  immediately  continue  with  a  DNA  extraction 
protocol.  

9.4  Minimum sample intake 

EURM-020  is  a  pure  fish  powder  originating  from  a  single  fish;  therefore,  the  minimum  sample 
intake  for  this  material  is  not  linked  to  the  within-unit  homogeneity.  Nevertheless,  to  obtain  a 
significant amount of DNA, it is recommended to use a minimum sample intake of 7 mg when using 
the extraction method used in this study.  

9.5  Use of the certified value 

The main purpose of this material is to assess method performance using PCR and/or sequencing, 
i.e.  for  checking  the  accuracy  of  analytical  results.  As  with  any  reference  material,  it  can  also  be 
used for validation studies. 

23 

 
10  Acknowledgements 

The authors would like to acknowledge the support received from various contributors of the JRC, 
Directorate F, among them are: O. De Rudder for the delivery of the raw material for the processing 
of this EURM, P. Conneely and M.-F. Tumba-Tshilumba for the KFT and PSA measurements, A. Lieven 
and  V.  Paracchini  for  sequencing  measurements  of  the  raw  material  for  this  EURM,  and 
F.  Vanderveken for the set-up of the required isochronous studies.  

Furthermore, the authors would like to thank L. Deprez and A. Held (JRC, Directorate F) for reviewing 
the  certification  report,  as  well  as  the  experts  of  the  Reference  Material  Review  Panel  "Biological 
Macromolecules  and  Biological/Biochemical  Parameters",  G.  Berben  (Centre  wallon  de  Recherches 
agronomiques, Gembloux, Belgium) and H. Hird (Manchester, UK) for their constructive comments.  

24 

 
11  References 

ISO 17034:2016, General requirements for the competence of reference materials producers, 
International Organization for Standardization, Geneva, Switzerland      

ISO Guide 35:2017, Reference materials – General and statistical principles for certification, 
International Organization for Standardization, Geneva, Switzerland 

ISO/IEC  17025:2005,  General  requirements  for  the  competence  of  testing  and  calibration 
laboratories, International Organization for Standardization, Geneva, Switzerland 

REGULATION (EU) No 1379/2013 OF THE EUROPEAN PARLIAMENT  AND OF THE COUNCIL of 
11 December 2013 on the common organisation of the markets in fishery and aquaculture 
products,  amending  Council  Regulations  (EC)  No  1184/2006  and  (EC)   No  1224/2009  and 
repealing Council Regulation (EC) No 104/2000 

J.  Barendse,  A.  Roel,  C.  Longo,  L.  Andriessen,  L.  M.  I.  Webster,  R.  Ogden  and  F. Neat,  DNA 
barcoding  validates  species  labelling  of  certified  seafood,  Current  Biology  29(6)  (2019) 
R198-R199 

COUNCIL REGULATION (EC) No 1224/2009 of 20 November 2009 establishing a Community 
control  system  for  ensuring  compliance  with  the  rules  of  the  common  fisheries  policy, 
amending  Regulations  (EC)  No  847/96,  (EC)  No  2371/2002,  (EC)   No   811/2004,  (EC)  No 
768/2005,  (EC)  No  2115/2005,  (EC) No  2166/2005,  (EC)   No  388/2006,  (EC) No  509/2007, 
(EC)  No  676/2007,  (EC)  No  1098/2007,  (EC)   No  1300/2008,  (EC)  No  1342/2008  and 
repealing Regulations (EEC) No  2847/93, (EC) No 1627/94 and (EC) No 1966/2006 

REGULATION  (EC)  No  178/2002  OF  THE  EUROPEAN  PARLIAMENT  AND  OF  THE  COUNCIL  of 
28 January  2002  laying  down  the  general  principles  and  requirements  of  food  law, 
establishing  the  European  Food  Safety  Authority  and  laying  down  procedures  in  matters  of 
food safety 

W.  M.  Brown,  M.  George  Jr.,  A.  C.  Wilson,  Rapid  evolution  of  animal  mitochondrial  DNA 
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(mitochondrial  cytochrome 

I  sequencing), 

1  

2  

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

25 

 
 
 
 
14 

R. D. Ward, T. S. Zemlak, B. H. Innes, P. R. Last and P. D. N. Hebert, DNA barcoding Australia’s 
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DNA 
doi:10.1111/j.1471-8286.2007.01748.x 

barcoding, 

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(2008)  8954–8961, 
doi:10.1021/jf800570r 

17   K.  A.  Mjelle,  B.  O.  Karlsen,  T.  E.  Jørgensen,  T.  Moum,  S.  D.  Johansen,  Halibut  mitochondrial 
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genomes  contain  extensive  heteroplasmic  tandem  repeat  arrays 
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involved 

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203-214, 

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Computational 

Journal 

Biology 

(2000) 

7(1-2) 

of 

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Indexing  for  Production  MegaBLAST  Searches",  Bioinformatics  24(16)   (2008)  1757-1764, 
doi:10.1093/bioinformatics/btn322 

20   S.  Ratnasingham,  P.  D.  N.  Hebert,  BARCODING:  BOLD:  The  Barcode  of  Life  Data  System 
355–364, 

(www.barcodinglife.org), 
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Molecular 

Ecology 

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Notes 

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21 

22 

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Molecular 
147-162, 
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Phylogenetics 

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125, 

and 

H.  van  Pelt-Heerschap,  A.  S.  Stein,  FishTrace:  Genetic  Catalogue,  Biological  Reference 
Collections  and  Online  Database  of  European  Marine  Fishes 
(www.fishtrace.org) 
https://fishtrace.jrc.ec.europa.eu/  and  submitted  to  NCBI  GenBank  (both  last  consulted 
11/12/2019) 

23  M.  Noren,  S.O.  Kullander,  M.  Kallersjo,  FishTrace:  Genetic  Catalogue,  Biological  Reference 
Collections  and  Online  Database  of  European  Marine  Fishes 
(www.fishtrace.org) 
https://fishtrace.jrc.ec.europa.eu/  and  submitted  to  NCBI  GenBank  (both  last  consulted 
11/12/2019) 

24 

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Canada's  commonly  encountered  marine  fishes,  Molecular  Ecology  Resources    13  (2) 
(2013)177-188, doi:10.1111/1755-0998.12043  

26 

27 

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S. A. Appleyard, CSIRO, Australian National Fish Collection, published in BOLD, The Barcode of 
Life Data System http://www.boldsystems.org/ (last consulted 11/12/2019) 

26 

 
 
 
 
28 

29 

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Barcode of Life Data System http://www.boldsystems.org/ (last consulted 11/12/2019) 

T. Knebelsberger, M. Landi, H. Neumann, M. Kloppmann, A. F. Sell, P. D. Campbell, S. Laakmann, 
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identification of the North European shelf fish fauna, Molecular Ecology Resources 14 (2014) 
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30 

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33 

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37 

38 

39 

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27 

 
 
Annexes 

Annex A: Summary of methods used 

Annex A.1: Primers for PCR-sequencing for the cytb and COI genes used for homogeneity 
and characterisation studies: 

Table 1: PCR and sequencing primers cytb 1) 

Primer name 

Sequence 5' – 3' 

AAA AAC CAC CGT TGT TAT TCA ACT A 

Forward  primer 
L14735  
Reverse 
H15149ad  
1) The expected size of the PCR product is 464 bp 

primer 

GCN CCT CAR AAT GAY ATT TGT CCT CA 

Table 2: PCR and sequencing primers COI 1) 

Primer 
PCR 

VF2_t1   

name, 

Sequence 5' – 3' 2) 3) 

TGTAAAACGACGGCCAGTCAA CCA ACC ACA AAG ACA TTG GCA C 

FishF2_t1  

TGTAAAACGACGGCCAGTCGA CTA ATC ATA AAG ATA TCG GCA C 

FishR2_t1   

FR1d_t1   

CAGGAAACAGCTATGACACT TCA GGG TGA CCG AAG AAT CAG AA 

CAGGAAACAGCTATGACACC TCA GGG TGT CCG AAR AAY CAR AA 

Primer 
sequencing 

name, 

Sequence 5' – 3' 

M13_F 

M13_R 

TGTAAAACGACGGCCAGT 

CAGGAAACAGCTATGAC 

Reference 

[10] 

[10] 

Reference 

[14, 13] 

[14, 13] 

[14, 13] 

[15, 13] 

Reference 

[35] 

[35] 

1) The expected size of the PCR product is 738 bp 
2) Primers with highlighted M13 tails  
3) One laboratory performed the sequencing of the homogeneity and characterisation samples by using a cocktail of two 
forward  or  two  reverse  primers  in  the  respective  forward  and  reverse  master-mix  compositions.  The  second  laboratory 
that  performed  the  sequencing  of  the  second  set  of  characterisation  samples  used  four  separate  PCR  master-mix 
compositions with single primers.    

28 

 
 
 
 
Annex A.2 Method for cytb specific PCR used for stability measurements: 

Table 1: Primer sequences cytb PCR 1) 

Primer 
PCR  

L14735 

name, 

Sequence 5' – 3' 

AAA AAC CAC CGT TGT TAT TCA ACT A 

H15149ad 

GCN CCT CAR AAT GAY ATT TGT CCT CA 

1) The expected size of the PCR product is 464 bp 

Table 2: Cycling conditions cytb PCR (C1000 Touch Thermal Cycler, Bio-Rad, NL) 

Section  

Hot start  

Denaturation 

Annealing 

Elongation 

Final elongation 

Cooling 

Temperature °C 

95 

95 

50 

72 

72 

10 

Time 

11 min 

45 s 

80 s 

90 s 

30 min 

∞ 

Reference 

[10] 

[10] 

Cycles 

1 

35 

1 

1 

Table 3: Master-mix composition for cytb PCR reaction 

Reagent 

Concentration  Concentration 
Final solution 
Stock 

µl per  
reaction 

Supplier 

Buffer  

10 X 

1 x 

5.00 

Primer F: L14735 
Primer R: H15149ad 
dNTPs 

7.5 μM 
7.5 μM 
10 mM 

0.5 μM 
0.5 μM 
250 μM 

3.33 1) 
3.33 1) 
1.25 

Polymerase 

5 U/μL 

0.05 U/μL 

0.50 

MgCl2 

25 mM 

3.0 mM 

6.00 

Water 

DNA 

~20 ng/μL  

~60 ng/rxn 

3.00 

27.59 1) 

AmpliTaq Gold PCR 
buffer, ThermoFisher 
scientific, Gent, BE 

dNTP mix, 10 mMol each, 
ThermoFisher scientific, 
Gent, BE 
AmpliTaq Gold DNA 
polymerase, 
ThermoFisher scientific, 
Gent, BE 
MgCl2, AmpliTaq Gold DNA 
polymerase kit, 
ThermoFisher scientific, 
Gent, BE 
Promega Benelux, Leiden, 
NL 
DNA concentration 
according Nanodrop 

Total volume (μl) 
1) For the stability study one PCR reaction was prepared using a cocktail of the forward and reverse primer together in 
one master-mix composition.  

50.00 

29 

 
 
 
 
  
  
  
  
 
 
  
  
 
  
  
 
Annex A.3 Method for COI specific PCR according standard operating procedure from the 
Labelfish Consortium used for stability measurements [13]: 

Table 1: Primer sequences COI PCR 1) 

Primer  name, 
PCR  

Sequence 5' – 3'  2) 

VF2_t1    

TGTAAAACGACGGCCAGTCAA CCA ACC ACA AAG ACA TTG GCA C 

FishF2_t1   

TGTAAAACGACGGCCAGTCGA CTA ATC ATA AAG ATA TCG GCA C 

FishR2_t1     

CAGGAAACAGCTATGACACT TCA GGG TGA CCG AAG AAT CAG AA 

FR1d_t1   

CAGGAAACAGCTATGACACC TCA GGG TGT CCG AAR AAY CAR AA 

1) The expected size of the PCR product is 738 bp 
2) Primers with highlighted M13 tails 

Table 2: Cycling conditions COI PCR (C1000 Touch Thermal Cycler, Bio-Rad, NL) 

Section  

Hot start  

Denaturation 

Annealing 

Elongation 

Final elongation 

Cooling 

Temperature °C 

94 

94 

52 

72 

72 

10 

Time 

2 min 

30 s 

40 s 

60 s 

10 min 

∞ 

Table 3: Master-mix composition for COI PCR reaction  

Reference 

[14, 13] 

[14, 13] 

[14, 13] 

[15, 13] 

Cycles 

1 

35 

1 

1 

Reagent 

Concentration  Concentration   µl per  
Stock 

Final solution 

reaction 

D(+)-Trehalose dihydrate  10 % 
10 X 
Buffer  

5 % 
1 x 

10.00 
2.00 

Forward  primers VF2_t1 
and FishF2_t1 

Reverse primers 
FishR2_t1 and FR1d_t1 
dNTPs 

10 μM 

0.1 μM each 

0.20 each 1) 

10 μM 

0.1 μM each 

0.20 each 1) 

10 mM 

200 μM 

Polymerase 

5 U/μL 

0.025 U/μL 

MgCl2 
Water 

DNA 

50 mM 

2.5 mM 

~20 ng/μL  

~60 ng/rxn 

3.00 

0.40 

0.10 

1.00 
2.70 1) 

Supplier  

Alfa Aesar ,VWR, Leuven, BE 
Reaction buffer, Bioline, 
GCbiotech, Waddinxveen, NL 

dNTP mix, Bioline, GCbiotech, 
Waddinxveen, NL 
Biotaq DNA polymerase, 
Bioline, GCbiotech, 
Waddinxveen, NL 
MgCl2, Bioline 
Promega Benelux, Leiden, 
NL   
DNA concentration according 
Nanodrop 

Total volume (μl) 
1) For the  stability  study,  one PCR reaction  was prepared using a  cocktail  of  the  two  forward  and  two  reverse  primers 
together in one master-mix composition.   

20.00 

30 

 
 
  
  
  
  
 
 
  
  
  
  
 
Annex B: Results of the homogeneity measurements 

Annex B1: Average extracted DNA concentration per bottle 

Homogeneity

]
L
µ
/
g
n
[
n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

180.0

160.0

140.0

120.0

100.0

80.0

60.0

40.0

20.0

0.0

145

250

413

541

710

771

988

1105 1252 1429

Bottle number

Figure 1:  Extracted  DNA  concentration [ng/μL] per bottle measured for the homogeneity study of 
EURM-020.  Each  data  point  represents  the  average  of  2  extraction  duplicates  from  each  bottle, 
N = 1,  n  =  2  with  error  bars  expressed  as  smeas.  Measurements  were  conducted  by  using  UV 
spectrophotometry (Nanodrop). 

31 

 
 
 
  
 
 
 
Annex B2: Results of the homogeneity measurements: cytb 

Table  1:  Homogeneity  results  EURM-020  of  the  species  identification  for  the  cytb  barcode  using 
BLASTN  2.9.0+  (NCBI).  Maximum  and  minimum  identity  results  of  the  sequence  of  the  extraction 
replicates  per  bottle,  N  =  10,  n  =  2,  compared  with  11  different  Hippoglossus  hippoglossus 
reference sequences in GenBank (NCBI). 

Bottle Number- 
replicate 

Species identification 

Maximum 
identity [%]  1) 

Minimum 
identity [%] 1) 

145-1 

145-2 

250-1 

250-2 

413-1 

413-2 

541-1 

541-2 

710-1 

710-2 

771-1 

771-2 

988-1 

988-2 

1105-1 

1105-2 

1252-1 

1252-2 

1429-1 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

99.8 

99.7 

99.8 

99.7 

99.7 

99.7 

99.8 

99.8 

99.7 

99.8 

99.8 

99.8 

99.8 

99.8 

99.7 

99.8 

99.7 

99.8 

99.8 

1429-2 

Hippoglossus hippoglossus 
1)  % Identity = Amount of identical nucleotides / minimum aligning length. 

99.8 

99.3 

99.2 

99.3 

99.0 

99.2 

99.2 

99.3 

99.3 

99.0 

99.3 

99.3 

99.3 

99.3 

99.3 

99.0 

99.3 

99.2 

99.3 

99.3 

99.3 

32 

 
 
Annex B3: Results of the homogeneity measurements: COI 

Table 1: Homogeneity results EURM-020 species identification for the COI barcode using the BOLD 
system  v4  identification  tool.  Maximum  and  minimum  similarity  results  of  the  sequence  of  the 
extraction  replicates  per  bottle,  N  =  10,  n  =  2,  compared  with  24  Hippoglossus  hippoglossus 
reference sequences in the BOLD Animal Public Record Barcode Database.  

Bottle Number-
replicate 

145-1 

145-2 

250-1 

250-2 

413-1 

413-2 

541-1 

541-2 

710-1 

710-2 

771-1 

771-2 

988-1 

988-2 

1105-1 

1105-2 

1252-1 

1252-2 

1429-1 

Species identification 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Maximum 
similarity [%] 1) 

Minimum 
similarity [% ] 1) 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.8 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

99.4 

1429-2 
1)  The statistics related to the aligned sequences in BOLD, is expressed in similarity percentage.   

Hippoglossus hippoglossus 

99.8 

99.4 

33 

 
Annex C:  Short-term stability study results 

Short-term stability EURM-020 at 4 °C

200.00

180.00

160.00

140.00

120.00

100.00

80.00

60.00

40.00

20.00

0.00

]
L
µ
/
g
n
[

n
o
i
t
a
r
t
n
e
e
c
n
o
c
A
N
D

0

1

2

3

4

5

[Week]

Figure 1:  Extracted  DNA concentration [ng/μL] of the samples included in the short-term stability 
study of EURM-020 at 4 °C. Average of 2 samples of each 2 extraction duplicates, N = 2, n = 2 with 
error bars expressed as smeas. Measurements performed by UV spectrophotometry (Nanodrop). 

Short-term stability EURM-020 at 18 °C

200.00

180.00

160.00

140.00

120.00

100.00

80.00

60.00

40.00

20.00

0.00

]
L
µ
/
g
n
[
n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

0

1

2

3

4

5

[Week]

Figure 2: Extracted DNA concentration [ng/μL] of the samples included in the short-term stability of 
EURM-020 at 18 °C. Average of 2 samples of each 2 extraction duplicates, N = 2, n = 2 with error 
bars expressed as smeas. Measurements performed by UV spectrophotometry (Nanodrop). 

34 

 
 
 
  
 
 
 
 
 
 
Annex C: Short-term stability study results 

Short-term stability EURM-020 at 60 °C

200.00

180.00

160.00

140.00

120.00

100.00

80.00

60.00

40.00

20.00

0.00

]
L
µ
/
g
n
[
n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

0

1

2

3

4

5

[Week]

Figure 3: Extracted DNA concentration [ng/μL] of the samples included in the short-term stability of 
EURM-020 at 60°C. Average of 2 samples of each 2 extraction duplicates, N = 2, n = 2 with error 
bars expressed as smeas. Measurements performed by UV spectrophotometry (Nanodrop). 

35 

 
 
 
 
 
Annex D: Long-term stability study results 

Long-term stability EURM-020 at 4 °C

200.00

180.00

160.00

140.00

120.00

100.00

80.00

60.00

40.00

20.00

0.00

]
L
µ
/
g
n
[
n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

0

3

6

9

12

15

[Month]

Figure 1: Extracted DNA concentration [ng/μL] of the samples included in the long-term stability of 
EURM-020  at  4°C.  Average  of  2  samples  of  each 2  extraction duplicates, N  =  2,  n =  2  with  error 
bars expressed as smeas. Measurements performed by UV spectrophotometry (Nanodrop). 

Long-term stability EURM-020 at -20 °C

200.00

180.00

160.00

140.00

120.00

100.00

80.00

60.00

40.00

20.00

0.00

]
L
µ
/
g
n
[

n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

0

3

6

9

12

15

[Month]

Figure 2: Extracted DNA concentration [ng/μL] of the samples included in the long-term stability of 
EURM-020 at -20°C. Average of 2 samples of each 2 extraction duplicates, N = 2, n = 2 with error 
bars expressed as smeas. Measurements performed by UV spectrophotometry (Nanodrop). 

36 

 
 
 
 
 
 
 
 
 
 
 
Annex E: Results of the characterisation measurements 

Annex E1: Taxon assignment and sequences  

Table 1: Taxon assignment characterisation samples per laboratory and assay.  

Laboratory  
code 

replicate 1 
Taxon assignment 

replicate 2 
Taxon assignment  

replicate 3 
Taxon assignment 

replicate 4 
Taxon assignment 

replicate 5 
Taxon assignment 

L1 cytb 
L2 cytb 
L1 COI 
L2 COI 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Hippoglossus hippoglossus 

Sequence of EURM-020, Hippoglossus hippoglossus, cytb barcode  

>EURM-020, Hippoglossus hippoglossus, cytb, 413 bp 
CAAGAACCCTAATGGCCAGTCTACGTAAATCCCACCCTCTTCTAAAAATCGCAAACGATGCTTTAGTCGACCTCC
CCGCCCCCTCTAATATCTCAGTTTGATGGAACTTTGGGTCTCTTTTAGGACTCTGTTTAATTACCCAAATTGCGA
CCGGCTTATTTCTAGCCATACACTACACATCAGACATTGCTACTGCCTTCACCTCCGTGGCCCACATCTGTCGAG
ACGTCAACTACGGCTGACTTATCCGAAGCATTCATGCCAACGGCGCATCATTCTTTTTCATTTGCCTCTACCTTC
ATATTGGCCGAGGACTATACTATGGCTCTTACCTCTATAAAGAAACATGAACTGTTGGGGTTATTCTTCTCCTTC
TCGTAATAATAACAGCCTTCGTTGGATACGTCCTCCCT  

The highlighted base shows the mismatch in all EURM-020 samples included in the characterisation 
study with the Hippoglossus hippoglossus reference sequences for cytb in GenBank (NCBI) used for 
the identification of EURM-020 (Table 3, Section 7).  

Sequence of EURM-020, Hippoglossus hippoglossus, COI barcode  

>EURM-020, Hippoglossus hippoglossus, COI, 652 bp 
CCTCTATCTCGTATTTGGTGCCTGAGCCGGAATAGTGGGGACAGGCCTAAGTCTGCTTATTCGGGCAGAACTAAG
CCAACCCGGGGCTCTCCTGGGAGACGACCAAATTTATAATGTGATCGTCACCGCACACGCCTTTGTAATAATCTT
TTTTATAGTAATACCCATTATGATTGGGGGGTTCGGAAACTGGCTTATTCCACTAATAATTGGGGCCCCAGACAT
GGCGTTCCCTCGAATGAATAATATGAGTTTCTGACTTCTTCCCCCCTCCTTTCTCCTCCTCTTAGCCTCTTCAGG
TGTTGAAGCCGGAGCAGGTACCGGATGAACCGTGTACCCCCCACTAGCTGGCAATTTAGCCCACGCCGGGGCATC
CGTAGACCTGACAATCTTCTCACTTCACCTTGCAGGAATTTCATCAATTCTGGGGGCAATTAACTTTATTACTAC
CATCATTAACATGAAACCCACAACAGTCACTATGTACCAAATTCCGTTATTTGTTTGAGCCGTTCTTATTACAGC
CGTACTTCTTCTTCTGTCCCTGCCCGTTTTAGCCGCAGGGATTACAATGCTACTAACAGACCGCAACCTTAACAC
GACCTTCTTTGACCCTGCCGGAGGAGGTGACCCCATTCTCTACCAACACCTA 

The highlighted base shows the mismatch in all EURM-020 samples included in the characterisation 
study with the Hippoglossus hippoglossus reference sequences for COI in the BOLD system used for 
the identification of EURM-020 (Table 4, Section 7).  

37 

 
 
Annex E2: Additional information 

Average extracted DNA concentration per bottle

200.0
180.0
160.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0

]
L
µ
/
g
n
[
n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

7

145

322

541

615

710

988

1134

1252

1343

Bottle number

Figure  1:  Extracted  DNA  concentration  [ng/μL]  of  the  samples  per  bottle  for  the  characterisation 
study  of  EURM-020.  Average  of  duplicate  extractions  N  =  1,  n  =  2,  with  error  bars  expressed 
as smeas. Measurements were performed by UV spectrophotometry (Nanodrop). 

38 

 
 
 
 
 
 
European Commission 

EUR 30097 EN – Joint Research Centre – Directorate F – Health, Consumers and Reference Materials 
Title:  

The certification of the genetic identification of a Hippoglossus hippoglossus (Atlantic halibut) 
fish powder: EURM®-020 

Author(s): A.M. Kortekaas, H. Leys, J. Seghers, S. Trapmann 
Luxembourg: Publications Office of the European Union 
2020 –38 pp. – 21.0 x 29.7 cm 
EUR – Scientific and Technical Research series – ISSN 1831-9424 
ISBN 978-92-76-16230-8 
doi:10.2760/170544 

 
 
 
 
 
 
 
As the Commission's in-house science service, the Joint Research Centre's mission is to provide EU policies 
with independent, evidence-based scientific and technical support throughout the whole policy cycle. 

Working in close cooperation with policy Directorates-General, the JRC addresses key societal challenges 
while stimulating innovation through developing new methods, tools and standards, and sharing its know-
how with the Member States, the scientific community and international partners. 

Key policy areas include: environment and climate change; energy and transport; agriculture and food 
security; health and consumer protection; information society and digital agenda; safety and security, 
including nuclear; all supported through a cross-cutting and multi-disciplinary approach. 

K
J
-
N
A
-
3
0
0
9
7
-
E
N
-
N

doi:10.2760/170544 

ISBN: 978-92-76-16230-8