SIST EN ISO 21571:2005/A1:2013

SLOVENSKI STANDARD
SIST EN ISO 21571:2005/A1:2013
01-junij-2013
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in
njihovih produktov - Izolacija nukleinskih kislin - Dopolnilo A1 (ISO
21571:2005/Amd 1:2013)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Nucleic acid extraction - Amendment 1 (ISO 21571:2005/Amd 1:2013)
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und
ihren Produkten - Nukleinsäureextraktion - Änderung 1 (ISO 21571:2005/Amd 1:2013)
Produits alimentaires - Méthodes d'analyse pour la détection des organismes
génétiquement modifiés et des produits dérivés - Extraction des acides nucléiques -
Amendement 1 (ISO 21571:2005/Amd 1:2013)
Ta slovenski standard je istoveten z: EN ISO 21571:2005/A1:2013
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN ISO 21571:2005/A1:2013 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 21571:2005/A1:2013

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SIST EN ISO 21571:2005/A1:2013


EUROPEAN STANDARD
EN ISO 21571:2005/A1

NORME EUROPÉENNE

EUROPÄISCHE NORM
March 2013
ICS 67.050
English Version
Foodstuffs - Methods of analysis for the detection of genetically
modified organisms and derived products - Nucleic acid
extraction - Amendment 1 (ISO 21571:2005/Amd 1:2013)
Produits alimentaires - Méthodes d'analyse pour la Lebensmittel - Verfahren zum Nachweis von gentechnisch
détection des organismes génétiquement modifiés et des modifizierten Organismen und ihren Produkten -
produits dérivés - Extraction des acides nucléiques - Nukleinsäureextraktion - Änderung 1 (ISO 21571:2005/Amd
Amendement 1 (ISO 21571:2005/Amd 1:2013) 1:2013)
This amendment A1 modifies the European Standard EN ISO 21571:2005; it was approved by CEN on 1 March 2013.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for inclusion of this
amendment into the relevant national standard without any alteration. Up-to-date lists and bibliographical references concerning such
national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This amendment exists in three official versions (English, French, German). A version in any other language made by translation under the
responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the
official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21571:2005/A1:2013: E
worldwide for CEN national Members.

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SIST EN ISO 21571:2005/A1:2013
EN ISO 21571:2005/A1:2013 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 21571:2005/A1:2013
EN ISO 21571:2005/A1:2013 (E)
Foreword
This document (EN ISO 21571:2005/A1:2013) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the
secretariat of which is held by DIN.
This Amendment to the European Standard EN ISO 21571:2005 shall be given the status of a national
standard, either by publication of an identical text or by endorsement, at the latest by September 2013, and
conflicting national standards shall be withdrawn at the latest by September 2013.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 21571:2005/Amd 1:2013 has been approved by CEN as EN ISO 21571:2005/A1:2013 without
any modification.
3

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SIST EN ISO 21571:2005/A1:2013

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SIST EN ISO 21571:2005/A1:2013
INTERNATIONAL ISO
STANDARD 21571
First edition
2005-02-15
AMENDMENT 1
2013-03-15
Foodstuffs — Methods of analysis for
the detection of genetically modified
organisms and derived products —
Nucleic acid extraction
AMENDMENT 1
Produits alimentaires — Méthodes d’analyse pour la détection
des organismes génétiquement modifiés et des produits dérivés —
Extraction des acides nucléiques
AMENDEMENT 1
Reference number
ISO 21571:2005/Amd.1:2013(E)
©
ISO 2013

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SIST EN ISO 21571:2005/A1:2013
ISO 21571:2005/Amd.1:2013(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2013
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
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Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2013 – All rights reserved

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SIST EN ISO 21571:2005/A1:2013
ISO 21571:2005/Amd.1:2013(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International
Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies
casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
Amendment 1 to ISO 21571:2005 was prepared by Technical Committee ISO/TC 34, Food products,
Subcommittee SC 16, Horizontal methods for molecular biomarker analysis.
© ISO 2013 – All rights reserved iii

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SIST EN ISO 21571:2005/A1:2013

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SIST EN ISO 21571:2005/A1:2013
ISO 21571:2005/Amd.1:2013(E)
Foodstuffs — Methods of analysis for the detection of
genetically modified organisms and derived products —
Nucleic acid extraction
AMENDMENT 1
Page v, Introduction
Delete the reference to ISO 21568.
Replace the reference to ISO 24276 with the following:
ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — General requirements and definitions
Page 1, Clause 2
Replace the reference to ISO 24276:—with the following:
ISO 24276:2006, Foodstuffs — Methods of analysis for the detection of genetically modified organisms
and derived products — General requirements and definitions
Delete the reference to Footnote 1) and the footnote itself.
Delete the reference to ISO 21568.
Page 2, 5.1.1, paragraph 3, line 2
Delete “(e.g. 3 000 particles at an LOD of 0,1 %) to allow a statistically valid conclusion to be made
(see ISO 21568)”.
Page 3, 5.1.2, paragraph 1, line 2
Replace “procedures described in ISO 24276” by “laboratory design specified in ISO 24276:2006, 5.3.2”.
Page 3, 5.1.2, paragraph 2
Replace by: “Laboratory samples shall be sufficiently homogeneous before reducing or grinding and
before taking the test portion.”
Page 3, 5.1.2, paragraph 4, lines 3 and 6
Delete “as specified in ISO 21568” and “as defined ISO 21568”.
Page 3, 5.1.2, paragraph 8, 1st sentence
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SIST EN ISO 21571:2005/A1:2013
ISO 21571:2005/Amd.1:2013(E)

Replace by “After such treatment, homogenize the whole laboratory sample.”
Page 4, 5.2.1
After paragraph 7, beginning “Re-suspend .”, insert an example as follows:
EXAMPLE A tris/EDTA (TE, 1× or 0,1×) buffer adjusted to pH 8,0 is appropriate for re-suspending or diluting
DNA.
Page 5, 5.3.1, paragraph 1
Add a new final sentence:
The total amount of DNA used in the PCR should be determined as well as the total amount of target
taxon DNA shall be considered, as non-target taxon DNA can affect the efficiency of the PCR.
Page 6,Clause 6, paragraph 2
Replace the phrase in parentheses by: “(e.g. if the analysis to be performed is PCR, the quality of the
extracted DNA should be assessed using adequate PCR controls).”
Page 9, A.1.1.6.5
2) 1)
Replace by and renumber the footnote.
Page 10, A.1.1.8
3) 2)
Replace by and renumber the footnote.
Page 19, A.1.4.2
Delete paragraph 2: “This method applied to microbial pellets or mycelium mats has been submitted for
[17]
interlaboratory validation exercises .”
Page 33, after A.5.1.8, add A.5.2
A.5.2 Guanidine chloroform method: Protocol for soybean lecithin
A.5.2.1 Purpose, relevance and scientific basis
Soybean lecithin is a frequent ingredient and is used in many food products as an emulsifier. Soybean
lecithin can be either produced using genetically modified (GM) or non-modified soybeans. The method
described here can be used to extract DNA present in the sample in order to perform subsequent PCR
analyses for detection of genetically modified DNA sequences derived from GM soybeans.
The method is based on Reference [44], and a very similar procedure has been validated in a collaborative
trial in Switzerland. In that study, for the purpose of interpretation, the quantity of extracted DNA was
spectrometrically determined (Reference [35], section 1.3). The Chemical and Veterinary Institute
Freiburg, Germany, conducted an additional collaborative trial with 12 participating laboratories where
the amount of extractable and amplifiable soybean DNA was determined by means of quantitative real-
time PCR. For the results, see Reference [45].
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SIST EN ISO 21571:2005/A1:2013
ISO 21571:2005/Amd.1:2013(E)

A.5.2.2 Scope
This method describes a procedure for DNA extraction from soybean lecithins in raw and cold-pressed
vegetable oils, respectively. If the DNA content of the sample material is low, the real-time PCR approach
is used for quantitation of the amount of DNA isolated from a test portion and for the calculation of the
practical limit of detection achievable in PCR analysis of the extracted DNA, respectively.
A.5.2.3 Validation status and performance criteria
A.5.2.3.1 Validation criteria
The method described in this subclause has been validated in a collaborative trial by determining
the quantity of extractable, amplifiable soybean DNA by means of quantitative real-time PCR. The
collaborative study was performed in accordance with the IUPAC protocol (Rererence [46]).
A.5.2.3.2 Robustness of the method
The method has been routinely used in enforcement and private GMO testing laboratories in Germany and
Switzerland for more than 10 years and no problems have been reported. Although specific robustness
data (e.g. by modifying method parameters) are not available, the experiences of the laboratories showed
that small variations in conditions does not interfere with the performance of the method.
A.5.2.3.3 Intralaboratory trial
The materials used in the collaborative trial study were tested in the method developers’ laboratory
regarding the intralaboratory precision of the method.
For estimation of the precision, five DNA extractions from each of five soybean lecithins were prepared
and measured by real-time PCR under repeatability conditions using a soybean lectin gene specific
[41]
method according to ISO 21570:2005, C.2. The results are given in Table A.6.
Table A.6 — Intralaboratory validation of the DNA extraction method with five soybean lecithin
samples (n = 5 extractions each)
Repeatability standard devia- Coefficient of variation of
Lecithin sample
Mean lectin copy No. tion, s repeatability, C
r V, r
No.
copy No. %
1 20 464 5 367 26
2 3 203 620 19
3 2 005 306 15
4 6 978 331 5
5 14 8 61
Prior to the collaborative trial, DNA was extracted from five commercial soybean lecithins and tested for
PCR inhibition. The DNA prepared from each sample was further diluted using TE buffer (1 volume DNA
diluted with 4 volumes of TE; 1 volume of first DNA dilution diluted with 4 volumes of TE, 1 volume of
second DNA dilution diluted with 4 volumes of TE). The difference between the calculated (extrapolated)
C -value of the undiluted DNA sample and the measured C -value of each DNA dilution was below 0,1,
t t
showing that DNA preparations were free of PCR-inhibitors.
A.5.2.3.4 Collaborative trial
A collaborative trial (validation study) was carried out in 12 laboratories (Reference [45]). Five
commercial soybean lecithin samples were used. Each laboratory received 15 coded samples and a
standard DNA for calibration. The samples were distributed in a way that each participant received
three identical samples of each of the five soybean lecithins. A single DNA extraction was performed per
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SIST EN ISO 21571:2005/A1:2013
ISO 21571:2005/Amd.1:2013(E)

sample by each laboratory. The returned results were assigned to the five different lecithin samples for
evaluation of the collaborative trial.
The extracted DNAs were tested in a real-time PCR amplifying a DNA sequence of the soybean lectin
[41]
gene according to the method in ISO 21570:2005, C.2. Standard-DNA for calibration was prepared
1)
3)
from soybean flour [ERM BF410a ] by using the Plant Mini Kit (Qiagen, Hilden/Germany) starting
with a CTAB extraction (see A.3). The concentration of DNA was estimated fluorimetrically with
3)
the PicoGreen dsDNA method (Reference [17]). DNA-standards were defined by the copy number
(cp) of haploid genome equivalents per microlitre. For the calculation, a haploid genome mass for
soybean of 1,13 pg was assumed. A dilution series was prepared ranging from about 50 000 cp/5 µl
3)
to 80 cp/5 µl. Seven laboratories used ABI real-time PCR equipment (ABI 7000, 7500, 7700), and five
3)
laboratories used the Light Cycler (Roche) with the following modifications to the protocol described
[41]
in ISO 21570:2005, C.2: PCR was performed in a 20 µl final volume containing QuantiTect Probe PCR
3)
Master Mix (Qiagen), primers GM1-F and GM1-R at 500 nmol/l each and 150 nmol/l of probe GM1. Real-
time PCR program was: initial step with 900 s at 95 °C, then 45 cycles with 10 s at 95 °C, 30 s at 60 °C
and 30 s at 72 °C. Fluorescence signal acquisition was done within the elongation step. The temperature
ramping rate was set to 2°C/s.
As a criterion for the suitability of the method, the practical limit of detection, LOD , for genetically
prac
modified soya was determined in accordance with ISO 24276. The value was calculated individually for
each sample using Formula (A.1):
LOD
abs
LOD %= ×100 (A.1)
prac
c
...