SIST EN ISO 21569:2005/A1:2013
(Amendment)Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Qualitative nucleic acid based methods - Amendment 1 (ISO 21569:2005/Amd 1:2013)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Qualitative nucleic acid based methods - Amendment 1 (ISO 21569:2005/Amd 1:2013)
This standard describes the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and the competency of the laboratory analysing foodstuffs for the presence of genetically modified organisms (GMOs) using the polymerase chain reaction (PCR). It relates to the general requirements for the specific amplification of target nucleic acid sequences (DNA) and for the confirmation of the identity of the amplified nucleic acid sequence.
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und ihren Produkten - Qualitative auf Nukleinsäuren basierende Verfahren - Änderung 1 (ISO 21569:2005/Amd 1:2013)
Produits alimentaires - Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés - Méthodes qualitatives basées sur l'utilisation des acides nucléiques - Amendement 1 (ISO 21569:2005/Amd 1:2013)
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in njihovih produktov - Kvalitativne metode na osnovi nukleinske kisline - Dopolnilo A1 (ISO 21569:2005/Amd 1:2013)
Ta standard opisuje zahteve za zagotavljanje kakovosti, ki naj bi jih laboratorij upošteval, da se zagotovita kakovost in celovitost podatkov ter kompetentnost laboratorija, ki ugotavlja prisotnost gensko spremenjenih organizmov (GMO) v živilih s polimerazno verižno reakcijo (PCR). Nanaša se na splošne zahteve za posebno povečanje ciljnih sekvenc nukleinske kisline (DNK) in za potrditev identitete povečane sekvence nukleinske kisline.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 21569:2005/A1:2013
01-junij-2013
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in
njihovih produktov - Kvalitativne metode na osnovi nukleinske kisline - Dopolnilo
A1 (ISO 21569:2005/Amd 1:2013)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Qualitative nucleic acid based methods - Amendment 1 (ISO
21569:2005/Amd 1:2013)
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und
ihren Produkten - Qualitative auf Nukleinsäuren basierende Verfahren - Änderung 1 (ISO
21569:2005/Amd 1:2013)
Produits alimentaires - Méthodes d'analyse pour la détection des organismes
génétiquement modifiés et des produits dérivés - Méthodes qualitatives basées sur
l'utilisation des acides nucléiques - Amendement 1 (ISO 21569:2005/Amd 1:2013)
Ta slovenski standard je istoveten z: EN ISO 21569:2005/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 21569:2005/A1:2013 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 21569:2005/A1:2013
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SIST EN ISO 21569:2005/A1:2013
EUROPEAN STANDARD
EN ISO 21569:2005/A1
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2013
ICS 67.050
English Version
Foodstuffs - Methods of analysis for the detection of genetically
modified organisms and derived products - Qualitative nucleic
acid based methods - Amendment 1 (ISO 21569:2005/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 - Qualitative
produits dérivés - Méthodes qualitatives basées sur auf Nukleinsäuren basierende Verfahren - Änderung 1 (ISO
l'utilisation des acides nucléiques - Amendement 1 (ISO 21569:2005/Amd 1:2013)
21569:2005/Amd 1:2013)
This amendment A1 modifies the European Standard EN ISO 21569:2005; it was approved by CEN on 14 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 21569:2005/A1:2013: E
worldwide for CEN national Members.
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SIST EN ISO 21569:2005/A1:2013
EN ISO 21569:2005/A1:2013 (E)
Contents Page
Foreword .3
2
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SIST EN ISO 21569:2005/A1:2013
EN ISO 21569:2005/A1:2013 (E)
Foreword
This document (EN ISO 21569:2005/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 21569:2005 shall be given the status of a national
standard, either by publication of an identical text or by endorsement, at the latest by October 2013, and
conflicting national standards shall be withdrawn at the latest by October 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 21569:2005/Amd 1:2013 has been approved by CEN as EN ISO 21569:2005/A1:2013 without
any modification.
3
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SIST EN ISO 21569:2005/A1:2013
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SIST EN ISO 21569:2005/A1:2013
INTERNATIONAL ISO
STANDARD 21569
First edition
2005-06-15
AMENDMENT 1
2013-04-01
Foodstuffs — Methods of analysis for
the detection of genetically modified
organisms and derived products —
Qualitative nucleic acid based methods
AMENDMENT 1
Produits alimentaires — Méthodes d’analyse pour la détection
des organismes génétiquement modifiés et des produits dérivés —
Méthodes qualitatives basées sur l’utilisation des acides nucléiques
AMENDEMENT 1
Reference number
ISO 21569:2005/Amd.1:2013(E)
©
ISO 2013
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SIST EN ISO 21569:2005/A1:2013
ISO 21569:2005/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
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ISO copyright office
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Published in Switzerland
ii © ISO 2013 – All rights reserved
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SIST EN ISO 21569:2005/A1:2013
ISO 21569:2005/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 21569:2005was prepared by Technical Committee ISO/TC 34, Food products,
Subcommittee SC 16, Horizontal methods for molecular biomarker analysis.
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SIST EN ISO 21569:2005/A1:2013
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SIST EN ISO 21569:2005/A1:2013
ISO 21569:2005/Amd.1:2013(E)
Foodstuffs — Methods of analysis for the detection of
genetically modified organisms and derived products —
Qualitative nucleic acid based methods
AMENDMENT 1
No attempt has been made in this amendment to update the footnote numbering to fit in with the scheme
adopted in ISO 21569:2005. The footnote numbers given are for use refer solely within this amendment.
Page v, Introduction, paragraph 1
Delete “— Sampling (ISO 21568)”.
Page 2, Clause 2, ISO 24276
Delete the footnote and update the entry to read:
ISO 24276:2006, Foodstuffs — Methods of analysis for the detection of genetically modified organisms
and derived products — General requirements and definitions
Page 2, 4.1, paragraph 2
Delete the existing text and insert the following.
A qualitative result shall clearly demonstrate the presence or absence of the genetic element under
study, relative to appropriate controls.
NOTE Detection limits and size of the test portion are critical aspects of a method.
Page 2, 7.3.3.3.3, paragraph 2
Delete “a representative” and insert “an appropriate” so that the text reads as follows.
Primers designed to detect taxon-specific target sequences should be shown to detect these
sequences reliably in an appropriate number of different members of the taxon.
Page 6, 8.1 a) and b)
In both cases, delete “ISO 24276:—”, and insert “ISO 24276:2006.
Page 7, 9.4
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ISO 21569:2005/Amd.1:2013(E)
Delete the existing text and insert the following.
Results within the same test portion shall be consistent. In case of +/− results for the two replicates,
repeat the two PCR for the respective test portion. If the two novel replicates are tested +/− or −/−,
the test portion is considered as negative.
Results from all test portions shall be consistent. When at least one test portion gives a positive
result and at least one gives a negative result, the analysis shall be repeated.
If at least one repetition of the procedure, beginning with the nucleic acid extraction, gives ambiguous
results such as a positive and a negative result, the report should state that the sample is negative
at the limit of detection (LOD).
Page 7, Clause 10, list item 2
Delete the existing text and insert the following.
— the specificity of the analytical method (event specific, construct specific, or screening method);
Page 23, Annex A
Insert A.5 and A.6 after the existing text.
A.5 Target taxon-specific method for the detection of DNAs derived from rice
A.5.1 Purpose, relevance and scientific basis
The GMO Detection Laboratory of Shanghai Jiao Tong University (GMDL-SJTU) organized a collaborative
study for validation of the applicability of a target taxon-specific method using the rice sucrose phosphate
synthase (SPS) gene as an endogenous gene for qualitative analysis of genetically modified (GM) or non-
GM rice. This study involved 12 laboratories from Spain, Korea, Lithuania, Slovenia, Japan, Italy, and China.
The operational procedure of the collaborative study comprised the following modules:
— qualitative PCR for validation of the heterogeneity of the SPS gene among rice cultivars for different
geographic and phylogenetic origins;
— qualitative PCR for validation of the species specificity of SPS gene for rice;
— qualitative PCR for evaluation of the LOD of the established SPS qualitative PCR assay.
The collaborative study was carried out in accordance with Reference [44].
The results of the collaborative study as well as the related protocol are given in A.5.3.
A.5.2 Principle
The method has been optimized for rice seeds and other processed products such as seed powder.
Applicability of the SPS gene was evaluated in this collaborative study using DNA samples extracted
from rice seeds and other plant materials.
The collaborative study organizer provided method-specific reagents (primers, probes, reaction master
mix), and the test DNA samples extracted from rice materials to collaborative study participants.
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A.5.3 Validation status and performance criteria
A.5.3.1 Robustness of the method
Robustness has been tested on the SPS gene qualitative PCR system for three different annealing
temperatures (i.e. 56 °C, 58 °C, and 60 °C), on three different DNA samples containing known amounts
of rice DNA (10 ng, 1 ng, 0,1 ng rice genome DNA samples) and with three repetitions per sample.
The qualitative PCR systems demonstrated the expected robustness and performed well at all three
annealing temperatures and three concentrations of the rice DNA samples.
1)
The SPS gene qualitative PCR system was also tested on different thermal cyclers (PTC-100, MJ
Research and instruments from Bio-Rad and Applied Biosystems), on three different reaction volumes
(25 μl, 30 μl, and 50 μl) and three repetitions per volume. The qualitative PCR systems had the expected
robustness and performed well on different thermal cyclers and with different reaction volumes.
A.5.3.2 Intralaboratory trial
The rice SPS gene has been described as being suitable for use as an endogenous reference gene in rice
identification and quantification (Reference [44]). The detailed technical information was modified
from Reference [44].
For sample preparation in the collaboration study, all the DNA samples were extracted by the GMDL-
SJTU using the CTAB method adopted from ISO 21571:2005, A.3. Spectrophotometric quantification
of DNA extracted was performed using a method adopted from ISO 21571:2005, B.1. After the DNA
quantification, a qualitative PCR using an 18S PCR system (Reference [45]) was carried out to provide
data about possible PCR inhibition.
The SPS gene PCR system was tested using rice genomic DNA by three researchers at the GMDL-SJTU.
The results were satisfactory; in particular, for qualitative PCR, the results show that the SPS gene is
specific for rice, and the LOD is about 0,1 %.
A.5.3.3 Collaborative trial
For the collaborative study, each participant received 12 rice DNA samples for heterogeneity testing; 10
DNA samples from plants other than rice plus one DNA sample from rice for species specificity testing; and
10 serially diluted rice samples for LOD evaluation. A negative and a positive control were also included.
The heterogeneity of the SPS gene among rice cultivars was evaluated using 12 rice cultivars from
different geographic and phylogenetic origins in China, such as Najing14, Taibei309, Shengnong265,
Jinyinbao, Minghui78, Huke3, Guangluai4, Zhe733, Hejiang19, Baizhehu, Xiangwanxian9 and Nipponbare.
The results returned from 12 laboratories showed that out of a total of 144 (12 × 12) rice DNA samples,
143 positive results were obtained using the SPS gene PCR system. This means that the false-negative
rate of the SPS gene PCR system for rice is 0,69 % (1/144) (see Table A.14). These data suggest that there
is low heterogeneity of the SPS gene in the target region.
The species specificity of the SPS gene was validated using a rice genome DNA sample (Guangluai4) and
10 other plant DNAs that were evolutionarily related to rice, common crops or model plants, such as
the fruit materials of bamboo (Phyllostachys spp.), green bristlegrass [Setaria viridis (L.) Beauv.], barley
(Hordeum vulgare), wheat (Triticum aestivum), foxtail millet (Setaria italica), rapeseed (Brassica napus),
tomato (Lycopersicon esculentum), potato (Solanum tuberosum), soya bean (Glycine max) and thale cress
(Arabidopsis thaliana). The results returned from 12 laboratories showed that out of a total of 120 (10 ×
12), non-rice plant DNA samples, 118 negative results were obtained using the SPS gene PCR system. This
means that the false-positive rate of the SPS gene PCR system for other 10 plant materials was 1,67 %
(2/120) (see Table A.14). These data suggest that the SPS gene is species specific for detection of rice.
1) Example of a suitable product available commercially. This information is given for the convenience of users of
this document and does not constitute an endorsement by ISO of this product.
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Table A.14 — The results of heterogeneity and of specificity testing of the qualitative PCR
Parameter (collaborative study of 2007) Value
No. laboratories 12
No. laboratories submitting results 12
No. samples per laboratory 22
No. accepted results 264
No. samples containing rice 144
No. samples not containing rice 120
False-positive results 2 (1,67 %)
False-negative results 1 (0,69 %)
The LOD of the SPS gene PCR system was validated using mixed powder containing maize and various
quantities of rice seed by means of qualitative PCR: all 12 laboratories detected the SPS gene in the DNA
sample extracted from mixed powder containing 0,1 % mass fraction or higher of rice, and two in 12
laboratories detected it from mixed powder containing 0,01 % mass fraction of rice. These data suggest
that the LOD of the SPS gene PCR system is as low as 0,1 % mass fraction (see Table A.15).
Table A.15 — The results of the LOD test of the qualitative PCR
Rice to maize mass fraction, m /m
rice maize
Parameter (collaborative study of
2007)
10 % 1 % 0,1 % 0,05 % 0,01 %
No. laboratories 12 12 12 12 12
No. laboratories submitting results 12 12 12 12 12
No. samples per laboratory 2 2 2 2 2
No. laboratories accepted results 12 12 12 12 12
Positive results 12 (100 %) 12 (100 %) 12 (100 %) 4 (33,33 %) 2 (16,67 %)
A.5.3.4 Molecular selectivity
A.5.3.4.1 General
For qualitative validation of the SPS gene as a specific rice gene, a 279 bp fragment of the conserved
region of the SPS gene was selected and amplified using specific primers.
A.5.3.4.2 Experimental
DNA samples extracted from 11 different plant materials (including rice) were analysed by the SPS gene
PCR system as described (Reference [44]). Among the 11 samples, only rice DNA gave positive results.
The other 10 samples (see A.5.3.3) gave negative results.
The DNA samples extracted from 12 different rice cultivars were analysed by the SPS gene PCR system
reported in Reference [44]. All 12 samples gave positive results.
A.5.3.4.3 Theoretical
The theoretical specificity of the SPS gene primer was assessed through a homology search using the
BLASTN 2.0MP-WashU program (Reference [82], search date: 2010-01-09). The 279 bp sequence used
as query is part of the NCBI accession number U33175 (nucleotides 1055–1333). The results of the basic
local alignment search tool (BLAST) confirmed the complete identity of the query sequence with rice
SPS gene sequence, and no homology with other genes and species.
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SIST EN ISO 21569:2005/A1:2013
ISO 21569:2005/Amd.1:2013(E)
A.5.4 Principle and summary
This methodology is a PCR procedure for the applicability of the SPS gene for use as a rice endogenous
gene in qualitative detection of GM or non-GM rice. Heterogeneity, species specificity of the SPS gene and
LOD were evaluated as part of the validation of this method. The 279 bp PCR product was visualized by
agarose gel electrophoresis.
A.5.5 Terms and definitions
[40]
For the purposes of this document, the terms and definitions of ISO 5725-1 and ISO 24276 apply.
A.5.6 Sample type and amounts
In the following, the data from the collaborative study are given as examples for sample types and
sample amounts adequate for this method.
DNA samples extracted from the seeds of 12 rice cultivars, 10 other plant materials (see A.5.3.3) and
the mixed powder containing different mass fractions of rice in maize seed power, were used in this
collaborative study.
The participants received the following samples.
— 12 DNA samples from 12 different rice cultivars that are widely planted in different region of
China (i.e. Najing14, Taibei309, Shengnong265, Jinyinbao, Minghui78, Huke3, Guangluai4, Zhe733,
Hejiang19, Baizhehu, Xiangwanxian9, and Nipponbare), 20 ng/μl, 50 μl each. These DNA samples
were used to validate the heterogeneity of the SPS gene among rice cultivars.
— 11 DNA samples from rice (Guangluai4) and 10 other plant materials which are related to rice
(i.e. bamboo, green bristlegrass, barley, wheat, foxtail millet) or common GM crops (i.e. rapeseed,
tomato, potato and soya bean) or model plants (i.e. thale cress), 20 ng/μl, 50 μl each. These DNA
samples were used to validate the species specific of the SPS gene in rice.
— 10 DNA samples from mixed powders of maize with different mass fractions of rice, 20 ng/μl, 50 μl
each. These DNA samples were double blind replicates of the series of five rice concentrations used
for testing the LOD of the SPS gene PCR system.
— Negative DNA target control (labelled N): salmon sperm DNA (20 ng/µl).
— Positive DNA target control (labelled P): rice (Guangluai4) genomic DNA (20 ng/µl). All the DNA
samples were purified using the CTAB method by the GMDL-SJTU. The negative and positive DNA
target controls were used for each PCR plate.
— Reaction reagents, primers for the SPS gene PCR system as follows:
— primer pair for conventional PCR: SPS-F/SPS-R;
— DNA dilution solution [0,1× tris–EDTA (TE), 1,2 ml].
A.5.7 Limit of detection and range of use
DNA was extracted from five mixed powder samples containing different amounts of rice. These samples
were analysed by the SPS PCR system as described (Reference [44]). Positive results were obtained with
samples containing mass fractions of 10 %, 1 %, and 0,1 % rice. The other two samples (containing mass
fractions of 0,05 % and 0,01 %) gave negative results.
According to the developed method, the relative LOD of the qualitative PCR method is about 0,1 % mass
fraction. The SPS gene PCR system can be used for specific detection and identification of rice materials
in other plant materials.
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ISO 21569:2005/Amd.1:2013(E)
A.5.8 Estimation of measurement uncertainty
The reproducibility of the method is given by the results of the collaborative trial (see A.5.3.3).
A.5.9 Interferences
In the studies performed, no additional information about interferences have been observed.
A.5.10 Physical and environmental conditions
See ISO 24276 for details. For example:.
— maintain strictly separated working areas for DNA preparation, PCR set-up, PCR amplification and
electrophoresis;
— any residual DNA should be removed from all equipment prior to its use;
— in order to avoid contamination, use filter pipette tips protected against aerosol;
— use only powder-free gloves and change them frequently.
A.5.11 Apparatus and equipment
A.5.11.1 Microcentrifuge.
A.5.11.2 Freezer operating at −20 °C and refrigerator operating at 4 °C.
A.5.11.3 Micropipettes.
A.5.11.4 Mixer, e.g. vortex mixer.
A.5.11.5 Microcentrifuge tubes, capacities: 0,2 ml, 1,5 ml, and 2,0 ml.
A.5.11.6 Tips and aerosol-resistant tips for micropipettes.
A.5.11.7 Rack for reaction tubes.
A.5.11.8 PVC or latex gloves.
A.5.11.9 DNA amplifying equipment (thermal cycler or equivalent apparatus).
A.5.11.10 Electrophoresis equipment, with power supply.
A.5.11.11 Imaging system for gel analysis.
A.5.11.12 Microwave oven (optional).
A.5.12 Reagents and materials
A.5.12.1 General
Unless otherwise stated, only reagents that conformed to the specifications of ISO 24276 and only
molecular biology grade water or water of equivalent purity were used.
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A.5.12.2 Qualitative PCR
A.5.12.2.1 PCR buffer (without MgCl ) 10×.
2
A.5.12.2.2 MgCl solution 25 mmol/l.
2
A.5.12.2.3 dNTP solution 2,5 mmol/l each.
A.5.12.2.4 Primer (see Table A.16).
A.5.12.2.5 DNA polymerase, thermostable.
A.5.12.3 Electrophoresis
For details see e.g. ISO 21571:2005, B.1.
A.5.12.3.1 Loading buffer (10 g/l sodium dodecyl sulfate, 500 g/l glycerol, 0,5 g/l bromophenol blue), 10×.
A.5.12.3.2 DNA size standard.
A.5.13 Sample collection, transportation, preservation, and storage
DNA solutions may be stored at 4 °C for a maximum of 1 week, or at −20 °C for long-term storage.
A.5.14 Preparation of test sample
Ensure that the test sample is representative of the laboratory sample, e.g. by grinding or homogenization.
Measures and operational steps to be taken into consideration are described in ISO 21571 and ISO 24276.
A.5.15 Instrument calibration
[41]
Instruments, e.g. thermal cyclers and pipettes should be calibrated as per ISO/IEC 17025.
A.5.16 Analysis steps
A.5.16.1 Preparation of the DNA for qualitative PCR
Extract DNA from the samples by using an adequate extraction method, e.g. ISO 21571:2005, A.3, CTAB
extraction method. Thaw, mix gently and centrifuge the DNA samples needed for the PCR run. Keep
thawed reagents at 1 °C to 4 °C on ice.
A.5.16.2 PCR reagents
A.5.16.2.1 Conventional PCR master mix
A conventional PCR reaction mixture containing the following: 1× PCR buffer, 200 µmol/l each of dNTPs,
2+
2,5 mmol/l Mg , 330 nmol/l forward/reverse primer, 1 unit Taq DNA polymerase.
A.5.16.2.2 Primers
See Table A.16.
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Table A.16 — Oligonucleotide primer sequences for qualitative PCR
Name Oligonucleotide DNA sequence (5’ to 3’)
Qualitative PCR primer sequence
SPS primer F TTg CgC CTg AAC ggA TAT
SPS primer R ggA gAA gCA CTg gAC gAgg
A.5.16.3 Procedure
A.5.16.3.1 General
The qualitative PCR for rice SPS gene was developed for a total volume of 30 µl per reaction mixture. The
use of 100 ng of template DNA per reaction well is recommended.
Thaw, mix gently and centrifuge the PCR master mix needed for the run. Keep thawed reagents at 1 °C
to 4 °C on ice.
Distribute 25 µl/tube of the master mixture to 200 µl PCR reaction tubes. Add 5 µl of DNA solution
samples, rice positive control, negative control, and blank control (H O) to the tubes, respectively.
2
Mix the PCR tubes gently, centrifuge in the microcentrifuge at 1 000 × g for 10 s.
Insert the plate into the instrument.
Run the PCR with qualitative PCR cycling conditions.
A.5.16.4 PCR controls
See 7.5 and ISO 24276.
A.5.16.5 Temperature–time programme
1) 1)
The PCR assay has been optimized for use in a PTC-100 (MJ Research) and an ABI 2720 (Applied
Biosystems) thermal cycler PCR machine. Although other PCR machines may be used, the thermal cycling
conditions may need to be verified. The qualitative PCR cycling parameters are indicated in Table A.17.
Table A.17 — Qualitative PCR temperature–time programme
Temperature Time
Step Stage No. cycles
°C s
1 Activation and initial denaturation 94 900 1×
2a Denaturation 94 30
2b Amplification Annealing 58 30 35×
2c Elongation 72 30
3 Final elongation 72 420 1×
A.5.16.6 Detection
After the PCR programme has finished, transfer 3 μl of 10× loading buffer to each reaction tube and mix
with the PCR products.
Load 10 μl of each PCR product on to electrophoresis gel (20 g/l agarose, 0,5 µg/ml ethidium bromide),
respectively.
Run the gel in the electrophoresis equipment under 5 V/cm, 20 min.
Record gel image with an UV gel documentation or similar system.
8 © ISO 2013 – All rights reserved
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SIST EN ISO 21569:2005/A1:2013
ISO 21569:2005/Amd.1:2013(E)
A fragment of 279 bp should be the specific product; other bands existing in the agarose electrophoresis
are unexpected products.
A.5.16.7 Accept or reject criteria
Method performance requirements used to evaluate the results from the collaborative study are as follows.
A fragment of 279 bp should be detected in the rice positive control (sample P), and no target fragment
should be detected in negative control (sample N) and blank. The detection of fragments with a size of
279 bp indicates that the sample DNA solution contains amplifiable DNA of SPS, and the result is positive,
otherwise the result is negative.
A.5.17 Sample identification
All samples should be identified unambiguously.
A.5.18 Interpretation and calculations of the results
The expected amplicon length of SPS is 279 bp in size.
A fragment of 279 bp should be detected in the rice positive control (sample P), and no target fragment
should be detected in negative control (sample N) and blank. The detection of fragments with a size of
279 bp indicates that the sample DNA solution contains amplifiable DNA of SPS, and the result is positive,
otherwise the result is negative.
A.6 Target taxon-specific method for the detection of components derived from
tomato
A.6.1 Purpose, relevance and scientific basis
The LAT52 gene encodes a heat-stable, glycosylated, cysteine-rich protein that is necessary for tomato
pollen development. The LAT52 detectio
...
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