SIST EN ISO 21570:2006/A1:2013
(Amendment)Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Quantitative nucleic acid based methods (ISO 21570:2005/Amd 1:2013)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Quantitative nucleic acid based methods (ISO 21570:2005/Amd 1:2013)
This International Standard provides the overall framework of quantitative methods for the detection genetically modified organisms (GMOs) in foodstuffs, using the polymerase chain reaction (PCR). It defines general requirements for the specific amplification of DNA target sequences, in order to quantify relative GMO-derived DNA content and to confirm the identity of the amplified DNA sequence. Guidelines, minimum requirements and performance criteria laid down in this International Standard intended to ensure that comparable, accurate and reproducible results are obtained in different laboratories. This International Standard has been established for food matrices, but is also applicable to other matrices, e.g. feed and plant samples from the environment. Specific examples of methods are provided in Annexes A to D.
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und ihren Produkten - Quantitative auf Nukleinsäuren basierende Verfahren (ISO 21570:2005/Amd 1:2013)
No scope available.
Produits alimentaires - Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés - Méthodes quantitatives basées sur l'utilisation des acides nucléiques (ISO 21570:2005/Amd 1:2013)
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in njihovih produktov - Kvantitativne metode na osnovi nukleinske kisline - Dopolnilo A1 (ISO 21570:2005/Amd 1:2013)
Ta mednarodni standard določa splošni okvir kvantitativnih metod za odkrivanje gensko spremenjenih organizmov (GMO) v živilih s polimerazno verižno reakcijo (PCR). Določa splošne zahteve za posebno povečanje ciljnih sekvenc DNK, da bi se količinsko opredelila relativna vsebnost DNK iz gensko spremenjenih organizmov in potrdila identiteta povečane sekvence DNK. Smernice, minimalne zahteve in merila učinkovitosti iz tega mednarodnega standarda so namenjeni zagotavljanju, da se v različnih laboratorijih pridobijo primerljivi, točni in ponovljivi rezultati. Ta mednarodni standard je bil pripravljen za živilske matrice, a se uporablja tudi za druge matrice, npr. za vzorce krme in rastlin iz okolja. Posebni primeri metod so opisani v dodatkih od A do D.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 21570:2006/A1:2013
01-junij-2013
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in
njihovih produktov - Kvantitativne metode na osnovi nukleinske kisline - Dopolnilo
A1 (ISO 21570:2005/Amd 1:2013)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Quantitative nucleic acid based methods (ISO 21570:2005/Amd
1:2013)
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und
ihren Produkten - Quantitative auf Nukleinsäuren basierende Verfahren (ISO
21570:2005/Amd 1:2013)
Produits alimentaires - Méthodes d'analyse pour la détection des organismes
génétiquement modifiés et des produits dérivés - Méthodes quantitatives basées sur
l'utilisation des acides nucléiques (ISO 21570:2005/Amd 1:2013)
Ta slovenski standard je istoveten z: EN ISO 21570:2005/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 21570:2006/A1:2013 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 21570:2006/A1:2013
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SIST EN ISO 21570:2006/A1:2013
EUROPEAN STANDARD
EN ISO 21570:2005/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 - Quantitative nucleic
acid based methods (ISO 21570:2005/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 - Méthodes quantitatives basées sur Quantitative auf Nukleinsäuren basierende Verfahren (ISO
l'utilisation des acides nucléiques (ISO 21570:2005/Amd 21570:2005/Amd 1:2013)
1:2013)
This amendment A1 modifies the European Standard EN ISO 21570:2005; it was approved by CEN on 11 April 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 21570:2005/A1:2013: E
worldwide for CEN national Members.
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SIST EN ISO 21570:2006/A1:2013
EN ISO 21570:2005/A1:2013 (E)
Contents Page
Foreword .3
2
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SIST EN ISO 21570:2006/A1:2013
EN ISO 21570:2005/A1:2013 (E)
Foreword
This document (EN ISO 21570:2005/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 21570:2005 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 21570:2005/Amd 1:2013 has been approved by CEN as EN ISO 21570:2005/A1:2013 without
any modification.
3
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SIST EN ISO 21570:2006/A1:2013
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SIST EN ISO 21570:2006/A1:2013
INTERNATIONAL ISO
STANDARD 21570
First edition
2005-11-01
AMENDMENT 1
2013-04-15
Foodstuffs — Methods of analysis for
the detection of genetically modified
organisms and derived products —
Quantitative nucleic acid based
methods
AMENDMENT 1
Produits alimentaires — Méthodes d’analyse pour la détection
des organismes génétiquement modifiés et des produits dérivés —
Méthodes quantitatives basées sur l’utilisation des acides nucléiques
AMENDEMENT 1
Reference number
ISO 21570:2005/Amd.1:2013(E)
©
ISO 2013
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SIST EN ISO 21570:2006/A1:2013
ISO 21570:2005/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
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Published in Switzerland
ii © ISO 2013 – All rights reserved
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SIST EN ISO 21570:2006/A1:2013
ISO 21570:2005/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 21570:2005 was 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 21570:2006/A1:2013
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SIST EN ISO 21570:2006/A1:2013
ISO 21570:2005/Amd.1:2013(E)
Foodstuffs — Methods of analysis for the detection of
genetically modified organisms and derived products —
Quantitative 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 21570:2005. The footnote numbers given are for use solely within this amendment.
Page 1, Clause 2
Update entries 1 to 3 as follows and delete footnote 1).
ISO 21569:2005 + AM1:2013, Foodstuffs — Methods of analysis for the detection of genetically modified
organisms and derived products — Qualitative nucleic acid based methods
ISO 21571:2005 + AM1:2013, Foodstuffs — Methods of analysis for the detection of genetically modified
organisms and derived products — Nucleic acid extraction
ISO 24276:2006 + AM1:2013, Foodstuffs — Methods of analysis for the detection of genetically modified
organisms and derived products — General requirements and definitions
Page 2, 7.1, last paragraph; Page 8, A.1.5.1; Page 15, B.1.5.1; Page 23, C.1.5.1; Page 31, C.2.5.1; Page 38,
C.3.5.1; Page 45, C.4.5.1; Page 53, C.5.5.1; Page 60, C.6.5.1; Page 68, C.7.5.1; Page 75, C.8.5.1; Page 83,
C.9.5.1; Page 90, D.1.5.1; Page 96, D.2.5.1
Delete “ISO 24276:—”, insert “ISO 24276:2006”.
Page 4, Clauses 8–10
Replace the existing text with the following.
8 Interpretation
The PCR result will be either a) or b).
a) Fit for quantification of the target sequence provided:
— the result is positive according to ISO 21569:2005, 8.1;
— the observable inhibition of the reaction is negligible;
— the analysis produces an unambiguous measurement value;
— the target sequence content is within the dynamic range of the method;
— the analysis is calibrated in an acceptable way (see 7.3).
b) Unfit for quantification of the target sequence if any of the conditions listed in a) are not fulfilled.
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ISO 21570:2005/Amd.1:2013(E)
Interpretation of ambiguous results within the same test portion: in case of +/− results for the two
replicates, repeat the two PCR for the relevant test portion. If the two novel replicates are tested +/− or
−/−, the test portion is considered as negative.
Interpretation of ambiguous results between two test portions: in case of ± results for the two test
portions of a sample, the extractions and analysis of two new test portions shall be performed. If again
the results are +/−, the sample is considered as negative according to ISO 24276:2006, 6.3.
The measurement uncertainty shall be sufficiently small to enable the laboratory to draw the
relevant conclusions.
Annexes A to D describe the measurement of the target DNA quantities. These quantities can be used
to calculate the GMO content. These calculations usually take into consideration relevant biological
factors, e.g. the homo- or heterozygosity of the target sequences.
If the GM target sequence content or the taxon-specific target sequence content is below the limit of
quantification, the result shall only be expressed qualitatively.
NOTE Stating that the GMO-derived DNA content is below the practical LOQ accompanied by a specification
of that LOQ is considered to be a qualitative expression of the result.
9 Expression of results
The results shall clearly state the quantity of the GM target sequence relative to the target taxon-specific
sequence. The results should also provide values for the measurement uncertainty, such as the standard
deviation or coefficient of variation. Furthermore, the LOD and LOQ of the method and the practical
LOD and LOQ should be reported. The indication that the result refers only to GMO targets should be
reported. In the case of quantitative screening analysis on complex matrices, it is recommended to
specify that the GMO signal can come from non target taxons.
The target sequences can or cannot be detected, or the quantity of at least one of them can be below the
limit of quantification. Table 1 describes the four alternative cases and the corresponding expression of
the result to be included into the test report.
The GMO-derived DNA content can also be reported as being above or below a specific value, taking into
account the measurement uncertainty.
10 Test report
The test report shall be written in accordance with ISO 24276 and ISO 21569 and shall contain at least
the following additional information:
a) the LOQ of the method and the matrix used to establish it;
b) the practical LOQ;
c) a reference to the method which has been used for the extraction of DNA;
d) a reference to the methods used for the amplification of the DNA target sequences;
e) the reference material used;
f) the results expressed according to Clause 9;
g) the PCR target and whether considered “event specific” or “construct specific” or “screening”;
h) the definition of the measurement uncertainty used.
NOTE For g) and h), information can figure in different documents (e.g. contract review, technical data sheets).
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SIST EN ISO 21570:2006/A1:2013
ISO 21570:2005/Amd.1:2013(E)
Table 1 — Expression of results
Result Expression of the result
Target taxon-specific sequence “For species X, DNA was not detected.”
is not detected.
Target taxon-specific sequence According to ISO 21569.
is detected but GM target
“For sample X, GM target sequence Y was not detected.
sequence is not detected.
The LOD of the method is x % determined with ABC (identify the reference
material).”
If it cannot be demonstrated that the test sample size and the amount of target
DNA included in the PCR is sufficient for the LOD to be applicable, then the fol-
lowing sentence shall be added:
“However, the amount of the target DNA extracted from species X can be/was
insufficient for the LOD to be applicable for this sample. The LOD of sample is
x %.” (Specify the unit used.)
NOTE The LOD of the sample is determined by the quantity of DNA of the species
included in the analytical reaction (copy number), and the ratio relative to the absolute
LOD of the GM target (copy number), and in the case of grain and seeds, the number of
grain or seeds in the portion that is ground.
The target taxon-specific For each GMO, state:
sequence and the GM target
“GMO (specify the GMO) derived DNA as determined by detection of (specify
sequence are both detected,
target sequence) derived from (specify species) was detected, below the prac-
but the quantity is below the
tical limit of quantification”
LOQ of at least one of the target
sequences. In addition, if applicable: “The practical limit of quantification is x %.” (Specify
the unit used.)
The target taxon-specific For each GMO, state:
sequence and the GM target
“The content of GMO (specify the GMO) derived DNA as determined by detec-
sequence are both detected
tion of (specify target sequence) derived from (specify species) is x ± u %”
meas
and the quantity is above the
where u is the measurement uncertainty. (Specify the unit used.)
meas
LOQ for both target sequences.
Page 11, Annex A
Add A.2 and A.3.
A.2 Target-taxon-specific method for the detection of DNA derived from rice
A.2.1 Principle
Rice SPS gene has been described as being suitable for use as an endogenous reference gene in GM rice
identification and quantification (Reference [59]). The GMO Detection Laboratory of Shanghai Jiao Tong
University (GMDL-SJTU) organized a collaborative trial for validation of the applicability of the rice
sucrose phosphate synthase (SPS) gene as an endogenous gene for quantitative analysis of genetically
modified (GM) or non-GM rice. The study involved 12 laboratories from Spain, Korea, Lithuania, Slovenia,
Japan, Italy, and China.
The operational procedure of the collaborative study comprised the following modules.
Quantitative real-time PCR for quantification of blind rice DNA samples used to construct standard
curves.
Quantitative real-time PCR for the quantification of blind rice DNA samples using the constructed
standard curves.
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ISO 21570:2005/Amd.1:2013(E)
The interlaboratory test was carried out in accordance with the following internationally accepted guidelines:
[51]–[56]
— ISO 5725;
— the IUPAC protocol for the design, conduct and interpretation of method-performance studies
(Reference [12]).
The results of the collaborative study as well as the related protocol are given in A.2.3.3.
A.2.2 Scope
The method has been optimized for rice grain and its processed products containing mixtures of
rice and other matrices, e.g. maize and soybean. The applicability of the SPS gene was tested through
collaborative trials using DNA samples extracted from rice grains.
A.2.3 Validation status and performance criteria
A.2.3.1 Robustness of the method
The robustness of the SPS gene quantitative real-time PCR system was tested by the method developer on
different temperature–time programmes (i.e. two-step and three step) and on three different DNA samples
containing known amounts of rice DNA (10 ng, 1 ng, 0,1 ng rice genome DNA samples). There were three
repetitions per sample. The quantitative real-time PCR systems had the expected ruggedness and worked
well at different temperature–time programmes and three concentrations of the rice DNA samples.
The quantitative PCR system for the SPS gene was also tested on different real-time PCR instruments
1)
1)
(Rotor gene 3000A, Corbett Research and ABI7700, Applied Biosystems), with three different
reaction volumes (20 µl, 25 µl, and 30 µl; three repetitions per volume). The quantitative real-time PCR
system demonstrated appropriate ruggedness, working well on the different real-time PCR instruments
and with the different reaction volumes.
A.2.3.2 Intralaboratory trial
For sample preparation, all the DNA samples were extracted using the cetyl(trimethyl)ammonium
bromide (CTAB) method adopted from ISO 21571. Spectrometric quantification of the amount of total DNA
extracted was performed using a method adopted from ISO 21571:2005, B.1. After the DNA quantification,
a quantitative real-time PCR run was carried out to provide data about possible PCR inhibition.
The SPS gene PCR system was tested by three researchers using the rice genome DNA, and gave
satisfactory results; in particular, in quantitative PCR, the bias was below 25 % over the dynamic range
(i.e. 0,05 ng to 1,00 ng).
A.2.3.3 Collaborative trial
Standard curves were constructed using serially diluted DNA samples extracted by the GMDL-SJTU
from four rice cultivars by means of quantitative PCR. The PCR efficiency, calculated from the slope of
−1/a
the standard curve as (10 − 1) × 100, where a is the slope, of the SPS gene PCR system ranging from
2
0,846 3 to 1,223 3, and the linearity (regression coefficient, R ) was on average equal to 0,997.
c
The results of the eight blind DNA samples are reported in Table A.5. These are evaluated with respect
to the method acceptance criteria and to the method performance requirements, as established by the
European Network of GMO laboratories (ENGL) and adopted by the European Reference Laboratory
for GM Food and Feed (EU-RL GMFF) (Reference [60]). In Table A.5, estimations of both repeatability
and reproducibility for each rice concentration level are reported, after identification and removal of
outliers according to Cochran’s test.
1) 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.5 — Results of quantitative real-time PCR
Blind samples
Parameter
0,5 ng 0,5 ng 1ng 1 ng 2 ng 5 ng 5 ng 10 ng
Laboratories returning
12 12 12 12 12 12 12 12
results
Samples per laboratory 1 1 1 1 1 1 1 1
Total data no. 108 108 108 108 108 108 108 108
Data excluded 4 2 0 8 0 0 0 0
Cochran’s Cochran’s Cochran’s
Reason for exclusion — — — — —
test test test
Mean value 0,407 8 0,412 1 0,814 6 0,734 4 1,857 5 5,261 0 5,445 7 10,889 6
Repeatability standard
0,058 3 0,071 9 0,143 5 0,111 0,336 2 0,936 4 1,142 7 1,867 7
deviation
Repeatability coefficient
14,29 17,44 17,62 15,11 18,10 17,80 20,98 17,15
of variation, %
Reproducibility standard
0,130 2 0,061 8 0,249 6 0,092 7 0,201 3 0,810 9 0,989 6 1,617 5
deviation
Reproducibility coef-
31,92 14,99 30,65 12,63 10,84 15,41 18,17 14,85
ficient of variation, %
Bias, absolute value 0,092 2 0,087 8 0,185 3 0,265 5 0,142 4 −0,261 0 −0,445 8 −0,889 6
Bias, % −18,44 −17,57 −18,54 −26,53 −7,12 5,22 8,92 8,90
A.2.3.4 Molecular selectivity
A.2.3.4.1 General
The primers and probe targeting the 81 bp SPS gene DNA fragment are listed in Table A.6.
Table A.6 — Oligonucleotide primers and probe sequences
Name Oligonucleotide DNA sequence (5′ to 3′)
Quantitative-real time PCR primer and probe sequence
SPS primer F TTgCgCCTgAACggATAT
SPS primer R CggTTgATCTTTTCgggATg
SPS probe HEX- TCCgAgCCgTCCgTgCgTC -TAMRA
A.2.3.4.2 Experimental
DNA samples extracted from 11 different plant materials (including rice) were analysed using the SPS
gene PCR method. Among the 11 samples, only rice DNA gave positive results. The 10 other samples
(i.e. bamboo, green bristlegrass, barley, wheat, foxtail millet, rapeseed, tomato, potato, soybean and
Arabidopsis) gave negative results.
DNA samples extracted from 12 different rice cultivars were analysed by the specific PCR method
developed for the detection of the SPS gene. All 12 samples gave positive results.
A.2.3.4.3 Theoretical
The theoretical specificity of the SPS gene primers and probe was assessed through a similarity search
using the BLASTN 2.0MP-WashU program (Reference [64], search date: 2010-01-09). The 81 bp sequence
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SIST EN ISO 21570:2006/A1:2013
ISO 21570:2005/Amd.1:2013(E)
used as query is part of the NCBI accession number U33175 (nucleotides 1055–1135). The results of the
blast search confirmed the complete identity of the query sequence with rice SPS gene sequence, and no
similarity with other genes and species.
A.2.4 Principle and summary
An 81 bp fragment of the SPS gene is amplified using two rice sps-specific primers. Accumulation
of PCR products is measured at the end of each PCR cycle (real-time) by means of a rice sps-specific
oligonucleotide probe labelled with two fluorescent dyes: FAM as reporter dye and TAMRA as quencher
1)
(see Table A.6). For that purpose, TaqMan® chemistry is employed. The fluorescence signal measured
crosses a user-defined threshold value after a certain number of cycles. This number is called the C
t
value. For quantification of the amount of rice sps-DNA in an unknown sample, the C value is converted
t
into a corresponding copy number value by comparison with a calibration curve whose C values are
t
directly linked with known copy numbers (regression analysis).
A.2.5 Terms and definitions
[51]
For the purposes of this clause, the terms and definitions of ISO 5725-1 and ISO 24276 apply.
A.2.6 Sample type and amounts
DNA samples extracted from the grains of four rice cultivars, were used to construct the standard
curves in this collaborative study. Then, eight blind samples were analysed using the four standard
curves constructed.
The participants received the following samples.
— Four DNA samples from different rice varieties (3M, Indica variety from US; Balilla, Japonica
variety from Italy; Guangluai, Indica variety from Southern China, and Shennong265, Chinese
Japonica variety), 50 ng/μl, 30 μl each. Each rice cultivar DNA was diluted and used to generate the
corresponding standard curve.
— Eight blind rice DNA samples from four different rice varieties with different concentrations (0 ng/µl
to ~50 ng/µl), 50 μl each.
— Negative DNA target control (labelled N): salmon sperm DNA (20 ng/µl).
— Positive DNA target control (labelled P): (Guangluai4) genomic DNA (20 ng/µl). All the DNA samples
were purified using the CTAB method by-GMDL-SJTU. The negative and positive DNA target controls
were used for each PCR plate.
— Primers and probes for the SPS gene PCR system (see Table A.6) and further reaction reagents as
follows:
— real-time PCR master mixture (1 ml × 6);
— DNA dilute solution (0,1× TE, 1,2 ml).
A.2.7 Limit of quantification (LOQ), range of use
According to the developed method, the absolute LOQ of the method is 0,01 ng/μl. The relative LOQ of
quantitative PCR has not been assessed in a collaborative trial.
A.2.8 Estimation of measurement uncertainty
The global uncertainty of the method is given by the results of the collaborative trial (see Table A.5).
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A.2.9 Interferences
The amount and the ability for amplification of the nucleic acid used as template for the real-time PCR
is of major importance for the sensitivity of the method. In addition to this general point, no specific
interferences are known for this method.
A.2.10 Physical and environmental conditions
The procedures require experience of working under sterile conditions.
Maintain strictly separated working areas for DNA extraction, PCR set-up and amplification.
Any residual DNA should be removed from equipment prior to its use.
In order to avoid contamination, filter pipette tips (A.2.11.6) protected against aerosol should be used.
Use only powder-free gloves (A.2.11.8) and change them frequently.
Clean laboratory benches and equipment periodically with sodium hypochlorite (10 % active chloride)
solution (bleach).
Pipettes should be calibrated regularly, if necessary.
A.2.11 Apparatus and equipment
Usual laboratory equipment and in particular the following.
A.2.11.1 Microcentrifuge.
A.2.11.2 Freezer maintained at −20 °C and refrigerator maintained at 4 °C.
A.2.11.3 Micropipettes.
A.2.11.4 Vortex mixer.
A.2.11.5 Tubes, of capacities 0,2 ml, 1,5 ml, 2,0 ml.
A.2.11.6 Tips and filter tips for micropipettes.
A.2.11.7 Rack for reaction tubes.
A.2.11.8 Vinyl or latex gloves.
A.2.11.9 Vacuum dryer suitable for drying DNA pellets,optional.
A.2.11.10 Real-time PCR system with plastic reaction vials suitable for fluorescence measurement.
1) 1)
A.2.11.11 Software: Sequence Detection System version 1.7 (Applied Biosystems Part No 4311876 )
or equivalent versions.
®1) 1)
A.2.11.12 Optical 96 well reaction plates, MicroAmp (Applied Biosystems Part No N801-0560 ).
®1) 1)
A.2.11.13 Optical adhesive covers, MicroAmp (Applied Biosystems Part No 4311971 ).
®1) 1)
A.2.11.14 Optical caps, MicroAmp (Applied Biosystems Part No. No 801-0935 ).
A.2.12 Reagents and materials
A.2.12.1 General
Unless otherwise stated, use only reagents that conform to the specifications of ISO 24276 and only
sterile distilled or demineralized water or water of equivalent purity.
© ISO 2013 – All rights reserved 7
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SIST EN ISO 21570:2006/A1:2013
ISO 21570:2005/Amd.1:2013(E)
A.2.12.2 DNA extraction
A.2.12.2.1 Cetyltrimethylammonium bromide (CTAB), ultrapure grade.
A.2.12.2.2 Tris-(hydroxymethyl)aminomethane hydrochloride (tris), molecular biology grade.
A.2.12.2.3 Ethylenediaminetetraacetic acid disodium salt (EDTA), titration 99,9 % mass fraction.
A.2.12.2.4 Ethanol, φ[CH CH OH] at least 96 % volume fraction.
3 2
A.2.12.2.5 Isopropanol, φ[CH CH(OH)CH ] at least 99,7 % volume fraction.
3 3
A.2.12.2.6 Chloroform, φ(CHCl ) at least 99 % volume fraction.
3
A.2.12.2.7 Sodium chloride, w(NaCl) at least 99 % mass fraction.
A.2.12.2.8 Sodium hydroxide, anhydrous, w(NaOH) at least 98 % mass fraction.
A.2.12.2.9 RNase A solution, 10 mg/ml.
A.2.12.2.10 Proteinase K solution, 20 mg/ml.
A.2.12.2.11 Dilution buffer: tris (A.2.12.2.2), 10 mmol/l, pH 9,0.
A.2.12.2.12 Hydrochloric acid, ρ(HCl) = 370 g/l.
A.2.12.2.13 Herring testes DNA, calf thymus DNA, or Lambda DNA.
A.2.12.3 Quantitative real-time PCR
®1)
TaqMan universal PCR master mix (1×) or suitable equivalent.
A.2.13 Sample collection, transport, 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.2.11.2). For quantitative real-time PCR set up, PCR reagents shall be kept thawed at 1 °C to 4 °C on ice.
A.2.14 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 detail in ISO 21571. For
the collaborative study, a total amount of 1 g ground rice noodles was used.
A.2.15 Instrument calibration
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Instruments, e.g. thermocyclers and pipettes shall be calibrated, e.g. according to ISO/IEC 17025.
A.2.16 Analysis steps
A.2.16.1 General
A.2.16.1.1 Preparation of the DNA for standard curve construction
Each of the 50 ng/μl rice DNA samples was diluted to 10 ng
...
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