ISO/TS 21569-8:2025

Technical
Specification
ISO/TS 21569-8
First edition
Horizontal methods for molecular
2025-04
biomarker analysis — Methods
of analysis for the detection of
genetically modified organisms and
derived products —
Part 8:
DNA extraction from alfalfa seeds
and real-time PCR based detection
methods for genetically modified
alfalfa events J101, J163 and KK179
Méthodes horizontales pour l'analyse moléculaire de
biomarqueurs — Méthodes d'analyse pour la détection des
organismes génétiquement modifiés et des produits dérivés —
Partie 8: Extraction d'ADN à partir de graines de luzerne et
méthodes de détection spécifiques à l'événement basées sur la
PCR en temps réel pour les lignées de luzerne génétiquement
modifiées J101, J163 et KK179
Reference number
© ISO 2025
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ii
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and materials . 2
5.1 General .2
5.2 Interlaboratory trial and control material .2
5.3 DNA extraction reagents and solutions .2
5.4 PCR reagents .3
6 Apparatus . 4
7 Procedure . 4
7.1 Preparation of test samples .4
7.2 Preparation of DNA extracts .4
7.2.1 General .4
7.2.2 DNA extraction from ground alfalfa seeds .5
7.3 PCR set-up .5
7.4 Temperature-time programme.6
8 Accept/reject criteria . 6
8.1 General .6
8.2 Identification .6
9 Validation status and performance criteria . 7
9.1 Sensitivity .7
9.2 Specificity .7
9.3 Robustness .7
9.4 Interlaboratory trial .8
9.4.1 General .8
9.4.2 Results of DNA extractions from ground alfalfa seed samples .9
9.4.3 False-positive rate/false negative rate .9
9.4.4 PCR efficiency and detection limit .10
9.5 Summary evaluation .11
10 Test report .11
Bibliography .12

iii
Foreword
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This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis.
A list of all parts in the ISO 21569 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
Technical Specification ISO/TS 21569-8:2025(en)
Horizontal methods for molecular biomarker analysis —
Methods of analysis for the detection of genetically modified
organisms and derived products —
Part 8:
DNA extraction from alfalfa seeds and real-time PCR based
detection methods for genetically modified alfalfa events
J101, J163 and KK179
1 Scope
This document specifies procedures for DNA extraction from alfalfa (Medicago sativa) seeds and for the
specific detection of the herbicide-tolerant alfalfa events J101 and J163 and the lignin-modified alfalfa event
KK179 in crop/plant/seed/grain test samples.
The detection methods are based on real-time PCR and are targeting the DNA transition sequences between
the alfalfa genome and the respective integrated gene construct. The methods can be applied for direct
event-specific identification or as a follow-up analysis, if sequences encoding the promoter of the Figwort
mosaic virus (P-FMV), the terminator of the nopaline synthase gene from Rhizobium radiobacter (T-nos), or
the construct CTP2/CP4-EPSPS (herbicide tolerance) were detected by screening analyses of test samples.
In this document, the methods were validated using ground alfalfa seeds and DNA extracted thereof.
The PCR methods are also applicable for the analysis of other matrices such as feed and foodstuffs. The
application of these PCR methods requires the extraction of an adequate amount of amplifiable DNA from
the relevant matrix.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 16577, Molecular biomarker analysis — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
ISO 21569, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Qualitative nucleic acid based methods
ISO 21571, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Nucleic acid extraction
ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — General requirements and definitions
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Principle
DNA is extracted from the test sample as described in this document or by applying another suitable method
with comparable performance characteristics (see ISO 21571). The DNA analysis consists of two parts:
a) verification of the amount, quality and amplifiability of the extracted DNA, by means of the real-time
[1]
PCR for the alfalfa-specific reference gene acc1;
b) analysis using the real-time PCR detection methods specific for the alfalfa events J101, J163 and KK179,
[2][3][4]
respectively.
Detection of PCR products is done using specific hydrolysis probes labelled with fluorescent dyes (FAM as
reporter dye and one or two quenchers) that bind the respective target sequences between the two primers
[5]
(so-called “TaqMan chemistry” ).
5 Reagents and materials
5.1 General
Chemicals of recognized analytical grade, appropriate for molecular biology shall be used, as a rule. The
water used shall be double distilled or PCR grade water, i.e. nuclease and nucleic acid free. For all operations
in which gloves are used, it should be ensured that these are powder-free. The use of aerosol-protected
pipette tips as protection against cross-contamination is recommended.
5.2 Interlaboratory trial and control material
1)
5.2.1 Ground alfalfa seeds, of a wild-type line and the events J101, J163 and KK179.
5.2.2 Plasmid DNAs, containing the target sequences of the J101, J163 and KK179 event-specific PCR
[2][3][4]
methods.
5.3 DNA extraction reagents and solutions
5.3.1 Hexadecyl-trimethyl-ammonium-bromide (CTAB) (C H BrN).
19 42
5.3.2 Glycogen, ρ(CHCL ) = 20 mg/l.
5.3.3 Potassium acetate solution, c(CH COOK) = 5 mol/l (storage at −20 °C).
5.3.4 Chloroform, φ (CHCl ) ≥ 99,5 %.
5.3.5 Hydrochloric acid, φ(HCl) = 37 %.
5.3.6 Isopropyl alcohol, φ[CH CH(OH)CH ] ≥ 99,5 %, (−20 °C; fresh aliquot).
3 3
5.3.7 Ethanol solution, φ(C H OH) = 70 %, (fresh aliquot).
2 5
1) This product is supplied by Forage Genetics Intl., LLC, West Salem, Wisconsin, USA. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of the supplier named.

5.3.8 Sodium chloride, c(NaCl) = 1,2 mol/l.
5.3.9 Tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl) buffer, c(C H ClNO ) =
4 12 3
0,1 mol/l (pH = 8,0).
5.3.10 Ethylenediaminetetraacetic acid disodium salt (Na -EDTA) solution, c(C H N O Na ) =
2 10 14 2 8 2
0,5 mol/l (pH = 8,0).
5.3.11 Proteinase-K solution, ρ = 10 mg/ml.
Do not autoclave. Store at −20 °C but avoid repeated freezing and thawing.
5.3.12 Polyvinylpolypyrrolidone (PVPP).
5.3.13 RNase A solution, ρ(RNase) = 10 mg/ml in c(HCl) = 0,01 mol/l (pH = 7,5) and c(NaCl) = 0,015 mol/l);
DNase free.
Store in aliquots at −20 °C.
5.3.14 CTAB lysis buffer, ρ(CTAB) = 20 g/l, c(NaCl) = 1,4 mol/l, c(Tris) = 0,1 mol/l, c(Na EDTA) = 0,02 mol/l.
Adjust pH to 8,0 with HCl.
5.3.15 CTAB precipitation buffer, ρ(CTAB) = 5 g/l, c(NaCl) = 0,04 mol/l, c(Tris) = 0,1 mol/l, c(Na EDTA) =
0,05 mol/l.
Adjust pH to 8,0 with HCl.
5.3.16 Elution buffer (0,1X TE buffer), c(Tris-HCl) = 1 mmol/l, c(EDTA) = 0,1 mmol/l (pH = 8,0).
5.4 PCR reagents
5.4.1 PCR master mix, contains thermostable DNA polymerase (for hot-start PCR), MgCl and
deoxyribonucleoside triphosphates (dNTPs).
5.4.2 TE buffer, c(Tris-HCl) = 10 mmol/l, c(EDTA) = 0,5 mmol/l (pH = 9,0).
5.4.3 Oligonucleotides (see Table 1).
Equivalent reporter dyes and/or (internal) quencher may be used for the probe if they can be shown to yield
similar or better results.
Table 1 — Oligonucleotides
Name DNA sequence of the oligonucleotide Final concentra-
tion in PCR
[1][2][3][4]
Transition sequence from alfalfa genome to respective insertion or acc1 gene as target sequences:
J101-F1 5′- gTC Atg TgT TTT gTA CTg ATC TTg Tg -3′ 400 nmol/l
J101-R1 5′- gAC CTg CAg AAg CTT gAT gg -3′ 400 nmol/l
J101-P 5′-(FAM)- ACT gAA ggC ggg AAA CgA CAA TCT gAT CC -(BHQ1)-3′ 200 nmol/l
J163-F1 5′- Cgg gAC AAg gTC ATC CAA ACT g -3′ 400 nmol/l
J163-R1 5′- ACC TTg TTg Agg CTT Tgg ACT g -3′ 400 nmol/l
J163-P 5′-(FAM)- TCT gCA ggT CCT gCT CgA gTg gAA gCT -(BHQ1)-3′ 200 nmol/l
KK179-F1 5′- CTT Agg gCA CTT gTT AgC ATT TTC -3′ 500 nmol/l
KK179-R1 5′- CCA TAT TgA CCA TCA TAC TCA TTg C -3′ 500 nmol/l
KK179-P 5′-(FAM)- Tgg CTT CAT /ZEN/ gTC Cgg gAA ATC TAC ATg g -(IowaBFQ)-3′ 200 nmol/l
Acc1-F 5′- gAT CAg TgA ACT TCg CAA AgT AC -3′ 150 nmol/l
Acc1-R 5′- CAA CgA CgT gAA CAC TAC AAC -3′ 150 nmol/l
Acc1-P 5′-(FAM)- TgA ATg CTC CTg TgA TCT gCC CAT gC -(BHQ1)-3′ 50 nmol/l
Key
® TM ®
FAM: 6-Carboxyfluorescein; BHQ 1: Black Hole Quencher 1; ZEN : additional internal quencher; IowaBFQ: Iowa Black
Fluorescent Quencher.
® TM ®
NOTE  BHQ 1, ZEN and Iowa Black Fluorescent Quencher are examples of suitable products available commercially. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of these
products.
6 Apparatus
Requirements concerning apparatus and materials shall be in accordance with ISO 21569. In addition to the
usual laboratory equipment, the following equipment shall be used.
6.1 Real-time PCR device, suitable for the excitation of fluorescent molecules and the detection of
fluorescence signals generated during PCR. In the interlaboratory trial, the real-time PCR devices listed in
Table 7 were used.
7 Procedure
7.1 Preparation of test samples
It should be ensured that the test sample used for DNA extraction is representative of the laboratory
sample, e.g. by grinding or homogenizing of the samples. Measures and operational steps to be taken into
consideration shall be as described in ISO 21571 and ISO 24276.
7.2 Preparation of DNA extracts
7.2.1 General
For the extraction of DNA from the test portion, the general instructions and requirements specified in
ISO 21571 should be followed. This extraction procedure was specifically chosen due to its ability to remove
PCR inhibitors.
NOTE The amount of extracted DNA can be quantified with a suitable method; for example, see ISO 21571.

7.2.2 DNA extraction from ground alfalfa seeds
7.2.2.1 Weigh out 2 % PVPP (5.3.12) (mass/volume based on 15 ml CTAB lysis buffer (5.3.14),
corresponding to 0,3 g) into a 50 ml tube and add 2 g of the sample material (ground alfalfa seeds).
7.2.2.2 Add 15 ml CTAB lysis buffer (5.3.14), close the tube, and shake thoroughly by hand. Mixing with a
test tube shaker is often not sufficient to achieve a homogeneous distribution of the PVPP (5.3.12).
7.2.2.3 Add 40 µl Proteinase K solution (5.3.11) and 20 µl RNase solution (5.3.13).
7.2.2.4 Incubate under shaking for 90 min at 65 °C; then centrifuge at 4 000g for 10 min; transfer 500 μl of
supernatant to a new 2 ml tube.
7.2.2.5 Add 300 μl pre-cooled (−20 °C) potassium acetate solution (5.3.3) and mix well on a test tube shaker.
7.2.2.6 Incubate for 5 min at −20 °C, then centrifuge at 16 000g for 10 min and transfer the supernatant to
a new 2 ml tube.
7.2.2.7 Add 700 μl chloroform (5.3.4), mix thoroughly on a test tube shaker and centrifuge at 16 000g
for 10 min.
7.2.2.8 Transfer the upper aqueous phase to a new 2 ml tube (interphase is highly instable; pipette slowly
with a 100 μl to 200 μl pipette).
7.2.2.9 Add two volumes of CTAB precipitation buffer (5.3.15), mix on a test tube shaker and incubate for
1 h at room temperature.
7.2.2.10 Centrifuge at 16 000g for 10 min, discard the supernatant, and dissolve the pellet in 350 μl NaCl
(5.3.8) solution.
7.2.2.11 Add 350 μl of chloroform (5.3.4), mix well on a test tube shaker and centrifuge at 16 000g for 10 min.
7.2.2.12 Transfer the upper phase to a new 2 ml
...