🍎 Qualitative detection of genetically modified organisms (GMO) and derived products in foodstuffs (also in feed and plant samples) by nucleic acid based methods — DNA extracted according to ISO 21571 is amplified by PCR (detection by electrophoresis or in real-time PCR, confirmation by hybridization, restriction digestion or sequencing) for taxon-specific, screening (e.g. the 35S promoter), construct-specific or event-specific sequences; with controls and criteria from ISO 24276, primer optimization (18–30 nucleotides, about 60 °C), an amplicon of 60–150 bp for highly degraded DNA; result “target sequence detected / not detected” with the limit of detection and reference material, PN-EN ISO 21569

Food chemistry PN-EN ISO 21569

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 21569:2005 in the French version (“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”, first edition of 15.06.2005, French version of 2006): contents, foreword, introduction, Clauses 1–10 and the beginning of Annex A up to A.1.5.3 (method specific for soya) — the sample ends on page 9. We read the European foreword in the sample of SIST EN ISO 21569:2005 (English text, up to 7.6). We do not read the rest of Annex A or Annexes B (screening methods), C (construct-specific methods) and D (event-specific methods); we know their titles from the contents. We read Amendment ISO 21569:2005/Amd 1:2013 in a separate iTeh sample (French text, “Amendement 1”, 2013-04-01): from the cover up to A.6.3.3 (page 11); we do not read the rest of the new clause A.6.

ACCORDING TO THE TEXT OF ISO 21569:2005. The standard was prepared by CEN/TC 275 “Food analysis — Horizontal methods” in collaboration with ISO/TC 34 (Vienna Agreement); according to the EN foreword, national standards had to adopt it by December 2005 at the latest. Introduction: testing for GM components consists of sampling (ISO 21568), extraction of nucleic acids (ISO 21571), their possible purification, quantification, dilution and analysis (e.g. PCR); quantitative methods — ISO 21570, general requirements and definitions — ISO 24276; qualitative detection establishes the presence or absence of the target sequence with suitable controls, within the limit of detection of the method and the analysed portion; the methods in Annexes A–D range from screening to construct- and event-specific methods; conformity with the standard may require a licence for PCR technology. Scope (1): qualitative detection of GMOs and derived products in nucleic acids extracted from the sample, mainly by PCR amplification; requirements for the specific detection and identification of target sequences and for confirming product identity; developed for food matrices, it may be applied to others (e.g. feed and plant samples from the environment). References (2): ISO 21571:2005 (extraction), ISO 24276 (general requirements and definitions, in preparation at the time of publication). Principle (4): each method must specify the target sequence; a qualitative result must clearly state the presence or absence of the genetic element, within the limit of detection and the test portion; amplification by DNA polymerase with primers in denaturation–annealing–extension cycles, after checking for absence of inhibitors; detection by gel electrophoresis or another technique, confirmation of identity e.g. by restriction enzymes, hybridization or sequencing; in real-time PCR amplification and detection are simultaneous. Reagents (5): solutions usually at about −20 °C, aliquoted to avoid freeze–thaw cycles and contamination; dNTPs (dATP, dCTP, dGTP and dTTP or dUTP — dUTP may affect analysis with restriction enzymes); PCR buffer, MgCl2 concentration specific to the method (in the annexes); thermostable polymerase; primers. Procedure (7): DNA sufficiently pure, with repeatable and reproducible quality and quantity for the method and matrix; the limit of detection depends on product processing and DNA degradation; inhibition controls always according to ISO 24276; conditions (MgCl2, thermal profile) are optimized for each primer pair; in an optimal reaction fewer than 40 cycles suffice to amplify 10 target molecules, and an optimized PCR should give a detectable product from 100 copies of template DNA in 40 cycles; hot-start PCR is recommended; validation values may not apply to other matrices and concentrations; product length matched to the DNA in the extract — for highly degraded DNA from processed food 60–150 bp, for raw materials up to about 250 bp; primers of 18–30 nucleotides, optimum annealing temperature about 60 °C, determined experimentally (estimated melting temperature — 65 °C with the relation sign illegible in the sample text), GC:AT as close to 50:50 as possible, minimal complementarity of 3′ ends and secondary structure; theoretical (database search, e.g. EMBL, GenBank) and experimental validation of specificity; targets: taxon-specific, construct-specific, event-specific sequences and screening elements; controls mandatory (ISO 24276), where possible certified reference materials as positive and negative controls; confirmation of the result with primers for another sequence (especially after screening) or by hybridization, digestion or sequencing; for sequences from naturally occurring organisms (virus, bacterium) it must be checked whether the DNA comes from a GMO — e.g. the 35S promoter comes from cauliflower mosaic virus (CaMV), so its detection may indicate a GMO or CaMV itself. Interpretation (8): positive — specific product detected and controls in accordance with Table 2 of ISO 24276; negative — no product detected with conforming controls; event-specific sequences may occur together in one GMO (stacked genes); ambiguous result — repetition. Expression of results (9): no “±”; a negative result never as “no GMO”; limit of detection related to at least one reference material and a relative value in the matrix (e.g. 100 ng of GTS 40-3-2 DNA at 0,01 %); negative result: “For sample X the target sequence Y was not detected. The LOD of the method is x %, determined using ABC”, and if it cannot be shown that the amount of target DNA suffices to reach the LOD — an additional sentence that the amount of DNA of the species may have been insufficient; positive result: “For sample X the target sequence Y was detected”, with GMO identity where applicable; results of two test portions must be consistent — if they differ, repetition, where possible with more DNA, with an inhibition control. Test report (10): according to ISO 24276, at least the LOD with the matrix, a description of specificity and results according to Clause 9. Annex A.1 (soya, lectin gene Le1, 118 bp amplicon, agarose gel electrophoresis): method validated in BgVV interlaboratory studies of 1997 (25 laboratories, 220 accepted results, 0 false positives and negatives) and 1998/1999 (27 laboratories, 60 results, 1 false positive and 1 false negative each — 2 %), with CTAB extraction according to ISO 21571:2005, A.3 (portion 100 mg); no amplification of DNA of other crops, beef and pork; absolute LOD not determined, detects at least 0,1 ng of soya DNA.

ACCORDING TO AMENDMENT ISO 21569:2005/AMD 1:2013. The amendment was prepared by ISO/TC 34 “Food products”, Subcommittee SC 16 “Horizontal methods for molecular biomarker analysis”. Introduction: sampling (ISO 21568) removed from the list of steps. The reference to ISO 24276 receives the date 2006 (also in 8.1). 4.1, paragraph 1 — new wording: a qualitative result must clearly indicate the presence or absence of the genetic element under study, examined with appropriate controls; note: the limit of detection and the size of the test portion are critical characteristics of a method. 7.3.3.3.3: primers for taxon-specific sequences must reliably detect these sequences in an “appropriate” (instead of “representative”) number of different members of the taxon. 9.4 — new wording: results from the same test portion must be consistent — with +/− results for the two replicates both PCRs are repeated, and if the new replicates give +/− or −/−, the test portion is considered negative; results from all test portions must be consistent — when at least one portion gives a positive and another a negative result, the analysis is repeated; if at least one repetition of the whole procedure starting from extraction gives an ambiguous result, the report should state that the sample is negative at the LOD. Clause 10: the report states the specificity of the method (event-specific, construct-specific or screening). Annex A, new A.5 — method specific for rice (sucrose phosphate synthase gene SPS, primers SPS F/SPS R, 279 bp amplicon, conventional PCR with electrophoresis): GMDL-SJTU interlaboratory study of 2007, 12 laboratories; 1 false negative in 144 rice samples (0,69 %) and 2 false positives in 120 samples of other plants (1,67 %); LOD about 0,1 % mass fraction of rice in maize flour (at 0,05 % and 0,01 % positive results only at some laboratories); CTAB extraction according to ISO 21571:2005, A.3; 30 µl reaction, 100 ng DNA per reaction recommended; 35 cycles with annealing at 58 °C; agarose gel 20 g/l with ethidium bromide 0,5 µg/ml, 5 V/cm, 20 min; DNA solutions at 4 °C for up to one week or at −20 °C. New A.6 — method specific for tomato (LAT52 gene), optimized for seeds, fruit, ketchup, juice and other products: interlaboratory study of 2007, 13 laboratories; 1 false negative in 156 (0,64 %) and 4 false positives in 130 (3,08 %), which according to the text may result from contamination during PCR; LOD in the intralaboratory study at least 0,1 %.

STATUS (as of 03.10.2026). In the PKN search (term “21569”, 03.10.2026) PN-EN ISO 21569:2007 (Polish version, “Introduces: EN ISO 21569:2005 [IDT], ISO 21569:2005 [IDT]”) has no withdrawal note; alongside it the supplementary element PN-EN ISO 21569:2007/A1:2013-07 (English version). Card: publication date 16.01.2007, 71 pages, KT 287 Biotechnology, ICS 67.050, “Replaces PN-EN ISO 21569:2005 – English version”, supplementary elements PN-EN ISO 21569:2007/A1:2013-07E, no withdrawal note. In the iTeh catalogue (read 03.10.2026) ISO 21569:2005 has the status “Published”, stage 90.92 (international standard to be revised), since 29.08.2024; as the revising document the card gives ISO/TS 21569-10:2026, and the iTeh search also shows the draft prEN ISO 21569-1.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read 03.10.2026). The number 21569 appears in 3 records at 3 laboratories: AB 465, AB 685, AB 868. In the current PCA documents (read 03.10.2026; AB 465 issue No. 31 of 23.12.2025, AB 685 issue No. 19 of 29.01.2026, AB 868 issue No. 21 of 06.03.2026) the standard appears in 3 lines at the same 3 laboratories — in the AB 685 line the number occurs 8 times (the standard, Amendment A1:2013 and six instructions IN-01–IN-06, issue 03 of 29.12.2024); in each document the number of page markers equals the number of PDF pages, without repetitions; a search of all PCA documents downloaded from the BIP on 03.10.2026 found no laboratories outside the database copy. Form “PN-EN ISO 21569” without year with procedure PBD-73.00.00 (AB 465), “PN-EN ISO 21569:2007” and “PN-EN ISO 21569:2007/A1:2013” with instructions (AB 685), “PN-EN ISO 21569” together with PN-EN ISO 21570 (AB 868). None of these items is suspended. The “Laboratories” tab (term “PN-EN ISO 21569”) shows all 3 laboratories — checked by a tab query on the labcoda.pl production server on 03.10.2026.

WHAT THE LABORATORIES TEST (items in PCA). AB 465: feed (flexible scope) — presence of genetic modifications, real-time PCR / PCR; AB 685: feed — presence of GMO-specific sequences: ctp2–cp4 epsps, nptII, bar, pFMV, pat, P-35S, T-nos, real-time PCR; AB 868: agricultural products and animal feed (flexible scope) — presence and content of genetic modifications, PCR, real-time PCR, together with PN-EN ISO 21570.

WHERE AN APPARENTLY CORRECT RESULT IS EASY TO GET. With a negative result: “no GMO” must not be written — only “target sequence Y not detected” with the LOD and reference material, and with an additional sentence where the DNA may have been insufficient (9.1, 9.2). With a screening element: the 35S promoter and other elements from naturally occurring organisms (CaMV) may come from an infection, not from a GMO — the presence of other DNA of that organism must be excluded (7.6); AB 685 tests P-35S. With processed DNA: degradation lowers detectability, and the amplicon should be 60–150 bp (7.1, 7.3.2). With controls: without an inhibition control and controls in accordance with ISO 24276 the result is neither positive nor negative (7.1, 8.1); two test portions must give consistent results (9.4). With the matrix: the standard was developed for food and mentions feed as a possible application (1) — all three laboratories test feed. With divergent results: according to Amendment A1:2013 a test portion with +/− results after repetition gives a negative result, and an ambiguous result of the repeated procedure is reported as negative at the LOD (9.4 as amended by A1) — without the amendment the text required consistency of two portions and repetition. With the edition: AB 685 applies Amendment A1:2013, while AB 465 and AB 868 cite the standard without year.

WHAT WE DO NOT GIVE. We do not quote the methods of Annexes B–D (screening, construct- and event-specific) or the rest of the soya method — they lie outside the sample. We have not read the rest of clause A.6 of Amendment A1:2013 or the content of ISO 24276. We do not give GMO labelling thresholds from legislation.

Method principle

DNA extracted from the sample (ISO 21571) is amplified by PCR with a pair of primers specific to the target sequence — taxon-specific, a screening element, construct-specific or event-specific — in cycles of denaturation, primer annealing and extension. The product is detected by gel electrophoresis or by fluorescence in real-time PCR and confirmed by hybridization, restriction digestion or sequencing. With controls in accordance with ISO 24276, the result reads “target sequence detected” or “not detected”, with the limit of detection related to a reference material.

Applications

• Qualitative detection of GMOs and derived products in food; possible application to feed and plant samples from the environment (1)
• Taxon-specific (e.g. soya lectin), screening, construct-specific and event-specific methods (Annexes A–D)
• Feed and agricultural products — items in the accreditation scopes of 3 laboratories in PCA (PCR, real-time PCR)

Key parameters

Standard

Standard number

PN-EN ISO 21569:2007

Title (PL)

Artykuły żywnościowe — Metody wykrywania organizmów zmodyfikowanych genetycznie i produktów pochodnych — Metody jakościowe oparte na kwasach nukleinowych

Title (EN)

Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Qualitative nucleic acid based methods

Step-by-step procedure

1. DNA extraction

   According to ISO 21571; purity, repeatable quantity, inhibition control (7.1). PN-EN ISO 21569:2007 without a withdrawal note in PKN (03.10.2026).

2. Choice of target and primers

   Taxon, screening element, construct or event; amplicon matched to DNA degradation (7.3, 7.4).

3. PCR with controls

   Optimization of MgCl2 and thermal profile, controls according to ISO 24276, reference materials (7.2, 7.5).

4. Detection and confirmation

   Electrophoresis or real-time PCR; confirmation by hybridization, restriction or sequencing; with 35S, exclusion of CaMV (7.6).

5. Result

   Positive or negative with conforming controls; wording with LOD; two portions consistent (8, 9).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Work with DNA and PCR reagents — protection against cross-contamination (aliquoting, separate areas)
• Electrophoresis — DNA stains and UV radiation according to safety data sheets

Frequently asked questions