🍎 Quantitative determination of genetically modified organisms (GMO) in foodstuffs (also in feed and plant samples) by PCR methods — real-time PCR or competitive PCR measures a GMO-specific sequence and an endogenous taxon-specific sequence, and the result is given as their ratio in percent; calibration with a suitable number of points and replicates (e.g. 4 × 2 or 6 × 1) using reference materials (preferably certified) or plasmid or synthetic dsDNA with equivalent behaviour; DNA from each test portion in at least two replicates; consistency criteria — deviations between dilutions ± 33 %, between test portions from −50 % to +100 % (ΔCt = 1); result “X ± uncertainty %” or qualitative when either sequence is below the LOQ, PN-EN ISO 21570

Food chemistry PN-EN ISO 21570

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 21570:2005 (English text, 2005 edition): contents, foreword, introduction, Clauses 1–10 and the beginning of Annex A up to Table A.3 (method for the maize adh1 gene) — the sample ends on page 7. We do not read the rest of Annex A or Annexes B (35S screening method for GTS 40-3-2 soya), C (nine construct-specific methods: GTS 40-3-2, Event176, MON 810, Bt11, GA21, T25) and D (event-specific methods for Bt11 and MON 810); we know their titles from the contents. We read Amendment ISO 21570:2005/Amd 1:2013 in a separate iTeh sample (Russian text, “Изменение 1”, 2013-04-15): from the cover up to A.2.10 (new method for rice); we do not read the rest of the amendment. We have not read the elements AC:2007 and Ap1:2007.

ACCORDING TO THE TEXT OF ISO 21570:2005. The standard was prepared by CEN/TC 275 “Food analysis — Horizontal methods” in collaboration with ISO/TC 34 (Vienna Agreement). Introduction: testing for GM components — sampling, extraction (ISO 21571), purification, quantification, dilution, analysis (PCR); qualitative methods — ISO 21569, general requirements — ISO 24276; conformity may require a licence for PCR technology. Scope (1): general framework for quantitative methods for detecting GMOs in food using PCR; requirements for the specific amplification of target sequences in order to determine the relative content of GMO-derived DNA and to confirm identity; developed for food matrices, it also applies to others, e.g. feed and plant samples from the environment. References (2): ISO 21569:2005, ISO 21571, ISO 24276 (in preparation), ISO Guide 32. Principle (4): determination of target sequences, each method specifying the sequences; competitive PCR or real-time PCR; the amount of the genetic element is expressed relative to a defined reference, calibrants and controls, within the dynamic range of the method and the test portion; analysis: amplification, detection and confirmation (simultaneous in real-time PCR), quantification against calibrants; usually the ratio (in percent) of the GMO sequence to an endogenous taxon-specific sequence, sometimes relative to the amount of matrix (e.g. GM microorganisms in food); calibrants related where possible to certified reference materials. Reagents (5): identical or equivalent to those given in the method, otherwise of molecular biology grade. Procedure (7): sequence concentration within the dynamic range of the method; note — a taxon-specific control run allows assessment of whether the DNA has sufficient quality, purity and quantity, especially from complex and highly processed matrices; DNA from each test portion is analysed in at least two replicates; controls according to ISO 24276, Table 1; allelic stability and the copy number of the sequence in varieties of different origin must be taken into account; calibration with a suitable number of points and replicates (e.g. four points in duplicate or six points in single determination), and calibration quality affects uncertainty; instead of genomic reference material, dilutions of a plasmid or synthetic dsDNA may be used if equivalent behaviour has been shown; stacked genes may lead to an overestimation of GMO content; LOQ according to ISO 24276; calculating content from copy numbers per haploid genome depends on homo- and heterozygosity; the ΔΔCt method is valid only when the amplification efficiencies of both assays are very similar; minimum consistency when the result is given in percent: within a test portion — measurements below the LOQ rejected and the largest deviation between dilutions and measurements equal to that expected from the dilution ± 33 %; between test portions — results must not differ by more than −50 % to +100 % (ΔCt = 1 in real-time PCR; e.g. 1,0 % and 2,0 % — acceptable, 0,9 % and 2,1 % — not); for trueness a reference material (preferably certified) for the event, with a similar matrix, must be selected and tested; in its absence — an in-house material with demonstrated stability, homogeneity and metrological traceability; uncertainty must meet the requirements for calibration (ISO Guide 32). Interpretation (8): a result is suitable for quantification when it is positive according to ISO 21569:2005, 8.1, inhibition is negligible, the value is unambiguous, the content is within the dynamic range and the calibration is correct; otherwise — not suitable; uncertainty must be small enough for conclusions to be drawn; when the content of the GM or taxon-specific sequence is below the LOQ, the result is given only qualitatively (the statement “below the practical LOQ” with its value is a qualitative result). Expression of results (9, Table 1): amount of the GM sequence relative to the taxon-specific sequence, with uncertainty (e.g. standard deviation), LOD and LOQ of the method and the practical LOD and LOQ; four cases — DNA of the species not detected; DNA of the species detected, GMO DNA not (with practical LOD); both detected but at least one sequence below the LOQ — “GMO DNA detected” (with practical LOQ); both above the LOQ — “the content of GMO DNA is X ± uncertainty %” (with unit); it may also be stated that the content is above or below a given value taking uncertainty into account. Test report (10): according to ISO 24276 and ISO 21569, additionally the LOQ of the method with the matrix, the practical LOQ, the DNA extraction method, the reference material, results according to Clause 9. Annex A.1 (maize adh1 gene, absolute quantification by real-time PCR, regression of Ct values against known copy numbers): primers ADH-FF3 and ADH-RR4 at 300 nmol/l each, probe ADH1-MDO (FAM/TAMRA) 200 nmol/l, 134 bp amplicon; MgCl2 25 mmol/l, dNTPs 2,5 mmol/l each; reaction volume 25 µl, template DNA up to 250 ng in 5 µl, 2X master mix 12,5 µl.

ACCORDING TO AMENDMENT ISO 21570:2005/AMD 1:2013. The amendment was prepared by ISO/TC 34, Subcommittee SC 16. References to ISO 21569, ISO 21571 and ISO 24276 changed to the 2005 and 2006 editions with their 2013 amendments; in 7.1 and in the method descriptions in the annexes “ISO 24276:—” is replaced by “ISO 24276:2006”. Clauses 8–10 receive new wording. Interpretation (8): a PCR result is suitable for quantification when it is positive according to 8.1 of ISO 21569, inhibition is insignificant, the measurement is unambiguous, the amount of the sequence is within the dynamic range and the calibration is appropriate (7.3) — otherwise it is not suitable; with +/− results in two replicates two PCRs are repeated, and if again +/− or −/−, the test portion is considered negative; with ± results from two test portions two new ones are extracted and tested, and with +/− again the test portion is considered negative (ISO 24276:2006, 6.3); uncertainty must be small enough for the laboratory to issue a justified conclusion; calculating the amount of GMO from the amount of DNA usually takes homo- and heterozygosity into account; when the GM or taxon-specific sequence is below the LOQ, the result is given only qualitatively, and the statement “below the practical LOQ” with its value is regarded as a qualitative expression. Expression of results (9): amount of the GM sequence relative to the taxon-specific sequence with uncertainty (e.g. standard deviation or coefficient of variation), LOD and LOQ of the method and practical values; the result refers only to the GMO targets; with a screening method and complex matrices it is recommended to note that the signal may come from non-target taxa; the content may be given as above or below a value taking uncertainty into account; Table 1 gives four wordings: no DNA of the species (“For species X, DNA was not detected”), no GM sequence (according to ISO 21569, with the LOD of the method and reference material, and if a sufficient amount of DNA cannot be shown — with an additional sentence and the LOD of the sample), sequence detected below the practical LOQ, quantitative result “x ± u %”. Test report (10): according to ISO 24276 and ISO 21569, at least the LOQ of the method with the material, practical LOQ, extraction method, amplification methods, reference material, result according to 9, PCR target and type of method (event-specific, construct-specific, screening), the way uncertainty was determined. Annex A, new A.2 — method specific for rice (sucrose phosphate synthase gene SPS, 81 bp fragment, real-time PCR with a TaqMan probe): GMDL-SJTU interlaboratory study, 12 laboratories, eight blind DNA samples of 0,5–10 ng; repeatability coefficient of variation 14,29–20,98 %, reproducibility 10,84–31,92 %, bias from −26,53 % to +8,92 %; within the dynamic range of 0,05–1,00 ng bias below 25 % in the intralaboratory study; absolute LOQ 0,01 ng/µl, relative LOQ not assessed; specificity: only rice positive among 11 plants, all 12 rice varieties positive; CTAB extraction according to ISO 21571; separate areas for extraction, PCR set-up and amplification, filter tips, powder-free gloves. In the Russian text, clause A.2.4 describes a probe labelled with FAM and quantification of “rice adh1 DNA”, while Table A.6 gives an SPS probe labelled with HEX — we do not resolve these discrepancies, as we have not read the English text of the amendment.

STATUS (as of 03.10.2026). In the PKN search (term “21570”, 03.10.2026) PN-EN ISO 21570:2007 (Polish version, “Introduces: EN ISO 21570:2005 [IDT], ISO 21570:2005 [IDT]”) has no withdrawal note; alongside it the supplementary element PN-EN ISO 21570:2007/A1:2013-06 (English version). Card: publication date 12.01.2007, 110 pages, KT 287 Biotechnology, ICS 67.050, “Replaces PN-EN ISO 21570:2006 – English version”, supplementary elements PN-EN ISO 21570:2007/AC:2007P, PN-EN ISO 21570:2007/Ap1:2007P and PN-EN ISO 21570:2007/A1:2013-06E, no withdrawal note. In the iTeh catalogue (read 03.10.2026) ISO 21570:2005 has the status “Published”, stage 90.60 (close of review), with Amendment 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 21570 appears in 3 records at 3 laboratories: AB 465, AB 704, AB 868. In the current PCA documents (read 03.10.2026; AB 465 issue No. 31 of 23.12.2025, AB 868 issue No. 21 of 06.03.2026, AB 704 issue No. 23 of 20.04.2026) the number appears in 3 lines at the same 3 laboratories; 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. The form is always “PN-EN ISO 21570” without year — with procedures PBD-74.00.00 and PBD-75.00.00 (AB 465), with “procedures developed by the laboratory” (AB 704) or together with PN-EN ISO 21569 (AB 868). None of these items is suspended. The “Laboratories” tab (term “PN-EN ISO 21570”) 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) — content of genetic modifications, real-time PCR; AB 704: agricultural products, food (foodstuffs) (flexible scope) — presence and content of GMOs, real-time PCR; AB 868: agricultural products and animal feed (flexible scope) — presence and content of genetic modifications, PCR and real-time PCR.

WHERE AN APPARENTLY CORRECT RESULT IS EASY TO GET. With the unit: the result is the ratio of the GM sequence to the taxon-specific sequence (usually in percent), not a mass fraction — the unit must be stated (4.3, Table 1). With the LOQ: when either of the two sequences is below the LOQ, no number is given — only a qualitative result with the practical LOQ (8). With consistency: two test portions must agree within −50 % to +100 % (1,0 and 2,0 % — yes, 0,9 and 2,1 % — no), and measurements in dilutions — within ± 33 % (7.5). With calculation: ΔΔCt only with very similar efficiencies of both reactions; copy number per haploid genome depends on zygosity; stacked genes overestimate the content (7.4). With calibration: calibrants related to certified reference materials, plasmid or synthetic dsDNA only after equivalence has been shown (4.3, 7.3). With the matrix: DNA from complex and highly processed matrices may be unsuitable for quantification — a taxon-specific control run is advisable (7.1). With divergent results: according to Amendment A1:2013 repeated +/− results make the test portion negative (8 as amended by A1). With the report: Amendment A1:2013 requires the practical LOQ, the reference material, the type of method and the way uncertainty was determined (10 as amended by A1). With the edition: all laboratories cite the standard without year, alongside in-house procedures; the PKN card lists three supplementary elements, and Amendment A1:2013 rewrites Clauses 8–10.

WHAT WE DO NOT GIVE. We do not quote the rest of the adh1 method or the methods of Annexes B–D — they lie outside the sample. We have not read the content of AC:2007, Ap1:2007, the rest of Amendment A1:2013 or ISO 24276. We do not give GMO labelling thresholds from legislation.

Method principle

In DNA extracted from the sample, two sequences are determined by real-time PCR (or competitive PCR): one specific to the GMO (screening element, construct or event) and an endogenous taxon-specific one. Copy numbers are obtained from calibration curves prepared from reference materials — preferably certified — and the GMO content is expressed as the ratio of the two sequences in percent, with uncertainty. A numerical result is given only when both sequences are above the LOQ and the measurements in test portions and dilutions meet the consistency criteria.

Applications

• Quantitative determination of GMOs in food; also in feed and plant samples from the environment (1)
• Example real-time PCR methods for maize (adh1, Event176, MON 810, Bt11, GA21, T25) and GTS 40-3-2 soya (Annexes A–D)
• Feed, agricultural products and food — items in the accreditation scopes of 3 laboratories in PCA (real-time PCR)

Key parameters

Standard

Standard number

PN-EN ISO 21570:2007

Title (PL)

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

Title (EN)

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

Step-by-step procedure

1. DNA

   Extraction according to ISO 21571; taxon-specific control run with processed matrices (7.1). PN-EN ISO 21570:2007 without a withdrawal note in PKN (03.10.2026).

2. Calibration

   Points and replicates over the working range, calibrants related to CRM (7.3).

3. Measurement

   GMO and taxon-specific sequences, each test portion in at least two replicates, controls according to ISO 24276 (7.1).

4. Consistency and interpretation

   ± 33 % in dilutions, −50 %…+100 % between portions; conditions of suitability for quantification (7.5, 8).

5. Result

   According to Table 1: X ± uncertainty % or a qualitative result with practical LOD/LOQ; LOD and LOQ of the method in the report (9, 10).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Work with DNA and PCR reagents — protection against cross-contamination
• Reagents according to the manufacturer’s safety data sheets

Frequently asked questions