PD ISO TS 21569-8-2025 PDF

Full title and description

PD ISO/TS 21569-8:2025 — 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. This document specifies validated procedures for extracting amplifiable DNA from alfalfa (Medicago sativa) seeds and for event-specific real-time PCR identification of the three named GM alfalfa events.

Abstract

This technical specification defines a DNA-extraction procedure for ground alfalfa seeds and event-specific real-time PCR assays that target the junction sequences between the alfalfa genome and the integrated constructs of herbicide-tolerant events J101 and J163, and lignin-modified event KK179. The methods are suitable for direct event identification and as confirmatory follow-up after initial screening (for example after detection of sequences such as P‑FMV, T‑nos or CTP2/CP4‑EPSPS). Validation was carried out on ground seed material; the PCR assays are also applicable to other matrices (foodstuffs, feed) provided sufficient amplifiable DNA is obtained.

General information

• Status: Published
• Publication date: 2 April 2025
• Publisher: International Organization for Standardization (ISO); available as a PD/TS deliverable and may be adopted by national standards bodies.
• ICS / categories: 67.050 (Food sciences — Methods of analysis and sampling)
• Edition / version: Edition 1 (2025)
• Number of pages: 12

Scope

Specifies procedures for extraction of amplifiable DNA from alfalfa seeds and event-specific real-time PCR detection methods for genetically modified alfalfa events J101, J163 and KK179. Applicable to crop/plant/seed/grain test samples and, with appropriate DNA extraction, to food and feed matrices. Methods target the genome–construct junctions and are intended for event-specific identification or as confirmatory analyses following screening tests.

Key topics and requirements

• Validated DNA extraction protocol optimized for ground alfalfa (Medicago sativa) seeds.
• Event-specific real-time PCR assays for alfalfa events J101, J163 and KK179 targeting transition (border) sequences.
• Use as direct identification or as follow-up confirmation after construct- or promoter/terminator-based screening.
• Performance and validation parameters established on seed material (limits of detection, specificity, controls, inhibition checks).
• Guidance on applicability to other matrices (feed, foodstuffs) and requirement for sufficient quality/quantity of amplifiable DNA.
• Requirements for appropriate positive/negative controls, assay acceptance criteria and interpretation of results.

Typical use and users

Used by molecular testing laboratories, official control and enforcement laboratories, seed testing facilities, food and feed testing services, plant breeding and biotechnology R&D groups, and regulatory authorities performing GMO detection, traceability and compliance testing. Also used by contract testing providers validating event-specific methods for clients in the agri‑biotech and seed sectors.

Related standards

Part of the ISO/TS 21569 series of horizontal methods for molecular biomarker analysis. Related deliverables include other parts of ISO/TS 21569 (screening and construct-specific real‑time PCR methods), the earlier ISO 21569:2005 family of documents on nucleic-acid based GMO detection, and other ISO/TC 34/SC 16 standards on food/feed GMO analysis and sampling. Laboratories implementing this part typically reference general PCR and quality standards used for nucleic acid testing and accreditation (e.g., ISO/IEC 17025) alongside the series.

Keywords

alfalfa, Medicago sativa, GMO, genetically modified organism, event-specific, J101, J163, KK179, DNA extraction, seed testing, real-time PCR, qPCR, molecular biomarker, food testing, feed testing

FAQ

Q: What is this standard?

A: It is PD ISO/TS 21569-8:2025, a Technical Specification that defines DNA extraction from alfalfa seeds and event-specific real-time PCR methods for detecting GM alfalfa events J101, J163 and KK179. It was published on 2 April 2025.

Q: What does it cover?

A: It covers validated procedures for extracting amplifiable DNA from ground alfalfa seed material and the design/usage of real-time PCR assays that detect the genome-to-construct junctions of the three specified GM alfalfa events. It also provides guidance on controls, validation parameters and applicability to other matrices such as food and feed.

Q: Who typically uses it?

A: Accredited testing laboratories, seed and food/feed testing services, regulatory and enforcement bodies, biotechnology developers, and research groups involved in GMO detection and verification.

Q: Is it current or superseded?

A: This document is current (published 2 April 2025) as Part 8 in the ISO/TS 21569 series. It does not by itself supersede more general or earlier ISO standards unless explicitly stated; rather it provides a part‑specific, validated method within the broader series of horizontal methods for GMO detection.

Q: Is it part of a series?

A: Yes — it is Part 8 of the ISO/TS 21569 series (Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products). The series includes screening, construct- and event-specific methods across multiple parts.

Q: What are the key keywords?

A: Alfalfa, GMO, event-specific, J101, J163, KK179, DNA extraction, seed testing, real-time PCR, qPCR, molecular biomarker.