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. DNA extraction from alfalfa seeds and real-time PCR based detection methods for genetically modified alfalfa events J101, J163 and KK179 | Status: Current

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.