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 mod...
- Standard No.
- ISO/TS 21569-8:2025
- Release Date
- 2025
- Published By
- International Organization for Standardization (ISO) IX / ISO
- Latest
- ISO/TS 21569-8:2025
- Introduction
Standard Technical Framework
Core Elements J101 Event J163 Event KK179 Event Limit of Detection (LOD95%) 3.6 copies 4.2 copies 7.0 copies Feature Sequence CQ857611/CQ857609 JA141099/JA141097 JA901749 Cq mean (15 copies/μl) 31.7 31.2 34.8
Key technologies for DNA extraction
The modified CTAB method is used as the standard, and the key steps include:
- Add 2% PVPP to remove polyphenol inhibitors
- Lyze at 65℃ for 90 minutes with Proteinase K digestion
- Precipitate non-DNA components with potassium acetate at -20℃
- Selectively precipitate DNA with CTAB buffer
- Dissolve DNA in 0.1X TE buffer preheated at 70℃
Validation data: Eleven inter-laboratory tests showed that the Cq value of the acc1 gene of the extracted DNA ranged from 16.9 to 23.0, meeting the quality requirement of ΔCq≤1.
Real-time fluorescence PCR system
Components J101 system J163 system KK179 system Primer concentration 400nM 400nM 500nM Probe labeling FAM-BHQ1 FAM-BHQ1 FAM-ZEN-IowaBFQ Amplification efficiency 95.8-106.3% 96.4-118.0% 78.1-103.5% Temperature program: initial denaturation at 95℃ for 3 min; 45 cycles (95℃ for 15 s, 60℃ for 60 s).
Method validation data
Validation by 11 European laboratories showed:
- Specificity: No cross-reaction to 12 non-target GM crops such as MON810
- Sensitivity: GM ingredients with a mass fraction of 0.1% can be stably detected
- Robustness: The Cq value difference between different equipment such as ABI7500/CFX96 is <3 cycles
Implementation recommendations
- Quality control requirements: Each batch must include a blank control, a positive control (containing the acc1 gene) and an inhibition control (1:4 diluted sample)
- Equipment calibration: It is recommended to use digital PCR to quantify the copy number of the standard
- Anti-pollution measures: Aerosol protection tips must be used, and the experiment must distinguish between the DNA extraction area and the PCR amplification area
- Data interpretation: The S-type amplification curve and Cq value ≤ 40 must be met at the same time
ISO/TS 21569-8:2025 Referenced Document
- 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 AMENDMENT 1
- ISO 21571 Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Nucleic acid extraction AMENDMENT 1
- ISO 24276 Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - General requirements and definitions; Amendment 1
ISO/TS 21569-8:2025 history
- 2025 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 mod...
