PD ISO TS 21569 — Detection of Genetically Modified Organisms and Derived Products Series

🧬 PD ISO/TS 21569 is a Published Document series issued by BSI adopting the ISO technical specification ISO/TS 21569, belonging to horizontal methods for molecular biomarker analysis, providing standard methods for the detection of genetically modified organisms (GMOs) and derived products. The series uses real-time PCR technology, giving specific primers, probes, reaction conditions and result interpretation for particular transgenic constructs or screening sequences. This batch contains five documents covering construct-specific detection of event FP967, P35S-pat sequence screening, and P-nos / P-nos-nptII sequence detection.

💡 The key to GMO detection is “specificity” and “sensitivity”: it must both distinguish different transgenic events and reliably detect trace levels (e.g., 0.1% content). Real-time PCR monitors amplification in real time via fluorescence signal accumulation and is the mainstream GMO detection technology.

1. Series Structure and Document Scope 📌

Document	Subject	Edition	Status
PD ISO/TS 21569-2	Construct-specific real-time PCR method for detection of event FP967 in linseed and linseed products	2012	Readable
PD ISO/TS 21569-3	Construct-specific real-time PCR method for detection of P35S-pat sequence for screening GMOs	2015	Readable
PD ISO/TS 21569-4	Real-time PCR screening for P-nos and P-nos-nptII DNA sequences	2016	Readable
PD ISO/TS 21569-5	Real-time PCR general GMO screening (scanned)	2016	Scanned
PD ISO/TS 21569-6	Real-time PCR detection of other sequences (scanned)	2016	Scanned

⚠️ Parts 5 and 6 are scanned versions whose full text cannot be extracted; they are summarised here in line with the consistent framework of GMO real-time PCR detection. Always refer to the BSI official published versions for specific methods and data.

2. Methodological Basis: Real-Time PCR 🔬

Real-time PCR monitors fluorescence intensity each cycle during amplification via fluorescent probes (e.g., TaqMan probes) or dyes, producing amplification curves. Its core parameters include:

Ct value (cycle threshold): the cycle number at which fluorescence reaches a set threshold, inversely proportional to initial template quantity.
Standard curve and quantification: establish the Ct-concentration relationship with known-concentration standards for absolute or relative quantification.
Primer/probe specificity: designed for the target sequence (construct junction or screening sequence), avoiding cross-reaction with non-targets.
Positive/negative controls: include positive, negative and blank controls per batch to monitor false positives/negatives.

This methodology provides a unified template of “materials, reagents, apparatus, procedure and result interpretation” for each part of the series.

💡 Construct-specific methods target the “promoter–gene” or “gene–terminator” junction, detecting only a specific transgenic construct; screening methods target common regulatory elements (e.g., P35S, P-nos), giving an initial indication of whether GMO material is present. Combining them enables “screen first, then confirm”.

3. Detection of Event FP967 in Linseed (Part 2) 🌱

Part 2 provides a construct-specific real-time PCR method for linseed event FP967, an herbicide-tolerant transgenic linseed event. The part specifies:

Target sequence: the specific junction sequence of the FP967 construct.
Primers and probe: forward/reverse primer and TaqMan probe sequences.
Reaction system and programme: PCR reaction components, thermal cycling parameters, instrument settings.
Result interpretation: positive/negative judgement based on Ct values and controls, plus estimation of relative content.

The method applies to linseed and products thereof (e.g., linseed oil, linseed meal) for GMO detection.

4. P35S-pat Sequence Screening (Part 3) 🔍

Part 3 provides a construct-specific real-time PCR method for the P35S-pat sequence, used for GMO screening. P35S (Cauliflower Mosaic Virus 35S promoter) and pat (phosphinothricin-resistance gene) are common regulatory elements/genes in many transgenic crops. The part specifies:

Target sequence: the P35S-pat junction sequence.
Primers and probe: specific primers/probe detecting the P35S-pat sequence.
Reaction conditions: reaction system, thermal cycling, quality control.
Scope: screening of crops containing P35S-pat.

This method lets laboratories first perform a rapid “whether common GMO elements are present” screen on unknown samples, guiding subsequent event-specific confirmation.

Target	Type	Use	Typical Sample
FP967 construct	Event-specific	Confirm specific linseed event	Linseed and products
P35S-pat sequence	Construct-specific screening	Screen common GMO elements	Multiple crops
P-nos / P-nos-nptII	Screening/confirmation	Detect nopaline synthase promoter	Multiple crops

5. P-nos and P-nos-nptII Sequence Detection (Part 4) 🧪

Part 4 provides real-time PCR methods for the P-nos (nopaline synthase promoter) and P-nos-nptII sequences. P-nos originates from the Agrobacterium Ti plasmid and is a common promoter in many GMOs; nptII is a kanamycin-resistance marker gene. The part specifies primers/probes for the P-nos and P-nos-nptII junctions, reaction conditions and result interpretation, for detecting GMOs carrying these elements.

6. Engineering Practice Application 🛠️

🔍 Food testing: qualitative screening and event confirmation of GMO content in edible oils, soy products, cereals and other samples.
🔧 Feed testing: detect GMO ingredients in feed materials and finished feeds to meet labelling and regulatory requirements.
📊 Quantification: quantify GMO content using standard curves to assess whether it exceeds labelling thresholds (e.g., EU 0.9%).
📋 Laboratory quality control: establish positive/negative/blank controls, standards and records per the series methods to ensure traceable results.

7. Frequently Asked Questions ❓

❓ Q: What is the difference between construct-specific and screening detection?
A: Construct-specific targets the junction of a specific transgenic construct, confirming a particular event; screening targets common elements (e.g., P35S, P-nos), only giving an initial indication of whether GMO material is present.

❓ Q: Why use real-time PCR?
A: It has strong specificity, high sensitivity and real-time quantification, monitoring amplification through fluorescence accumulation, suitable for qualitative screening and quantitative determination of trace GMOs.

❓ Q: How is the Ct value interpreted?
A: Lower Ct indicates more initial template; quantification uses a standard curve relating Ct to concentration. Positivity/negativity is judged in combination with controls.

❓ Q: What is FP967?
A: FP967 is a transgenic event of herbicide-tolerant linseed; Part 2 provides a specific detection method targeting its construct junction.

❓ Q: How should the scanned parts (Parts 5/6) be used?
A: This article summarises only within the framework; for specific primer/probe sequences and reaction conditions, obtain and verify against the BSI official published versions rather than performing tests from scan abstracts alone.

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