GMOMETHODS: EU DATABASE OF REFERENCE METHODS

Qualitative PCR method for detection of Figwort Mosaic Virus 35S

promoter
Element specific Last updated 12/6/2017

1. GENERAL INFORMATION

Target genetic element Figwort Mosaic Virus 35S promoter (P-FMV)

PCR Assay Simplex Real Time
Detection TaqMan®

Compendium Reference QL-ELE-00-015

2. VALIDATION DATA

Collaborative trial coordinator Federal Office of Consumer Protection and Food Safety (BVL)

Test material applied in collaborative trial DNA

Materials used for calibration/controls plasmid DNA solution

Tested GM Events

Event Name Unique Identifier Crop Name

GT73 (RT73) MON-00073-7 Brassica napus

MON89788 MON-89788-1 Glycine max

Collaborative Trial Description

Each participant received 18 DNA blind samples including non-GM oilseed rape DNA, non-GM soybean DNA or DNA
samples containing 10 or 50 copies of the p-FMV target sequence from oilseed rape event GT73 or soybean event
MON89788. The laboratories also received reaction reagents, a plasmid standard solution and primers and probes.
The participants prepared a serial dilution down to the theoretical value of 0.1 target copy per reaction for the
determination of the limit of detection (LOD). Each DNA sample and dilution of the standard DNA were tested using
the P-FMV procedure. The blind DNA samples were analysed in a single PCR reaction. The standard dilution's having
up to 50 target copies per reaction were analysed in triplicates while the standards dilution from 20 to 0.1 target copies
were analysed in ten replicates (total of 160 tests each) for the LOD determination.

Method Performance

LOD Relative not reported LOD Absolute 5 HGE

Values determined in the collaborative trial

False positive (%) 0%

False negative (%) 1%

Test Level (%)

0 20 50
Specificity (%) 100%
Sensitivity (%) 98% 100%

Unit of Measurement Test Level Target copy N.

Comment
Element specific procedures are targeting sequences that often, but not always, are found in GMOs. For this reason
it is necessary to verify a positive P-FMV result. A further investigation using construct-specific or event-specific
methods should be carried out.

3. REFERENCES

Official Compendium of Analytical Methods according to § 64 LFGB (Food and Feed law) (2014). Food Analysis,
Detection of a DNA sequence of the FMV-promoter (pFMV) in foodstuffs with real-time PCR. BVL L 00.00-148,
Beuth, Berlin Koln

ISO/TS 21569-5:2016 Horizontal methods for molecular biomarker analysis -- Methods of analysis for the detection
of genetically modified organisms and derived products -- Part 5: Real-time PCR based screening method for the
detection of the FMV promoter (P-FMV) DNA sequence

4. PRIMERS AND PROBES SEQUENCES

GM-target(s) Figwort Mosaic Virus 35S promoter (P-FMV)

Primer Forward 5'-CAAAATAACGTGGAAAAGAGCT-3'

Target element P-FMV

Primer Reverse 5'-TCTTTTGTGGTCGTCACTGC-3'

Target element P-FMV

Amplicon length 78 bp

Probe 5'-FAM-CTGACAGCCCACTCACTAATGC-BHQ1-3'
 5. PCR REACTIONS SETUP
GM-target(s)

Reagent Final Concentration

QuantiTect Multiplex PCR No-Rox Mastermix (Qiagen) 1x

Primer Fw 0.34 µmol/L

Primer Rev 0.34 µmol/L

Probe 0.120 µmol/L

Double-distilled sterile water #

Template DNA max

Final Volume 25 µL

6. AMPLIFICATION CONDITIONS

GM-target(s)

Stage Temperature Time NoCycles

Activation/Initial Denaturation 95°C 900” 1

Denaturation 95°C 15”

Annealing & Extension 60°C 60”

Denaturing, Annealing & Extension 45