Eurofins GeneScan Technologies GmbH

Engesserstr. 4, D-79108 Freiburg

T | +49 - 761 - 50 38-200
F | +49 - 761 - 50 38-111
kits@eurofins.com
www.eurofins.com/kits

Validation Sheet
GMOScreen RT 35S/IPC and NOS/IPC and FMV/IPC
(LR/HR+)
Cat. # 5421220303
Module GS-P-09.035 and GS-P-09.036 and GS-P-09.037

Validation was carried out on the following real-time PCR machine(s):

Validation/Verification performed on:

ABI 7500 (Applied Biosystems®) ,
ABI 7500 Fast (in standard mode)
MX3000P /MX3005P (Agilent)
ABI 7700 (Applied Biosystems)
ABI 7900 (Applied Biosystems)

The following parameters have been validated/verified and the respective performance criteria have been fulfilled:

Validation Setup/
Parameter
Performance criteria
Property of a method to respond exclusively to the
characteristic or analyte of interest. Specificity testing of GMO-
specific PCR systems.

• Test:
Test of GM materials:
1% GM content (100 ng/rxn), 3 reactions each,
and non-GM material: 100 ng/rxn, 3 rxns each

35S positive set:

Soy: RRS, A5547-127, DP-356043
Specificity Maize: Bt10, Bt11, Bt176, TC1507, DAS-59122-7, Liberty Link
T25, NK603, MON863, MON88017, MON810
Canola: T45, Topas 19/2
Cotton: RoundUpReady, Bollgard I, Bollgard II, LL25
Rice: LL62

nos positive set:

Soy: RRS
Maize: GA21, MIR604, 3272, Bt10, Bt11, MON863,
MON88017, NK603
Cotton: RoundUpReady, Bollgard I, Bollgard II, LibertyLink
(LL25), VipCot102
Canola: MS8, RF3, MS1, RF1, RF2
Potato: EH92

FMV positive set:

pGSE611, MON89788 soybean, RR cotton,
RT73 rapeseed, H7 sugarbeet; MON89034 maize

page 1 of 4
 Eurofins GeneScan Technologies GmbH
Engesserstr. 4, D-79108 Freiburg
T | +49 - 761 - 50 38-200
F | +49 - 761 - 50 38-111
kits@eurofins.com
www.eurofins.com/kits

Negative set 35S, nos:

Cotton: 281-24-236 x 3006-210-23
Non-GM materials:
Soy, maize, canola, cotton, rice, sugar beet, potato

Negativ set for FMV:

GM material:
RR soybean, LL soybean (5547-127), LL soybean (2704-12),
DP-305423-2 soybean, DP-356043-5 soybean, MON810
maize, Bt176 maize, Bt11 maize, GA21 maize, T25 maize,
Specificity (continued) MON863 maize, NK603 maize, MIR604 maize, MON88017
maize, 3272 maize, DAS-59122-7 maize, Bt10 maize, TC1507
maize, MS8 rapeseed, MS1 rapeseed, Rf3 rapeseed, Rf1
rapeseed, Rf2 rapeseed, T45 rapeseed, Topas 19/2
rapeseed, Bollgard cotton, Bollgard II cotton, LL25 cotton,
281-24-236x3006-210-12 cotton, EH92 potato, LL62 rice
Non-GM material:
wheat, rye, oat, barley, sorghum, potato, tomato, maize,
soybean,rice

• Acceptance criteria:
-Positive reaction in the appropriate positive set only
-No amplification in the negative set and non-GM materials
-IPC valid in all reactions

• Acceptance criteria fulfilled

b. BLAST database search with amplicon sequence

• Acceptance criteria:
No 100% match with other sequences than target shall be
found

• Acceptance criteria fulfilled

page 2 of 4
 Eurofins GeneScan Technologies GmbH
Engesserstr. 4, D-79108 Freiburg
T | +49 - 761 - 50 38-200
F | +49 - 761 - 50 38-111
kits@eurofins.com
www.eurofins.com/kits

Parameter Validation Setup/ Performance criteria

The absolute LOD is defined as ≤ 10 copies by proofing

single-copy sensitivity and taking into account a notable safety
factor.

For all systems:

Limit of detection, absolute • Test:
(LODabs) 12 reactions at 0.693 copies target per reaction (plasmid
DNA)
.
• Acceptance criteria:
4-9 positives in 12 reactions

• Acceptance criteria fulfilled

Addition of inhibiting substances at different concentrations to

the mix. Analysis of plasmid or genomic standard DNA at 10
copies target per reaction. Highest inhibitor concentration shall
result in total inhibition of PCR systems.
Determination of the inhibitor concentration where inhibition
occurs for PCR systems.

all systems:
IPC reliability • Test:
Test: 5 inhibitor concentration levels and 0.1 x TE as
reference, each in duplicates, Inhibitors to be tested: e.g.
strongly inhibited DNA extracts

• Acceptance criteria:
Inhibition of IPC system not later than GMO specific
PCR system.

• Acceptance criteria fulfilled

page 3 of 4
 Eurofins GeneScan Technologies GmbH
Engesserstr. 4, D-79108 Freiburg
T | +49 - 761 - 50 38-200
F | +49 - 761 - 50 38-111
kits@eurofins.com
www.eurofins.com/kits

Parameter Validation Setup/ Performance criteria

Test of DNA isolated from real-life laboratory test samples.

Alternatively DNA samples with a modified DNA extraction
procedure enhancing inhibitory effects. Such modifications
may be extended incubation times, reduced washing steps,
decreased elution volumes etc. Matrices most relevant for the
assay type shall be selected.

• Test:
1. Negative or low copy positive real-life test samples
showing partial or complete inhibition:

For all PCR systems:

44 samples ( ABI Prism 7900), 12 samples (ABI 7500 Fast,
MX3005 and ABI Prism 7700) in double preparations, 1 rxn
each prep spiked with 10 copies target DNA.
Robustness/ Matrix-effects
2. Negative real-life test samples exhibiting no inhibitory
Effects

For all PCR systems:

46 samples ( ABI Prism 7900), 12 samples (ABI 7500 Fast,
MX3005 and ABI Prism 7700) in double preparations, 1 rxn
each prep, no spiking with positive material

• Acceptance criteria:

1) result is positive or IPC indicates inhibition.

2) result is negative and IPC indicates no
inhibition.(Samples exhibiting particularly low GM
contents may result in +/+ or +/- results with high CT
values)

• Acceptance criteria fulfilled

Proper function of the PCR assay after storage of the PCR

reagents as supplied under several storage conditions.

• Test:

Storage conditions to be tested:

1x freeze/thaw (reference variant), 5x freeze/thaw, 10 cycle:
35°C/4°C, 1 min each or 3d @ RT. Test of 3 or 6 reactions
Stability NTC and 3 or 6 reactions positive (10 cp/rxn) for each storage
condition.

• Acceptance criteria:
All NTCs:
dRn increase < 50 % threshold value
All positives:
CT Shift<1 and dRn decrease <33% compared to reference
variant
• Acceptance criteria fulfilled

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