Mericon™ Quant GMO Handbook

February 2011
mericon™ Quant GMO Handbook
For detection and quantification of genetically

modified organisms in food or animal feed
samples using real-time PCR
Sample & Assay Technologies

QIAGEN Sample and Assay Technologies
QIAGEN is the leading provider of innovative sample and assay technologies,
enabling the isolation and detection of contents of any biological sample. Our
advanced, high-quality products and services ensure success from sample to
result.
QIAGEN sets standards in:
 Purification of DNA, RNA, and proteins
 Nucleic acid and protein assays
 microRNA research and RNAi
 Automation of sample and assay technologies
Our mission is to enable you to achieve outstanding success and
breakthroughs. For more information, visit www.qiagen.com.
Contents
Kit Contents 4
Storage 5
Product Use Limitations 5
Product Warranty and Satisfaction Guarantee 5
Quality Control 5
Technical Assistance 6
Safety Information 6
Introduction 7
Principle 7
Assay-specific information 10
Equipment and Reagents to Be Supplied by User 14
Important Notes 15
General precautions 15
Relevant assay controls 15
Internal control calibration for real-time cyclers 16
 Detection and Quantification of GMO DNA by Real-Time PCR
without ROX 17
 Detection and Quantification of GMO DNA by Real-Time PCR
with ROX 23
 Analyzing the Results 29
Troubleshooting Guide 33
References 36
Ordering Information 37
mericon Quant GMO Detection Assays Handbook 02/2011 3

Kit Contents
mericon Quant GMO Detection Assays
See Table 1 for specific kits
Number of reactions 48
Yellow PCR Assay GMO* 4 x 12 reactions
Orange PCR Assay Reference System* 4 x 12 reactions
Red Quant Control DNA† 12 reactions
Green Standard DNA 48 reactions
Blue Multiplex PCR Master Mix‡ 1040 μl
QuantiTect® Nucleic Acid Dilution Buffer 1.5 ml
RNase-Free Water 1.9 ml
50x ROX Dye Solution 45 μl
Handbook 1
* Contains target-specific primers and probes, as well as the internal control (IC).
†
Quantification Control DNA: Contains DNA of the target GMO and reference species in a
defined ratio.
‡
Contains HotStarTaq® Plus DNA Polymerase, dedicated multiplex real-time PCR buffer, and
dNTP mix (dATP, dCTP, dGTP, dTTP).
Table 1. mericon Quant GMO Detection Assays
Product name Catalog no.

mericon Quant RR Soy (48) 291514
For specific detection and quantification of DNA
from Roundup Ready soy
mericon Quant MON 810 (48) 291524
For specific detection and quantification of DNA
from MON 810 corn
* For more detailed information, see “Assay-specific information”, page 10.
4 mericon Quant GMO Detection Assays Handbook 02/2011

Storage
mericon Quant GMO Detection Assays are shipped on dry ice. Multiplex PCR
Master Mix and ROX dye should be stored at –20°C immediately upon receipt,
in a constant-temperature freezer. All remaining not reconstituted kit
components should be stored at 2–8°C and protected from light. Stored under
these conditions and handled correctly, assay performance remains unaffected
until the date of expiration printed on the quality control label inside the kit box
or envelope.
Reconstituted reagents of mericon Quant GMO Detection Assays should be
dispensed into aliquots to avoid more than 5 freeze–thaw cycles, and stored at
2–8°C for short-term storage (1 month) or –20°C for long-term storage.
Product Use Limitations

mericon Quant GMO Detection Assays are intended for molecular biology
applications in food, animal feed, and pharmaceutical product testing. These
products are not intended for the diagnosis, prevention, or treatment of a disease.
Product Warranty and Satisfaction Guarantee

QIAGEN guarantees the performance of all products in the manner described
in our product literature. The purchaser must determine the suitability of the
product for its particular use. Should any product fail to perform satisfactorily
due to any reason other than misuse, QIAGEN will replace it free of charge or
refund the purchase price. We reserve the right to change, alter, or modify any
product to enhance its performance and design. If a QIAGEN product does not
meet your expectations, simply call your local Technical Service Department or
distributor. We will credit your account or exchange the product — as you wish.
Separate conditions apply to QIAGEN scientific instruments, service products,
and to products shipped on dry ice. Please inquire for more information.
A copy of QIAGEN terms and conditions can be obtained on request, and is
also provided on the back of our invoices. If you have questions about product
specifications or performance, please call QIAGEN Technical Services or your
local distributor (see back cover or visit www.qiagen.com).
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each
lot of Quant GMO Detection Assays is tested against predetermined
specifications to ensure consistent product quality.

Technical Assistance
At QIAGEN, we pride ourselves on the quality and availability of our technical
support. Our Technical Service Departments are staffed by experienced
scientists with extensive practical and theoretical expertise in sample and assay
technologies and the use of QIAGEN products. If you have any questions or
experience any difficulties regarding mericon Quant GMO Detection Assays or
QIAGEN products in general, please do not hesitate to contact us.
QIAGEN customers are a major source of information regarding advanced or
specialized uses of our products. This information is helpful to other scientists as
well as to the researchers at QIAGEN. We therefore encourage you to contact
us if you have any suggestions about product performance or new applications
and techniques.
For technical assistance and more information, please see our Technical
Support Center at www.qiagen.com/Support or call one of the QIAGEN
Technical Service Departments or local distributors (see back cover or visit
www.qiagen.com).
Safety Information
When working with chemicals, always wear a suitable lab coat, disposable
gloves, and protective goggles. For more information, please consult the
appropriate material safety data sheets (MSDSs). These are available online in
convenient and compact PDF format at www.qiagen.com/Support/MSDS.aspx
where you can find, view, and print the MSDS for each QIAGEN kit and kit
component.
24-hour emergency information

Emergency medical information in English, French, and German can be
obtained 24 hours a day from:
Poison Information Center Mainz, Germany
Tel: +49-6131-19240

Introduction
mericon Quant GMO Detection Assays are a ready-to-use system for the
detection and quantification of specific DNA constructs from genetically
modified organisms (GMO) in food, animal feed, and pharmaceutical products
using real-time polymerase chain reaction (PCR). These assays perform
optimally on the Rotor-Gene® Q but have also been validated for block thermal
cyclers. The Multiplex PCR Master Mix included in each kit contains QIAGEN
proprietary technology including HotStarTaq Plus DNA Polymerase, patented
multiplex PCR technology such as Factor MP, and fast cycling technology
including Q-Bond®. Multiplex PCR Master Mix is also highly tolerant to PCR
inhibitors.
Each mericon Quant Assay contains an optimized mixture of PCR primer sets for
a GMO-specific and a species-specific (Reference System) target sequence,
each with an internal control (IC), plus probes labeled with distinct fluorescent
dyes. In both systems, the test sample is detected with FAM reporter (495/520
nm), and the internal control is detected with MAX™ NHS Ester reporter (MAX;
524/557 nm). In addition, each kit includes a Quantification Control DNA, a
Standard DNA, and all the reagents necessary to perform the analysis.
Principle
Detection of genetically modified organisms (GMO) by PCR is based on the
amplification of a genetic construct used in GMOs. In real-time PCR, the
amplified product is detected via target-specific fluorescent probes that bind to
the amplified product. Accumulation of PCR product results in an increased
fluorescent signal from the bound probes. Monitoring the fluorescence
intensities during the PCR run (i.e., in real time) allows the detection of the
accumulating PCR product without having to re-open the reaction tubes
afterward.
The probes of mericon PCR Assays are sequence-specific oligonucleotides with a
fluorophore and a quencher moiety attached. The fluorophore is at the 5' end
of the probe, and the quencher moiety is located at the 3' end. If the target
DNA sequence is present, the probe is cleaved by the 5'3' exonuclease activity
of HotStarTaq Plus DNA Polymerase during the extension phase of PCR. This
separates the fluorophore and the quencher moiety resulting in a detectable
fluorescence that is proportional to the amount of accumulated PCR product.

Quantification of the target GMO is carried out based on the alignment of the
PCR results from a GMO-specific PCR system and a Reference PCR system. The
GMO assay targets either a genetic construct commonly used in a desired
GMO or a DNA sequence that is specific for a particular GMO (e.g., Roundup
Ready soy). The Reference assay targets the plant species of the GMO (e.g.,
soy). Standard curves for both systems form the basis of the quantification
experiment. They are generated with a defined dilution of the Standard DNA,
which contains a defined ratio of GMO and Reference DNA.
The PCR primer sets for each mericon Quant GMO assay are highly specific.
Targets are verified bioinformatically and experimentally. Cross-reactivity is
tested with a continuously growing panel of selected targets for each mericon
PCR Assay. Each assay can detect down to 10 target copies in a reaction.
Dedicated mericon sample preparation solutions are available from QIAGEN
for a broad range of starting materials. These solutions were developed to
complement mericon PCR Assays, and provide a complete and efficient
workflow for food safety testing.
HotStarTaq Plus DNA Polymerase

HotStarTaq Plus DNA Polymerase is a modified form of QIAGEN Taq DNA
Polymerase. It is provided in an inactive state and has no enzymatic activity at
ambient temperature. This prevents formation of misprimed products and
primer–dimers during the reaction setup and first denaturation step.
Competition for reactants by PCR artifacts is therefore avoided, enabling high
PCR specificity and accurate quantification. The enzyme is first activated by a 5-
minute, 95°C incubation step at the start of the reaction, which enables rapid
and convenient setup at room temperature. In addition, the concentration of the
polymerase in the master mix is optimized to allow short extension times in the
combined annealing/extension step of each PCR cycle.
Multiplex PCR Master Mix

The Multiplex PCR Master Mix is specifically developed for fast-cycling,
multiplex, real-time PCR using sequence-specific probes. A novel additive in the
buffer, Q-Bond, allows short cycling times on standard cyclers and on fast
cyclers with rapid ramping rates. Q-Bond increases the affinity of HotStarTaq
Plus DNA Polymerase for short single-stranded DNA, reducing the time required
for primer/probe annealing to a few seconds. The buffer also contains Factor MP,
which facilitates multiplex PCR. This synthetic factor increases the local
concentration of primers and probes at the DNA template and stabilizes
specifically bound primers and probes, allowing efficient annealing and
extension. In addition, the Multiplex PCR Buffer is carefully formulated to be
highly tolerant to inhibitors commonly present in food.

QuantiTect Nucleic Acid Dilution Buffer
QuantiTect Nucleic Acid Dilution Buffer is an optimized solution to dilute the
nucleic acids used as the standards and the quantification or positive controls
for mericon PCR Assays. The buffer stabilizes DNA standards and controls
during dilution and reaction setup and prevents loss of nucleic acids on plastic
surfaces, such as tubes or pipet tips. The buffer is ready to use and is free of
DNases. Proper use of the buffer enables safe and accurate dilution of the
small amounts of nucleic acids typically used as controls for analysis of nucleic
acids. Aliquots of diluted standards and quantification or positive controls can
be stored in QuantiTect Nucleic Acid Dilution Buffer at –15 to –30°C for up to 6
months. Repeated freezing and thawing should be avoided.
ROX Dye Solution, 50x

ROX Dye Solution is used with certain real-time cyclers to compensate for non-
PCR-related variations in fluorescence detection. It is provided in a 50x solution
for your convenience. It is not needed for Rotor-Gene Q, LightCycler® systems
from Roche®, SmartCycler® instruments from Cepheid, and Bio-Rad. It is
necessary for most instruments from Applied Biosystems®, and is optional for
Stratagene® cyclers from Agilent. Instructions for using the dye are provided in
the protocol “Protocol: Detection and Quantification of GMO DNA by Real-
Time PCR with ROX” on page 23.
Primer/probe mix with internal control

Each mericon PCR Assay includes rigorously designed primers and probes in a
carefully balanced mix that amplify a target sequence and an internal control
(IC) with high specificity. This internal control provides information regarding
the presence of inhibitors in tested samples and the overall success of the PCR.
MAX NHS Ester is employed as the reporter dye for the internal control. With
excitation/emission maxima of 524/557 nm and a non-fluorescent quencher
(Iowa Black®), MAX dye has a spectral profile comparable to HEX®, JOE®, or
VIC®, and can be used with most real-time cyclers.

Assay-specific information
mericon Quant RR Soy Kit

The mericon Quant RR Soy Kit is designed for the specific detection and
quantification of Roundup Ready soy DNA in food, animal feed, and
pharmaceutical products.
Limit of detection of the GMO and Reference System assays

The GMO-specific RR Soy Assay and the Reference System Assay (soy assay) of
the mericon Quant RR Soy Kit can both detect down to 10 copies of target DNA
in a reaction.
Specificity of the GMO and Reference System assays

The GMO-specific RR Soy Assay exhibits high specificity for Roundup Ready soy.
No cross-reactivity was observed with other plant or animal species, GMOs, or
genetic constructs not found in Roundup Ready soy (Table 2), using 50 ng of
tested DNA.
Table 2. Results of cross-reactivity experiments for the RR Soy Assay
Genetic construct, Genetic construct,

GMO, or species Result GMO, or species Result
Roundup Ready soy + Bt11 corn –
Bt176 corn – Lambda DNA –
StarLink corn – LL rape –
CaMV – GA21 corn –
Roundup Ready canola – T25 corn –
MON 810 corn – NK603 corn –
Potato – Rice –
Pig – Rye –
Wheat – Barley –
Cattle – Sheep –
* Cross-reactivity experiments are ongoing. For up-to-date information, visit

www.qiagen.com/mericonQuantGMO.

The Reference System Assay (soy assay) exhibits high specificity for soy and
Roundup Ready soy. No cross-reactivity was observed with other plant or
animal species or GMOs (Table 3), using 50 ng of tested DNA.
Table 3. Results of cross-reactivity experiments for the Reference System

Assay for the Quant RR Soy Kit

Soy + Roundup Ready soy +
Potato – Cattle –
Horse – Goat –
Rye – Corn –
Barley – Pig –
Rice – Sheep –
Canola – Wheat –
Roundup Ready rice – CaMV –
T25 corn – CGH351 corn –
NK603 corn – Bt176 corn –
Bt11 corn – MON 810 corn –


mericon Quant MON 810 Kit
The mericon Quant MON 810 Kit is designed for the detection and quantification
of MON 810 corn in food, animal feed, and pharmaceutical products.
Limit of detection of the GMO and Reference System assays

The GMO-specific MON 810 Assay and the Reference System Assay (corn
assay) of the mericon Quant MON 810 Kit can both detect down to 10 copies
of target DNA in a reaction.
Specificity of the GMO and Reference System assays

The GMO-specific MON 810 Assay exhibits high specificity for MON 810 corn.
No cross-reactivity was observed with other plant or animal species, GMOs, or
genetic constructs not found in MON 810 corn (Table 4), using 50 ng of tested
DNA.
Table 4. Results from cross-reactivity experiments for the MON 810 Assay

MON 810 corn + 356043 soy –
Bt176 corn – EH92-527 potato –
Bt11 corn – H7-1 sugar beet –
MIR604 corn – Soy –
3272 corn – Barley –
NK603 corn – Potato –
TC1507 corn – Rice –
MON88017 corn – Wheat –
MON863 corn – Rye –
59122 corn – Cattle –
98140 corn – Pig –
Roundup Ready soy – Sheep –
305423 soy – Goat –


The Reference System Assay (corn Assay) exhibits high specificity for corn and
MON 810 corn. No cross-reactivity was observed with other plant or animal
species or GMOs (Table 5), using 50 ng of tested DNA.
Table 5. Results from cross-reactivity experiments for the Reference

System Assay for the Quant MON 810 Kit

Corn + Sheep –
Soy – Goat –
Canola – Horse –
Rice – Hazelnut –
Potato – Pecan nut –
Rye – Macadamia –
Wheat – Almond –
Barley – Celery –
Cattle – Mustard –
Pig – Lupine –


Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable
gloves, and protective goggles. For more information, consult the appropriate
material safety data sheets (MSDSs), available from the product supplier.
 Nucleic acid isolation kit. We recommend the DNeasy® mericon Food Kit
(see Ordering Information)
 Pipets (adjustable)*
 Sterile pipet tips with filters
 Rotor-Gene Q or other real-time PCR instrument* with fluorescence
detection for approximately 520 nm (FAM fluorescence) and approximately
560 nm (for yellow dyes).
 PCR plastics for the thermal cycler to be used.
For Rotor-Gene Q: Strip Tubes and Caps, 0.1 ml, for use with 72-well
rotor (cat. no. 981103 or 981106) or PCR Tubes, 0.2 ml, for use with
36-well rotor (cat. no. 981005 or 981008)
 For Rotor-Gene Q: Loading Block 72 x 0.1 ml Tubes, cat. no. 9018901, or
Loading Block 96 x 0.2 ml Tubes, cat. no. 9018905
 Tube rack
 Microcentrifuge*
 Vortexer*
 Ice bucket with ice or cooling block
* Ensure that instruments have been checked and calibrated according to the manufacturer’s
recommendations. Use of yellow dye to detect the internal control of the mericon PCR Assays
requires calibration on some instruments.

Important Notes
General precautions
The user should always pay attention to the following:
 Use gloves as well as sterile pipet tips with filters.
 All materials and media possibly containing DNA of the tested GMO
should be autoclaved for 20 min at 120°C.
 Store and extract positive materials (specimens, positive controls, and
amplicons) separately from all other reagents, and add them to the
reaction mix in a spatially separated facility.
 Thaw all components thoroughly at room temperature (15–25°C) before
starting an assay.
 When thawed, mix the components (by pipetting repeatedly up and down
or by pulse vortexing), and centrifuge briefly.
Relevant assay controls
Internal control
Each vial of the GMO and Reference System PCR assays contains an internal
control to monitor successful PCR reaction and to detect possible PCR inhibition.
Standard dilutions
A Standard DNA is provided with each mericon Quant GMO Kit to prepare
standard curves for the GMO and Reference System. It contains DNA of the
target GMO, as well as of the reference species. For the Quant RR Soy Kit, the
GMO:Reference ratio of the Standard DNA is1:1. For the Quant MON 810 Kit,
the GMO:Reference ratio of the Standard DNA is 1:0.57. The Standard DNA is
diluted in defined steps and dilutions measured in the relevant copy number
range for the GMO and for the reference species with both assay systems.
Standard dilutions are integrated into the PCR reaction setup (see Table 9, page
20, and Table 16, page 26).

Quantification Control
The Quantification Control serves as a positive control for the quantification
experiment. It contains DNA of the target GMO, as well as of the reference
species. In all mericon Quantification Controls, the GMO:Reference DNA ratio
is 1±0.2%. During a quantification experiment, verification of this ratio ensures
preparation of accurate standard curves and correct performance of
quantification calculations. The Quantification Control is integrated into the PCR
reaction setup (see Table 9, page 20, and Table 16, page 26).
Negative PCR control

Negative controls should be included in each analysis run to check for possible
contamination of the mericon Assays during reaction setup. Instead of adding
sample DNA to a reaction vial containing Multiplex PCR Master Mix, add the
same volume of QuantiTect Nucleic Acid Dilution Buffer or RNase-free water.
Internal control calibration for real-time cyclers

MAX NHS Ester (MAX) dye is used to detect the internal control of mericon PCR
Assays. Table 6 lists common thermal cyclers with their calibration requirement
and the detection channel or filter set for this dye. Refer to the manufacturer’s
manual of the thermal cycler to be used for detailed calibration instructions.
Table 6. Calibration requirements and detection channel for MAX NHS

Ester (MAX) dye
Filter suitable for

MAX NHS Ester
Thermal cycler Dye calibration* detection
Rotor-Gene Q
Not required Yellow
Rotor-Gene 6000
Applied Biosystems models
7000, 7300, 7500, 7700, Required for new
VIC
7900HT, StepOne™, instruments†
StepOnePlus™
Stratagene (Agilent) models Filter set 535/550 nm
Not required
Mx3005P®, Mx3000P® (HEX, JOE, VIC)
* For information on detection channel settings for instruments not listed in Table 6, contact
QIAGEN Technical Services.
†
If the instrument is new, a dye calibration for the individual channels (e.g., VIC) of the real-
time cycler must be performed. See the manufacturer’s manual for details on calibration.

Protocol: Detection and Quantification of GMO DNA
by Real-Time PCR without ROX
Important points before starting
 Before beginning the procedure, read “Important Notes”, page 15.
 Take time to familiarize yourself with the Rotor-Gene Q or other real-time
PCR instrument to be used before starting the protocol. See the instrument
user manual.
Things to do before starting

 Prepare the mericon PCR Assay GMO (tube with yellow lid).
Add 130 μl Multiplex PCR Master Mix (tube with blue lid) to each vial of
mericon PCR Assay GMO (yellow lid). Mix by pipetting up and down 5 times
or vortexing, and centrifuge briefly.
Note: If the reconstituted PCR Assay GMO will not be used entirely in one
assay run, make appropriate aliquots to avoid more than 5 freeze–thaw
cycles, and store the aliquots at 2–8°C for short-term storage (1 month) or
–20°C for long-term storage.
 Prepare the mericon PCR Assay Reference System (tube with orange lid)
Add 130 μl Multiplex PCR Master Mix (tube with blue lid) to each vial of
mericon PCR Assay Reference System (orange lid). Mix by pipetting up and
down 5 times or vortexing, and centrifuge briefly.
Note: If the reconstituted PCR Assay Reference System will not be used
entirely in one assay run, make appropriate aliquots to avoid more than
5 freeze–thaw cycles, and store the aliquots at 2–8°C for short-term
storage (1 month) or –20°C for long-term storage.
 Dissolve the dried Quantification Control DNA (tube with red lid). Add
150 μl of QuantiTect Nucleic Acid Dilution Buffer to the vial and mix by
pipetting up and down 5 times or vortexing. Centrifuge briefly.
Note: If the dissolved Quantification Control will not be used entirely in one
 Dissolve the dried Standard DNA (tube with green lid). Add 120 μl of
QuantiTect Nucleic Acid Dilution Buffer to the vial (Standard 1) and mix by
Use this solution for the preparation of defined Standard DNA dilutions.
Assign the copy numbers given in Table 7 for the Quant RR Soy Kit and
Table 8 (page 18) for the Quant MON 810 Kit to the standard dilutions.

Table 7. Preparation of the Standard DNA dilution series for the
Quant RR Soy Kit
Copy no. per Copy no. per

RRS PCR Reference
Standard Dilutions reaction PCR reaction
Standard DNA dissolved in
Standard 1 – 81,920
120 μl QT NA Dilution Buffer
10 μl Standard 1 + 70 μl
Standard 2 10,240 10,240
QT NA Dilution Buffer
Standard 3 1280 1280
Standard 4 160 160
Standard 5 20 –
Table 8. Preparation of the Standard DNA dilution series for the

Quant MON 810 Kit
Copy no per Copy no per

MON 810 Reference
Standard Dilutions PCR reaction PCR reaction
Standard 2 10,240 5,837
Standard 3 1280 730
Standard 4 160 91
Standard 5 20 –

Note: If the reconstituted Standard DNA (Standard 1) will not be used
storage (1 month) or –20°C for long-term storage.
 Before each use, all reagents need to be thawed completely, mixed (by
repeated pipetting up and down or quick vortexing), and centrifuged briefly.
PCR reaction assignment

Assign Standard, Quantification Control, and sample reactions to the respective
assay systems as shown in Table 9 (page 20). Standard dilutions as well as
Quantification Control and negative template controls (NTC) are assayed in
duplicates. Sample extraction should be carried out at least twice, and extracted
DNA used in PCR reactions with both assay systems at 2 different concentrations
(e.g., undiluted and 1:4 diluted).

Table 9. Assignment of Standard, Quantification Control, and sample
reactions
Number of Number of
GMO PCR Reference System
Component replicates PCR replicates
Standard 1 – 2
Standard 2 2 2
Standard 3 2 2
Standard 4 2 2
Standard 5 2 –
Quantification Control DNA 2 2
NTC 2 2
Sample extraction 1, dilution 1 1 1
Procedure
1. Set up the Standard, Quantification Control, and sample reactions
according to Table 10, page 21. Keep all samples and reaction tubes
on ice during setup.
If using the Rotor-Gene Q, place the desired number of PCR tubes or strips
into the adapters of the cooling block for the Rotor-Gene Q.

Table 10. Setup of Standard, Quantification Control, and sample
reactions for the GMO and Reference System mericon PCR Assays
Standard Quantification Negative

Component dilutions Control Samples Control
Reconstituted mericon
Assay (GMO or 10 μl 10 μl 10 μl 10 μl
Reference System)
Respective Standard
10 μl – – –
dilution
– 10 μl – –
DNA
Sample DNA – – 10 μl –
QuantiTect Nucleic
Acid Dilution Buffer or – – – 10 μl
RNase-free water
Total volume 20 μl 20 μl 20 μl 20 μl
2. Close the PCR tubes or strips and place them in the reaction chamber
of the thermal cycler, securing them according to the instrument
manual.
If using the Rotor-Gene Q, make sure that the locking ring is placed on top
of the rotor to prevent accidental opening of the tubes during the run.
3. Program the thermal cycler. If using the Rotor-Gene Q or Rotor-Gene
6000, use the cycling protocol in Table 11 (page 22). For all other
real-time cyclers, use the cycling protocol in Table 12 (page 22).
Note: For information on instrument detection settings for the MAX NHS
Ester dye (MAX) used to detect the internal control of mericon Assays, see
Table 6 on page 16.
4. For the Rotor-Gene Q or Rotor-Gene 6000 make sure that ‘Perform
Optimisation Before 1st Acquisition’ in the Gain Optimisation menu
is activated.
5. Start the PCR run.
6. Proceed to the protocol “Analyzing the Results” on page 29.

Table 11. Cycling protocol for Rotor-Gene Q or Rotor-Gene 6000
Step Time Temperature Comments

Initial PCR Activation of HotStarTaq
5 min 95°C
activation step Plus DNA Polymerase
3-step cycling:
Denaturation 15 s 95°C
Data collection at 60°C
Annealing 15 s 60°C
Extension 10 s 72°C
Number of cycles 45
Detection Reporter Excitation/emission Channel
Target FAM 495/520 nm Green
Internal control MAX 524/557 nm Yellow
Table 12. Cycling protocol for real-time cyclers other than Rotor-Gene Q
or Rotor-Gene 6000

5 min 95°C
3-step cycling:
Annealing 23 s* 60°C
Number of cycles 45
Target FAM 495/520 nm Green (FAM)
Internal control MAX 524/557 nm Yellow (VIC)†
* For some instruments, the shortest annealing time possible is longer than 23 s (in the range
of 32 s). Use the shortest annealing time permitted by the instrument.
†
See Table 6 (page 16) for information on instrument-specific detection channel or filter set.

Protocol: Detection and Quantification of GMO DNA
by Real-Time PCR with ROX
For certain real-time thermal cyclers, the use of a ROX passive reference dye
during the PCR is necessary to compensate for variations in the fluorescence
signal that are not related to the PCR.
Important points before starting

 Before beginning the procedure, read “Important Notes”, page 15.
 Take time to familiarize yourself with the real-time PCR instrument to be
used before starting the protocol. See the instrument user manual.
Things to do before starting

 Prepare the PCR Assay GMO (tube with yellow lid) and the PCR Assay
Reference System (tube with orange lid) by adding Multiplex PCR Master
Mix (tube with blue lid) and ROX dye to the tubes of both Assay systems
according to Table 13. Mix by pipetting up and down 5 times or vortexing,
and centrifuge briefly.
Table 13. Components to add to the mericon Assay
Multiplex PCR
Thermal cycler ROX dye Master Mix
Applied Biosystems models
7000, 7300, 7700, 7900HT, 10.4 μl per vial 130 μl per vial
StepOne, StepOnePlus
Applied Biosystems model 7500 5.2 μl per vial 130 μl per vial
Rotor-Gene models, Stratagene
Mx models, LightCycler 480,
ROX reference dye not necessary
SmartCycler models, Bio-Rad
instruments
Note: If the reconstituted mericon Assays will not be used entirely in one

 Dissolve the dried Quantification Control DNA (tube with red lid). Add
150 μl of QuantiTect Nucleic Acid Dilution Buffer to the vial, and mix by
Note: If the dissolved Quantification Control will not be used entirely in one
 Dissolve the dried Standard DNA (tube with green lid). Add 120 μl of
QuantiTect Nucleic Acid Dilution Buffer to the vial (Standard 1), and mix by
Use this solution for the preparation of defined Standard DNA dilutions.
Assign the copy numbers given in Table 14 for the Quant RR Soy Kit and
Table 15 (page 25) for the Quant MON 810 Kit to the standard dilutions.
Table 14. Preparation of the Standard DNA dilution series for the Quant
RR Soy Kit

RRS PCR Reference
Standard Dilutions reaction PCR reaction
Standard 2 10,240 10,240
Standard 3 1280 1280
Standard 4 160 160
Standard 5 20 –

Table 15. Preparation of the Standard DNA dilution series of the Quant
MON 810 Kit

MON 810 Reference
Standard Dilutions PCR reaction PCR reaction
Standard 2 10,240 5,837
Standard 3 1280 730
Standard 4 160 91
Standard 5 20 –
Note: If the reconstituted Standard DNA (Standard 1) will not be used

storage (1 month) or –20°C for long-term storage
 Before each use, all reagents need to be thawed completely, mixed (by
repeated up and down pipetting or by quick vortexing), and centrifuged
briefly.

PCR reaction assignment
Assign the Standard, Quantification Control, and sample reactions to the
respective assay systems as shown in Table 16. Standard dilutions, as well as
Quantification Control and negative template controls (NTC) are assayed in
duplicates. Sample extraction should be carried out at least twice, and the
extracted DNA used in PCR reactions with both assay systems at 2 different
concentrations (e.g., undiluted and 1:4 diluted).
Table 16. Assignment of Standard, Quantification Control, and sample

reactions
Number of GMO Number of Reference

Component PCR replicates System PCR replicates
Standard 1 – 2
Standard 2 2 2
Standard 3 2 2
Standard 4 2 2
Standard 5 2 –
Quantification Control DNA 2 2
NTC 2 2
Procedure
1. Set-up the Standard, Quantification Control, and sample reactions
according to Table 17 or Table 18 (page 27), depending on the
thermal cycler to be used. Keep all samples and reaction tubes on ice
during setup.

reactions for Applied Biosystems models 7000, 7300, 7700, 7900HT,
StepOne, and StepOnePlus

Assay (GMO or 10.8 μl 10.8 μl 10.8 μl 10.8 μl
Reference System
Respective Standard
9.2 μl – – –
dilution
– 9.2 μl – –
DNA
Sample DNA – – 9.2 μl –
QuantiTect Nucleic
Acid Dilution Buffer or – – – 9.2 μl
RNase-free water

reactions for Applied Biosystems model 7500

Assay (GMO or 10.4 μl 10.4 μl 10.4 μl 10.4 μl
Reference System
Respective Standard
9.6 μl – – –
dilution
– 9.6 μl – –
DNA
Sample DNA – – 9.6 μl –
QuantiTect Nucleic
Acid Dilution Buffer or – – – 9.6 μl
RNase-free water

2. Close the PCR tubes or strips, and place them in the reaction
chamber of the thermal cycler, securing them according to the
instrument manual.
3. Program the real-time cycler according to Table 19.
Note: For information on instrument detection settings for the MAX NHS
Ester dye (MAX) used to detect the internal control of mericon Assays, see
Table 6 on page 16. Start the PCR run.
4. Start the PCR run.
5. Proceed to the protocol “Analyzing the Results” on page 29.
Table 19. Cycling protocol for real-time cyclers using ROX reference dye

5 min 95°C
3-step cycling:
Annealing 23 s* 60°C
Number of cycles 45
Target FAM 520 nm Green (FAM)
Internal control MAX 520 nm Yellow (VIC)†
* For some instruments, the shortest annealing time possible is longer than 23 s (in the range
of 32 s). Use the shortest annealing time permitted by the instrument.
†
See Table 6 (page 16) for information on instrument-specific detection channel or filter set.

Protocol: Analyzing the Results
Qualitative GMO detection
Qualitative determination of the presence or absence of DNA from a genetically
modified organism (GMO) is carried out based on the amplification of the
target sequence by the GMO-specific mericon PCR assay. Amplification is
visualized in real time on the amplification plot generated by the application
software of the real-time PCR instrument used. A positive result is visible as a
final point on the fluorescence curve that lies clearly above the threshold.
Figures 1–3 are examples of possible outcomes, which are summarized in
Table 20 (page 30).
Green channel Yellow channel

Normalized fluorescence
Cycle Cycle
Figure 1. The sample is negative for the tested GMO or genetic construct. The 3 sample
curves in the green channel (left) are at the baseline and below a preset threshold. The
corresponding curves of the internal control in the yellow channel (right) are above the
threshold, indicating that the PCR was successful.
Green channel
Yellow channel
Cycle Cycle
Figure 2. The sample is positive for the tested GMO or genetic construct. The 3 sample
curves in the green channel (left) and the corresponding curves of the internal control in the
yellow channel (right) are above a preset threshold indicating the presence of target DNA in
the sample and a successful PCR.

Green channel Yellow channel
Cycle Cycle
Figure 3. The PCR is inhibited. No amplification of the three samples in the green channel
(left) or the internal control in the yellow channel (right). All curves lie along the baseline and
do not exceed a preset threshold.
Table 20. Summary of possible outcomes
Amplification of Amplification of
internal control sample Result
+ + Sample is positive
+ – Sample is negative
– – PCR failed (inhibition)
Partial inhibition of the PCR due to the presence of detectable but tolerable
concentrations of inhibitors in the samples is typically indicated by a shift of the
Internal Control to higher cycle values. As a guideline, the uninhibited Internal
Control should give a stable cycle value between 28 and 32. A cycle value
above 33 indicates inhibition.
In the event of PCR inhibition, dilute the extracted samples 1:10 with QuantiTect
Nucleic Acid Dilution Buffer or RNase-free water and repeat the test.
If the DNA template concentration in the PCR reaction is very high, a shift of the
Internal Control to lower cycle values might occur, which does not influence its
sensitivity toward PCR inhibitors or amplification of the target DNA.

Quantitative GMO detection
Quantification of the genetically modified organism (GMO) is carried out based
on the alignment of the PCR results from the GMO-specific PCR system and the
Reference PCR system. Results are obtained according to the reaction scheme
illustrated in Table 9, page 20, or Table 16, page 26.
Standard dilutions are measured in the relevant concentration range for
detection of the GMO and overall plant species content of the sample. The
GMO DNA normally represents only a small fraction of the overall sample
DNA. It is measured at the lower end of the copy number range (Table 7 and
Table 8, page 18, or Table 14 and Table 15, pages 24 and 25). The plant
species DNA normally represents the major part of the sample DNA. It is
measured at the upper end of the copy number range (Table 7 and Table 8,
page 18 or Table 14 and Table 15, pages 24 and 25). Cycle values of the
standard dilutions obtained with each assay system are plotted against the
logarithm of the respective copy numbers to provide a separate standard curve
for the GMO system and the Reference system. Cycle values of the unknown
samples are compared with both standard curves to determine the copy
numbers of the GMO and Reference species in the unknown sample. From the
ratio of the determined sample copy numbers for both systems (Reference
system set at 100%), the percentage of GMO in the unknown sample is
calculated.
If possible, it is recommended to use the software of the applied real-time cycler
for data analysis. From the acquired CT values of the standard dilutions, the
instrument normally generates standard curves and calculates slope, as well as
goodness-of-fit of the linear regression. An accurate GMO quantification relies
on the precision of the standard curves. Accuracy of the quantification
experiment is monitored via the Quantification Control. The Quantification
Control contains a defined GMO:Reference ratio of 1±0.2%. Quantification
results are representative if the GMO content of the Quantification Control is
determined within a 0.8–1.2 % range.
GMO quantification is carried out with DNA samples of two separate DNA
extractions, applying two different DNA dilutions to verify reproducibility and
consistency of the calculated GMO content. If the GMO content of both
dilutions strongly differs, one or both assay systems are influenced by PCR
inhibitors. In this case, the quantification experiment must be repeated with
additional sample dilutions until the GMO contents align.
Table 21 (page 32) is an example of possible data obtained from a
representative RR Soy quantification experiment.

Table 21. Example of possible data outcome and calculated GMO
content from a representative RRS quantification experiment
Component GMO Copy no. Ref Copy no. % GMO

18.86
Standard 1 – – 81,920 –
18.55
23.35 21.83
Standard 2 10,420 10,420 –
23.29 21.74
25.78 24.60
Standard 3 1250 1250 –
26.10 24.86
29.30 27.87
Standard 4 160 160 –
29.22 27.71
32.90
Standard 5 20 – – –
32.92
R2 value
0.9942 – 0.9988 – –
(linearity)
y-axis intercept 37.29 – 35.15 – –
Slope standard
–3.55 – –3.34 – –
curve
PCR efficiency 91% – 99% – –
Quant. Control
30.15 102 21.64 10,986 0.93%
replicate 1
Quant. Control
29.89 121 21.56 11,608 1.04%
replicate 2
Extraction 1,
27.75 485 20.67 21,425 2.26%
undiluted
Extraction 1,
29.64 143 22.51 6,035 2.37%
1:4 diluted
Extraction 2,
25.60 1,954 18.64 86,686 2.25%
undiluted
Extraction 2,
27.00 789 20.02 33,518 2.35%
1:4 diluted

Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may
arise. For more information, see also the Frequently Asked Questions page at
our Technical Support Center: www.qiagen.com/FAQ/FAQList.aspx. The
scientists in QIAGEN Technical Services are always happy to answer any
questions you may have about either the information and protocols in this
handbook or sample and assay technologies (for contact information, see back
cover or visit www.qiagen.com).
Comments and suggestions

No signal with the Quantification Control
a) The selected For data analysis, select the green channel (FAM) for the
fluorescence samples and the yellow channel (VIC or corresponding
channel for PCR filter set) for the internal control. Please see the cycling
data analysis protocols in Table 11 or Table 12 (page 22), or in Table
does not 19 (page 28). Refer to the manufacturer’s manual of the
comply with the cycler to be used.
protocol
b) Incorrect Compare the temperature profile with the protocol. See
programming the cycling protocols in Table 11 or Table 12 (page 22),
of the real-time or in Table 19 (page 28). Refer to the manufacturer’s
PCR instrument manual of the cycler to be used.
c) Incorrect Check that reactions were set up according to Table 10
configuration of (page 21) or Table 17 or Table 18 (page 27). Repeat the
the PCR PCR, if necessary.
d) Kit or kit See “Storage” (page 5). Check the storage conditions and
components the expiration date (see the kit label) of the reagents and
incorrectly use a new kit, if necessary.
stored
e) The mericon Check the expiration date (see the kit label) of the
PCR Assay has reagents and use a new kit, if necessary.
expired
Determination of a GMO content of the Quantification Control, which is
not in the 1±0.2% range
a) Standard curves Make sure that the Standard DNA dilutions were strictly
are not prepared according to Table 7 or Table 8 (page 18) or
prepared Table 14 or Table 15 (pages 24 and 25). Provided copy
correctly numbers refer to final copy numbers per PCR reaction.

b) Standard curves For preparation of the standard curves CT values (y-axis)
are not plotted are plotted against the logarithm of the copy numbers
or read-off (x-axis) provided in Table 7 or Table 8 (page 18) or
properly Table 14 or Table 15 (pages 24 and 25). The data
correlation needs to follow a linear regression. Copy
numbers correlating with CT values of the Quantification
Control should be read off the standard curves by the
real-time cycler software or alternatively calculated via the
linear equation corresponding to the data fit.
c) Quality of the Accuracy of the standard curves is verified via the
standard curves Quantification Control, which contains a GMO:Reference
is low ratio of 1±0.2%. Standard curves are correctly prepared
if the Quantification Control is determined within a 0.8–
1.2 % range.
Deviation of determined GMO content of sample dilutions from one
DNA extraction.
Inhibition of one Repeat the quantification experiment with additional
of both PCR sample dilutions until the GMO contents align.
systems
Weak or no signal in the internal amplification control
a) The PCR Check that PCR conditions match the cycling protocols in
conditions do Table 11 or Table 12 (page 22), or Table 19 (page 28).
not comply with Repeat the PCR with corrected settings, if necessary.
the protocol
b) The PCR was Use the recommended DNA isolation method and closely
inhibited follow the manufacturer’s instructions. QIAGEN offers
dedicated sample preparation kits developed to
complement mericon PCR Assays, and provide a complete
and efficient workflow for food safety testing.
c) Kit or kit See “Storage” (page 5). Check the storage conditions and
components the expiration date (see the kit label) of the reagents and
incorrectly use a new kit, if necessary.
stored
d) The mericon Check the expiration date (see the kit label) of the
PCR Assay has reagents and use a new kit, if necessary.
expired

Signals present for the negative controls
a) Contamination Repeat the PCR with new reagents in replicates.

occurred during If possible, close the PCR tubes directly after addition of
PCR setup the sample to be tested.
Make sure to pipet the positive controls last.
Make sure that work space and instruments are
decontaminated at regular intervals.
b) Contamination Repeat the extraction and PCR of the sample to be tested
occurred during using new reagents.
extraction Make sure that work space and instruments are
decontaminated at regular intervals.

References
QIAGEN maintains a large, up-to-date online database of scientific
publications utilizing QIAGEN products. Comprehensive search options allow
you to find the articles you need, either by a simple keyword search or by
specifying the application, research area, title, etc.
For a complete list of references, visit the QIAGEN Reference Database online
at www.qiagen.com/RefDB/search.asp or contact QIAGEN Technical Services
or your local distributor.

Ordering Information
Product Contents Cat. no.
mericon Quant RR Soy For 48 reactions: PCR Assay RR Soy, 291514
(48) PCR Assay Reference System, Quant
Control DNA, Standard DNA, Multiplex
PCR Master Mix, QuantiTect Nucleic
Acid Dilution Buffer, RNase-Free Water,
50x ROX Dye Solution
mericon Quant MON For 48 reactions: PCR Assay MON 810, 291524
810 (48) PCR Assay Reference System, Quant
Control DNA, Standard DNA, Multiplex
PCR Master Mix, QuantiTect Nucleic
Acid Dilution Buffer, RNase-Free Water,
50x ROX Dye Solution
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mericon Screen Nos Kit For 24 reactions: PCR Assay Screen 291043
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mericon RR Soy (24)* For 24 reactions: PCR Assay RR Soy, 291113
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triple Kit (24)* Campylobacter triple, Positive Control,
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mericon sample preparation kits
DNeasy mericon Food 50 QIAquick® Spin Columns, 69514
Kit (50) Proteinase K, buffers
mericon DNA Bacteria Fast Lysis Buffer 69525
Kit (100)
mericon DNA Bacteria 50 Pathogen Lysis Tubes L, Fast Lysis 69534
Plus Kit (50) Buffer
Instrumentation
Rotor-Gene Q 5plex Real-time PCR cycler with 5 channels Inquire
(green, yellow, orange, red, crimson),
laptop computer, software, accessories,
1-year warranty on parts and labor
For up-to-date licensing information and product-specific disclaimers, see the

respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and
user manuals are available at www.qiagen.com or can be requested from
QIAGEN Technical Services or your local distributor.

Notes

Notes

Trademarks: QIAGEN®, DNeasy®, QIAquick®, HotStarTaq®, mericon™, Q-Bond®, QuantiTect®, Rotor-Gene® (QIAGEN Group); Mx3005P®,
Mx3000P®, Stratagene® (Agilent Technologies); SmartCycler® (Cepheid); Roche® (Hoffman-La Roche, Inc.); Iowa Black®, MAX™ (Integrated DNA
Technologies, Inc.); Applied Biosystems®, FAM®, HEX®, JOE®, StepOne™, StepOnePlus™, VIC® (Life Technologies); LightCycler® (Roche Group).
Developed by: ifp Institut für Produktqualität, Teltowkanalstr. 2, 12247 Berlin, Germany. www.produktqualitaet.com
Limited License Agreement
Use of this product signifies the agreement of any purchaser or user of the mericon Quant GMO Assays to the following terms:
1. The mericon Quant GMO Assays may be used solely in accordance with the mericon Quant GMO Handbook and for use with components
contained in the Kit only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this
Kit with any components not included within this Kit except as described in the mericon Quant GMO Handbook and additional protocols
available at www.qiagen.com.
2. Other than expressly stated licenses, QIAGEN makes no warranty that this Kit and/or its use(s) do not infringe the rights of third-parties.
3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited
above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court
costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit
and/or its components.
For updated license terms, see www.qiagen.com.
NOTICE TO PURCHASER: LIMITED LICENSE
The purchase price of this product includes a limited, non-transferable license under some or all of US. Patents Nos. 5,210,015, 5,487,972,
5,476,774, 5,219,727, and 5,804,375 or corresponding patent claims outside the United States, owned by Roche Molecular Systems, Inc. or F.
Hoffmann-La Roche Ltd, and US. Patents Nos. 5,538,848, 5,723,591, 5,876,930, 6,030,787, and 6,258,569 and corresponding patent claims
outside the United States, owned by Applera Corporation, to use only this amount of product to practice the 5' nuclease process solely in the Food
Testing Applications Field, including reporting results of purchaser’s activities for a fee or other commercial consideration and also for the
purchaser’s own internal research. No right under any other patent claims (such as apparatus or system claims in US. Patent No. 6,814,934) and no
right to use this product for any other purpose, is hereby granted expressly, by implication or by estoppel. Further information on purchasing licenses
may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Center Drive, Foster City, California 94404, USA.
License Probe manufactured by Integrated DNA Technologies, Inc. under license from Applied Biosystems. License Probe incorporates IDT’s Iowa
Black quencher technology and is accompanied by a limited research use only license from IDT.
© 2011 QIAGEN, all rights reserved.

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February 2011

mericon™ Quant GMO Handbook

For detection and quantification of genetically

Sample & Assay Technologies

mericon Quant GMO Detection Assays Handbook 02/2011 3

Table 1. mericon Quant GMO Detection Assays

Product name Catalog no.

* For more detailed information, see “Assay-specific information”, page 10.

4 mericon Quant GMO Detection Assays Handbook 02/2011

Product Use Limitations

Product Warranty and Satisfaction Guarantee

mericon Quant GMO Detection Assays Handbook 02/2011 5

24-hour emergency information

6 mericon Quant GMO Detection Assays Handbook 02/2011

mericon Quant GMO Detection Assays Handbook 02/2011 7

HotStarTaq Plus DNA Polymerase

Multiplex PCR Master Mix

8 mericon Quant GMO Detection Assays Handbook 02/2011

ROX Dye Solution, 50x

Primer/probe mix with internal control

mericon Quant GMO Detection Assays Handbook 02/2011 9

mericon Quant RR Soy Kit

Limit of detection of the GMO and Reference System assays

Specificity of the GMO and Reference System assays

Table 2. Results of cross-reactivity experiments for the RR Soy Assay

Genetic construct, Genetic construct,

* Cross-reactivity experiments are ongoing. For up-to-date information, visit

10 mericon Quant GMO Detection Assays Handbook 02/2011

Table 3. Results of cross-reactivity experiments for the Reference System

Genetic construct, Genetic construct,

* Cross-reactivity experiments are ongoing. For up-to-date information, visit

mericon Quant GMO Detection Assays Handbook 02/2011 11

Limit of detection of the GMO and Reference System assays

Specificity of the GMO and Reference System assays

Genetic construct, Genetic construct,

* Cross-reactivity experiments are ongoing. For up-to-date information, visit

12 mericon Quant GMO Detection Assays Handbook 02/2011

Table 5. Results from cross-reactivity experiments for the Reference

Genetic construct, Genetic construct,

* Cross-reactivity experiments are ongoing. For up-to-date information, visit

mericon Quant GMO Detection Assays Handbook 02/2011 13

14 mericon Quant GMO Detection Assays Handbook 02/2011

Relevant assay controls

mericon Quant GMO Detection Assays Handbook 02/2011 15

Negative PCR control

Internal control calibration for real-time cyclers

Table 6. Calibration requirements and detection channel for MAX NHS

Filter suitable for

16 mericon Quant GMO Detection Assays Handbook 02/2011

Things to do before starting

mericon Quant GMO Detection Assays Handbook 02/2011 17

Copy no. per Copy no. per

Table 8. Preparation of the Standard DNA dilution series for the

Copy no per Copy no per

18 mericon Quant GMO Detection Assays Handbook 02/2011

PCR reaction assignment

mericon Quant GMO Detection Assays Handbook 02/2011 19

20 mericon Quant GMO Detection Assays Handbook 02/2011

Standard Quantification Negative