hygiena GMO MON863 Maize Detection Kit Instruction Manual

: June 1, 2024
: Hygiena

Table of Contents

hygiena GMO MON863 Maize Detection Kit
Introduction
Principle of PCR detection
Contents
Additional Materials, Reagents and Devices Required
General Precautions
Sampling and handling
Protocol
Data analysis
Troubleshooting
Supplementary Information
Change Index
References
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hygiena GMO MON863 Maize Detection Kit

Introduction

Many countries worldwide have implemented legislation for the use, cultivation and labeling of foodstuffs containing genetically modified organisms (GMOs). These regulations allow the usage of GMOs under certain conditions, often including a defined threshold for labeling or where the import and use of GMOs is prohibited. Thus, reliable methods for the detection and identification of GMOs in food and feed are required.
With the foodproof® SL GMO product line, Hygiena® Diagnostics offers a wide range of easy and reliable assays for the detection of GMOs. The foodproof SL GMO Detection Kits allow fast, safe and easy detection in food and feed samples.

Intended Use
The foodproof SL GMO MON863 Maize Detection Kit is designed to detect the GM- maize MON863 event-specific gene in various processed foods, raw materials, feed, seeds, etc.
This kit provides a real-time PCR Master Mix with enzyme components and the specific primer/probe set for rapid testing by real-time PCR, as well as the Internal Control (IC) system for reliable results.

Principle of PCR detection

The foodproof SL GMO MON863 maize detection assay is a qualitative, duplex real-time PCR test for the detection of the GM maize MON863-specific gene and the Internal Control (IC) using specific primers and probes labeled with fluorescent dyes. The target sequences are detected through FAM and HEX (VIC) channels, respectively.
The primer and probe mixture provided is based on the so-called TaqMan® principle. During PCR amplification, forward and reverse primers hybridize to the target DNA. A fluorogenic probe is included in the same reaction mixture, which consists of an oligonucleotide labeled with a 5’-reporter dye and a downstream 3’-quencher. During PCR amplification, the probe is cleaved and the reporter dye and quencher are separated. The resulting increase in fluorescence can be detected through a range of real-time PCR platforms.
The monitoring of the fluorescence intensities during the real-time PCR allows the detection of accumulating product without re-opening the reaction tubes after the PCR run.
The kit minimizes contamination risk and contains all reagents needed for detection (except for PCR-grade H2O).

Internal Amplification Control
This kit contains the Internal Positive Control (IC) as PCR inhibition Control. The IC allows the user to detect and control possible PCR inhibition. The IC reagents are included in the primer/probe mixture and the IC is co- amplified with target DNA from the sample. The results can be visualized in the HEX (VIC) channel.
Carry-over prevention using UNG system
The foodproof SL GMO MON863 Maize Detection Kit utilizes the UNG system. Carry-over contamination between PCR reactions can be prevented by including uracil-N-glycosylase (UNG) in the reaction mix. UNG can only prevent carry- over from PCR reactions that include deoxyuridine triphosphate (dUTP) in the original PCR reaction.

Reagent	Cap Label	Volume	Description
2x real-time PCR Master Mix	****

Additional Materials, Reagents and Devices Required

Disposable powder-free gloves and laboratory coat
Pipettors (0.5 to 10 μL, 2 to 20 μL, 20 to 200 μL, 200 to 1,000 μL)
Sterile aerosol-barrier pipette tips
Ice or benchtop cooler
Vortex mixer
Clean bench area or PCR box
Tabletop centrifuge with rotor for 2 mL reaction tubes
Real-time thermal cycler with FAM and HEX (VIC) detection channels
Disposable polypropylene microtubes for PCR
PCR-grade H2O
For DNA Extraction: foodproof Sample Preparation Kit III foodproof Magnetic Preparation Kit III

General Precautions

Store extracted positive material (samples, controls and other amplicons) away from all other reagents and add it to the reaction mix in a separate area.
Thaw all components thoroughly on ice before starting the experiment.
When thawed, mix the components and centrifuge briefly.
Do not pipette by mouth.
Do not eat, drink, smoke, apply cosmetics or handle contact lenses in laboratory work areas.
Do not use a kit beyond its expiration date.
Safety Data Sheets (SDS) can be found at www.hygiena.com/documents.
Use disposable gloves, laboratory coats and eye protection while handling samples and reagents.
Thoroughly wash hands afterward.
Dispose of all samples and unused reagents in compliance with local regulations.
Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with Biosafety Level 2 or other appropriate biosafety practices.
Clean and disinfect all sample or reagent spills using a disinfectant such as 0.5% sodium hypochlorite, or other suitable disinfectant.
Avoid contact of specimens and reagents with the skin, eyes and mucosa. If contact occurs with skin, eyes or mucosa, immediately flush with water and seek medical attention.
Use of this product should be limited to personnel trained in laboratory DNA amplification techniques.
To avoid carry-over contamination with PCR product or control DNA, please note the following points:
1. Be careful not to contaminate the Primer/Probe Mixture and 2x real-time PCR Master Mix with other PCR products or Control DNA through pipetting. To prevent contamination, the use of aerosol-barrier tips is recommended.
2. Open and close all sample tubes carefully. Avoid splashing or spraying PCR samples.
3. It is important to have designated lab areas where PCR reactions are set up, preferentially separated in space from the areas where PCR reactions are analyzed.
4. The laboratory process must be one-directional; it should begin in the Extraction Area and move to the Amplification and Detection Area. Do not transport samples, equipment and reagents to the areas where you performed previous steps.

Sampling and handling

Sample Collection
Various processed food, raw material, feed and seed samples are routinely examined.
Sample Storage
The assay sensitivity can be reduced if you routinely freeze the samples before testing or store them for an extended period of time. Avoid repeated freezing and thawing of samples, which may lead to DNA degradation and decreased sensitivity.
Nucleic Acid Extraction
Carry out DNA isolation according to the extraction kit’s product instructions. For more information, please see www.hygiena.com

Protocol

DNA Isolation
Hygiena Diagnostics provides sample preparation kits suitable for all kinds of foods and raw materials.
(See 5. “Additional Required Materials, Reagents and Devices”)
Preparing the PCR
To prevent the risk of contamination with foreign DNA, we recommend that all experiment steps be performed in a PCR cleanroom or separated environment area. Aerosol-barrier pipette tips are recommended for each step.
Thawing the Kit Components
The use of ice or a benchtop cooler is recommended during experiments to maintain enzyme activity.
Prepare Reaction Master Mix
Each reaction has a total volume of 25 μL; the volume of the DNA sample is 5 μL.

Prepare the reaction mixture according to Table 2 below.
Table 2: PCR reaction mixture

Composition	Volume
Primer / Probe Mixture	4 µL
2x real-time PCR Master Mix	12.5 µL
PCR-grade H2O	3.5 µL
Total	20 µL

Add 5 μL of extracted DNA sample into the tube.

Prepare Control Amplification Reactions

Positive control amplification: Add 5 μL of Control DNA instead of sample DNA.
Negative control amplification: Add 5 μL of PCR-grade H2O instead of sample DNA

Mixing
Mix the reagents in the PCR reaction tubes by tapping a minimum of 5 times. Briefly centrifuge the tubes to remove any air bubbles or drops inside the cap.

Amplification

Program your real-time PCR instrument according to the manufacturer’s manual.
Create a temperature-time profile on your instrument as follows in Table 3.

Table 3: Temperature Time Profile

Temperature	Time	Cycle
50 °C*	2 min	1
95 °C	10 min	1
95 °C	15 seconds	****

45

60 °C**| 1 min

*The UDG activation step inhibits contamination by PCR product.
**Detect the fluorescence at this step.

Data analysis

The fluorescence curves are analyzed in FAM and HEX (VIC) fluorescence detection channels (see Table 4).
You can predict the presence or absence of the target gene in your samples by analyzing the real-time PCR results.

Table 4: Specific Detection on Fluorescence Channel

Target Gene	Fluorophore
MON863	FAM
IC	HEX (VIC)

Interpretation of Results

The signal is considered to be positive if the corresponding fluorescence accumulation curve crosses the threshold line.
Results are accepted as relevant if both positive and negative amplification controls pass.
IC: When amplifying a target sample with a high copy number, the IC may not produce an amplification plot. This does not invalidate the test and should be interpreted as a positive experimental result.

Table 5: Interpretation of Results

| Positive Control| Negative Control| MON863| IC| Interpretation
---|---|---|---|---|---
FAM| HEX (VIC)
Case 1| +| –| +| +| MON863 gene is detected.
Case 2| +| –| +| -*
Case 3| +| –| –| +| MON863 gene is not detected.
Case 4| +| –| –| –| Invalid result; retest
Case 5| +| +| +/-| +/-
Case 6| –| +/-| +/-| +/-

*Detection of the Internal Amplification Control in the respective channel is not required for a positive result.
A high copy number of the target gene can lead to reduced or absent Internal Amplification Control signal.

Troubleshooting

Situation	Possible cause	Recommendation
Negative control samples are positive.	Carry-over contamination	Exchange all

critical solutions.

Repeat the analysis of all tests with fresh aliquots of all reagents.

Take measures to detect and eliminate the source of contamination.

No signal is detected for amplification positive controls.| Incorrect programming of the real-time PCR instrument.| The PCR should be repeated after checking the programming of instruments, storage conditions and the expiration date.
The kit reagents have expired.
Kit components have not been stored according to the manufacturer’s instructions.
· Incorrect PCR reaction

· Pipetting errors

· Omitted reagents

| The PCR should be repeated after checking for correct pipetting scheme and reaction setup.
No signal is detected for IC in HEX (VIC) channel and MON863-specific gene in FAM channel.| PCR inhibitors are present at a high concentration.| DNA extraction should be repeated.

Stability and Storage
Store the kit at -15 to -25 °C through the expiration date printed on the label.

Specifications

Sensitivity Limit of detection (LOD) at 0.1%
Specificity 100% exclusivity for non-target genes

Quality control
In compliance with the Federal State Institution of Science “Central Research Institute of Epidemiology” ISO 13485 certified Quality Management System, each lot of foodproof SL GMO MON863 Maize Detection Kit has been tested against predetermined specifications to ensure consistent product quality.

Ordering information

Product	Order No.	# Tests
foodproof SL GMO MON863 Maize Detection Kit	KIT230214	50 reactions
foodproof Sample Preparation Kit III	KIT230174	50 reactions

Supplementary Information

Ordering Information
Hygiena Diagnostics offers a broad range of reagents and services. For a complete overview and for more information, please visit our website at www.hygiena.com.

Trademarks
foodproof®, microproof®, vetproof®, ShortPrep®, StarPrep®, RoboPrep® and LyoKit® are registered trademarks of Hygiena Diagnostics GmbH. Hygiena® is a registered trademark of Hygiena. Other brand or product names are trademarks of their respective holders.
Other brand or product names are trademarks of their respective holders.

Contact and Support
If you have questions or experience problems with this or any other product of Hygiena Diagnostics GmbH, please contact our Technical Support staff (www.hygiena.com/support). Our scientists commit themselves to providing rapid and effective help. We also want you to contact us if you have suggestions for enhancing our product performance or using our products in new or specialized ways. Such customer information has repeatedly proven invaluable to us and the worldwide research community.

Reference Number
The reference number and original Hygiena Diagnostics GmbH article number: Z 720 12