hygiena foodproof SL Duck Species Detection Kit Instruction Manual
- June 1, 2024
- Hygiena
Table of Contents
foodproof® SL Duck Species Detection Kit
Revision A, March 2024
Product No. KIT230228
Kit for 50 reactions for a maximum of 48 samples
Store at -15 to -25 °C
For food testing purposes.
FOR IN VITRO USE ONLY
Introduction
To assure a high level of food and feed safety, accurate animal species
identification and the detection of adulterants are two of the greatest
challenges facing food and feed products companies today. Therefore, the need
for scientifically valid species identity methods is increasingly important.
Although a number of traditional morphological, microscopic and chemical
methods have commonly been used for species identity testing, technologies
using DNA offer reliable alternative methods that can provide increased
precision in differentiating closely related species, as well as identifying
intentional and accidental adulterants and contaminants.
The Animal Species detection kits are designed for use by food and feed
producers, dairies, marketers of these products, as well as regulators and
auditors of final food and feed quality and safety. It is also intended to be
used to verify that ruminant feed and feed supplements are properly labeled
and do not contain ruminant materials.
Intended Use
The foodproof® SL Duck Species Detection Kit is designed to detect the
specific gene for duck species in feed and food. 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 assay, as well as the Internal Control (IC)
system for reliable results.
Principle of PCR detection
The foodproof SL Duck Species Detection Kit is a qualitative, duplex real-time
PCR test for the detection of a duckspecific gene and the Internal Control
(IC) using specific primers and probes labeled with the luorescent dyes.
The target sequences are detected through the FAM and HEX (VIC) channels,
respectively.
The primer and probe mixture provided exploits 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 on 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 reopening the reaction tubes after
the PCR run, such as electrophoresis. The kit minimizes contamination risk and
contains all reagents needed for detection (except for PCR-grade H2
3.1 Internal Amplification Control
This kit contains the Internal Control (IC) as PCR inhibition Control. The IC
allows the user to determine 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 samples. The results can be visualized in the HEX
(VIC) channel.
Contents
This kit is intended for 50 reactions, including controls.
Table 1: Kit Contents
Reagent | Cap Label | Volume | Description |
---|
2x real-time PCR
Master Mix| 2xM| 500 IA| Buffer containing dNTPs, MgC12and Tag DNA polymerase
Primer / Probe Mix| P| 200 pl| Primer/ probe mixture:
•Duck specific primer and probe
•IC-specific primer and probe
•IC DNA
Control DNA| C| 50 ul_| Positive control DNA
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 H2
- For DNA Extraction: foodproof Sample Preparation Kit
General precautions
-
Store extracted positive material (samples, controls and other amplicons) away from all other reagents and add 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
7.1 Sample Collection
Various processed food, raw material, feed and seed samples are routinely
examined.
7.2 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.
7.3 Nucleic Acid Extraction
Carry out DNA isolation according to the extraction kit’s product
instructions. For more information, please see
www.hygiena.com.
Protocol
8.1 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”).
8.2 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.
8.2.1 Thawing the Kit Components
The use of ice or a benchtop cooler is recommended during experiments to
maintain enzyme activity.
8.2.2 Prepare Reaction Master Mix
Each reaction has a total volume of 20 µL; the volume of the DNA sample is
6 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 | 10 µL |
Total | 14 µL |
Add 6 µL of extracted DNA sample into the tube.
8.2.3 Prepare Control Amplification Reactions
- Positive control amplification: Add 6 µL of Control DNA instead of sample DNA.
- Negative control amplification: Add 6 µL of PCR-grade H2O instead of sample DNA.
8.2.4 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.
8.3 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 |
---|---|---|
95 °C | 10 min | 1 |
95 °C | 15 seconds | 35 |
61 °C* | 40 seconds |
- 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 result.
Table 4: Specific Detection on Fluorescence Channel
Target Gene | Fluorophore |
---|---|
Duck specific gene | FAM |
IC | HEX (VIC) |
9.1 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
| PositiveControl| Negative Control| FAM| HEX (VIC)| Interpretation
---|---|---|---|---|---
| | | Duckspecific gene| IC
Case 1| +| –| | +| The duck-specific gene is detected in a sample.
Case 2| +| –| +| _*| The duck-specific gene is detected in a sample.
Case 3| +| –| –| +| The duck-specific gene is not detected in a sample.
Case 4| +| –| –| –| Invalid result/retest.
Case 5| +| +| +/-| +/-
Case 6| –| +| +/-| +/-
Case 7| –| –| +/-| +/-
- 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 on HEX (VIC) channel and duckspecific gene on 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
10 GE Limit of detection (LOD) -
Specificity
100% exclusivity for about 100 non-specific species DNAs
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 Duck species Detection Kit has been tested against predetermined specifications to ensure consistent product quality.
Ordering information
Product | Order No. | # Tests |
---|---|---|
foodproof SL Duck Species Detection Kit | KIT230228 | 50 reactions |
foodproof Sample Preparation Kit III | KIT230174 | 50 reactions |
Supplementary Information
15.1 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.
15.3 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.
15.4 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.
15.5 Reference Number
The reference number and original Hygiena Diagnostics GmbH article number:
Z 730 07
Change Index
Version 1, July 2021
First version of the package insert.
Revision A, March 2024
Rebranding and new layout.
Z 730 07 20 -> INS-KIT230228-RevA
Hygiena®
Camarillo, CA 93012
USA
diagnostics.support@hygiena.com
Manufactured by
Hygiena Diagnostics GmbH
Hermannswerder 17
14473 Potsdam
Germany
www.hygiena.com
References
Read User Manual Online (PDF format)
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