Hygiena foodproof® SL Clostridium perfringens Detection Kit
Revision B, December 2024
PCR kit for the qualitative detection of Clostridium perfringens DNA using real-time PCR instruments.
Product No. KIT230202
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
Visit: www.hygiena.com
1. Introduction
Clostridium perfringens (C. perfringens) is a spore-forming gram-positive bacterium that is found in many environmental sources as well as in the intestines of humans and animals. C. perfringens is commonly found on raw meat and poultry. It prefers to grow in conditions with very little or no oxygen; under ideal conditions, it can multiply very rapidly.
Clostridium perfringens is one of the most common causes of food poisoning in the United States. According to some estimates, this type of bacteria causes nearly a million illnesses yearly. Cooking kills the growing C. perfringens cells that cause food poisoning, but not necessarily the spores. If cooked food is not promptly served or refrigerated, the spores can grow and produce new cells. C. perfringens infections often occur when foods are prepared in large quantities and are then kept warm for a long time before serving. That's why outbreaks of these infections are usually linked to institutions (such as hospitals, school cafeterias, prisons, and nursing homes) or events with catered food.
2. Intended Use
The foodproof® SL Clostridium perfringens Detection Kit is designed to detect the specific sequence of the α-toxin gene for Clostridium perfringens in various food sources and environmental samples. This kit provides a real-time PCR Master Mix with enzyme components and a specific primer/probe set for rapid testing by real-time PCR assay, as well as the Internal Control (IC) system for reliable results.
3. Principle of PCR detection
foodproof SL Clostridium perfringens detection assay is a qualitative, duplex real-time PCR test for detection of a pathogen-specific gene (α-toxin) and the Internal Control (IC) using specific primers and probes labeled with fluorescent 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 reaction cycles 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).
3.1 Internal Amplification Control
This kit contains the Internal Positive 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 sample. The results can be visualized in the HEX (VIC) channel.
4. Contents
This kit is intended for 50 reactions, including controls.
| Reagent | Cap Label | Volume | Description |
|---|---|---|---|
| 2x Real-time PCR Master Mix | 2xM | 500 µL | Buffer containing dNTPs, MgCl2 and Taq DNA polymerase |
| Primer / Probe Mixture | P | 200 µL | Primer/ probe mixture:
|
| Control DNA | C | 50 µL | Positive control DNA |
5. Additionally required materials, reagents and devices
- 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 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 microcentrifuge tubes for PCR
- PCR-grade H2O
- For DNA Extraction: foodproof® StarPrep® Two Kit or equivalent
6. 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 techniques of DNA amplification.
- To avoid carry-over contamination with PCR product or control DNA, please note the following points:
- Be careful not to contaminate the Primer/Probe Mixture and 2x real-time PCR Master Mix with other PCR products or Control DNA during pipetting. To prevent contamination, the use of aerosol-barrier tips is recommended.
- Open and close all sample tubes carefully. Avoid splashing or spraying PCR samples.
- 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 run/analyzed.
- 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 areas where previous steps were performed.
7. Sampling and handling
7.1 Sample Collection
Various food source samples, environmental samples and cultured bacteria 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.
8. 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.
| Composition | Volume |
|---|---|
| Primer / Probe Mixture | 4 µL |
| 2x Real-time PCR Master Mix | 10 µL |
| Total | 14 µL |
2. Add 6 µL of extracted DNA sample into the tube. (Total volume is 20 µL)
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 them a minimum of 5 times. Briefly centrifuge the tubes to remove air bubbles and drops from the inside of 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.
| Temperature | Time | Cycle |
|---|---|---|
| 95 °C | 10 min | 1 |
| 95 °C | 15 sec | 40 |
| 60 °C * | 1 min |
* Detect the fluorescence at this step.
9. 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.
| Target Gene | Fluorophore |
|---|---|
| α-toxin | 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.
| Positive Control | Negative Control | α-toxin | IC | Interpretation | |
|---|---|---|---|---|---|
| Case 1 | + | - | + | + | α-toxin gene is detected. |
| Case 2 | + | - | + | -* | |
| Case 3 | + | - | - | + | α-toxin gene is not detected. |
| Case 4 | + | - | - | - | |
| Case 5 | + | + | +/- | +/- | Invalid result; retest |
| Case 6 | - | +/- | +/- | +/- |
* Detection of the Internal Amplification Control in the respective channel is not required for positive results. A high copy number of the target gene can lead to reduced or absent Internal Amplification Control signal.
10. Troubleshooting
| Situation | Possible cause | Recommendation |
|---|---|---|
| Negative control samples are positive. | Carry-over contamination |
|
| No signal is detected for amplification positive controls. | Incorrect programming of the real-time PCR instrument. The kit reagents have expired. Kit components have not been stored according to the manufacturer's instructions. |
|
| No signal is detected for IC in HEX (VIC) channel and α-toxin gene in FAM channel. | Incorrect PCR reaction Pipetting errors Omitted reagents PCR inhibitors are present at a high concentration. |
|
11. Stability and Storage
Store the kit at -15 to -25 °C through the expiration date printed on the label.
12. Specifications
- Sensitivity: The limit of detection (LOD) is 10 to 100 genetic equivalents (GE).
- Specificity: 100% exclusivity for approximately 100 non-target strains
13. Quality control
In compliance with Federal State Institution of Science “Central Research Institute of Epidemiology” ISO 13485 certified Quality Management System, each lot of foodproof SL Clostridium perfringens Detection Kit has been tested against predetermined specifications to ensure consistent product quality.
14. Ordering information
| Product | Order No. | # Tests |
|---|---|---|
| foodproof SL Clostridium perfringens Detection Kit | KIT230202 | 50 reactions |
| foodproof StarPrep Two | KIT230177 | 96 reactions |
15. Supplementary Information
15.1 Ordering Information
Hygiena Diagnostics is offering 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.
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 700 08
16. Change Index
Version 1
First version of the package insert.
Revision A, February 2024
Rebranding and new layout. Z 700 08 20 -> INS-KIT230202-RevA
Revision B, December 2024
Removal of 'clinical material' from matrix description. Z 700 08 20 -> INS-KIT230202-REVB
Company Information
Hygiena®
Camarillo, CA 93012
USA
diagnostics.support@hygiena.com
Manufactured by
Hygiena Diagnostics GmbH
Hermannswerder 17
14473 Potsdam
Germany
www.hygiena.com
File Info : application/pdf, 10 Pages, 347.33KB
DocumentDocumentReferences
Innovative Diagnostic Solutions for Hygiene & Food Safety | Hygiena- Innovative Diagnostic Solutions for Hygiene & Food Safety | Hygiena
- Document Library | Hygiena
- Technical Support | Hygiena
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