PORCINE DETECTION KIT
UPro Real-time PCR PORCINE DETECTION KIT
Collaborating Partner
Identification of meat species present in food samples is an important step for verifying the origin and traceability of raw materials, as well as for controlling the quality of the handling process in the production of a food or medicine product. UPro Real-time PCR Porcine Detection Kit is a kit developed by PT Prodia Diagnostic Line in collaboration with the Faculty of Pharmacy, Padjadjaran University. This product is designed to specifically detect the DNA content of pig, wild boar, and other pig species in food and medicine samples.
- Real-time based testing, no electrophoresis required (end point analysis).
- High specificity and sensitivity for detecting even tiny amounts of porcine DNA contaminants.
- The testing process until the interpretation of the results is fast and real time.
- UPro is designed as a multiplex test to specifically detect pig and wild boar DNA content and is equipped with internal controls specifically designed to detect mammalian and avian DNA.
The principle of the test is based on PCR amplification of unique species-specific sequences found only in the pig and wild boar genomes. Bioinformatics analysis followed by careful primer design ensured the detection of all strains at once and excluded the detection of other species. Furthermore, the short amplicon size (~100bps) overcomes the problem of DNA degradation, thereby reducing the possibility of false negatives. The probe detection system in this kit uses FAM® for target DNA and the internal control uses VIC/HEX®.
Positive control
The kit contains positive control porcine DNA for PCR control. Each time a kit is used, at least one positive control reaction must be included in the process. These positive samples indicate that the primer and probe sets detected the species desired. If no amplification is observed in the positive control, the test results are invalid and must be repeated. Caution must be taken to ensure that the positive control does not contaminate other kit components which could cause a false positive result.
Negative control
To validate positive findings, a negative control must be included each time the kit is used. A negative result indicates that the reagent was not contaminated during the process.
Internal controls
The signal from the universal sequence serves as an internal control indicating a valid biological template extraction process. The negative result indicates that the amount of animal material is insufficient to perform an accurate test.
The UPro Porcine Real-time PCR Kit is designed to detect specific DNA sequences of porcine species found in food and health products. This kit contains all the necessary enzyme components and primer/probe sets for porcine DNA amplification.
| Product | 50 Reagents |
|---|---|
| UPro Porcine 2x Mastermix | 1 tube, 500 μl |
| UPro Porcine Primer Mix 1 (target) | 1 tube, 50 μl |
| UPro Porcine Primer Mix 2 (internal control) | 1 tube, 50 μl |
| UPro Porcine Probe 1 (target) | 1 tube, 25 μl |
| UPro Porcine Probe 2 (internal control) | 1 tube, 25 μl |
| Nuclease-free Water | 1 tube, 1000 μl |
| Positive Control | 1 tube, 150 μl |
| Negative Control | 1 tube, 150 μl |
| Procedure Kit | 1 |
The kit should be stored at -20°C. Freeze/thaw cycles should be kept to a minimum, and can be accomplished by making aliquots of kit components. The kit is stable until the stated expiration date.
Prepation
• Operating steps are performed in the PCR clean room or separate environmental area.
• Use different filter tips for each step.
• The reagent must be cold temperature when carrying out the steps
Reagent preparation
• Determine the total number of Real-time PCR reactions, including positive and negative controls.
• Perform 3-second spin-downs for each reagent in the kit.
• For each DNA sample prepare the reaction mixture according to Table 2 below:
| Component | Volume Steps |
|---|---|
| UPro Porcine 2x Mastermix | 10 μl |
| UPro Porcine Primer Mix 1 (target) | 1 μl |
| UPro Porcine Primer Mix 2 (internal control) | 1 μl |
| UPro Porcine Probe 1 (target) | 0.5 μl |
| UPro Porcine Probe 2 (internal control) | 0.5 μl |
| DNA Template (1-10 ng) or Positive control | 2 - 5 μl |
| Nuclease-free Water | Up to a total volume of 20 μl |
| Total | 20 μl |
• Prepare a DNA template sample.
• Pipette 2-5 μl of template DNA into each tube. For negative control tubes, use 2-5 μl of nuclease-free water or up to a total volume of 20 μl.
• Spin down momentarily to collect all the reagents at the bottom of the tube.
Perform PCR according to the protocol in Table 4 below:
| Cycle | Steps | Time | Temperature |
|---|---|---|---|
| 1 | Initial denaturation | 10 minutes | 95° |
| 45 | Denaturation | 15 seconds | 95° |
| Sticking | 60 seconds | 60° |
| No | Ct Value | Interpretasi | ||||
|---|---|---|---|---|---|---|
| Sampel (FAM) | Sampel (VIC/HEX) | Kontrol Positif (FAM) | Kontrol Positif (VIC/HEX) | Kontrol Negatif | ||
| 1 | <40 | <40 | <40 | <40 | No Ct / >40 | Porcine-specific genes were detected in the samples |
| 2 | Tidak ada Ct / >40 | <40 | <40 | <40 | No Ct / >40 | Porcine-specific genes were not detected in the samples |
| 3 | <40 / Tidak ada Ct / >40 | <40 / Tidak ada Ct / >40 | Tidak ada Ct / >40 | <40 | No Ct / >40 | Invalid, do PCR again |
| 4 | <40 / Tidak ada Ct / >49 | <40 / Tidak ada Ct / >40 | <49 | No Ct | No Ct / >40 | Invalid, do PCR again |
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