Salmonellosis is a food-borne infectious disease occurring worldwide that is caused by the Gram-negative zoonotic pathogen Salmonella spp. and recognized as a major microbial hazard in human and animal food, including pet food, animal feed, raw materials, and ingredients. The test kit «Salmonella enteritidis qPCR kit» is designed for multiplexed detection of the specific to pathogenic Salmonella DNA (InvA gene) and genotyping for Salmonella enteritidis species in food and environmental samples, according to an FDA-validated protocol, and the Internal Control (16 S) gene as sample processing control by real-time polymerase chain reaction (PCR) with fluorescent detection of the analysis results.
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Salmonella enteritidis qPCR kit
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Test123
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Clinical significance
Salmonella is one of the most common food-borne pathogens and causes an estimated 2–4 million cases of salmonellosis annually. Various serotypes of Salmonella have been associated with raw meats, poultry, eggs, milk and dairy products, fish, shrimp, etc. To date, over 2500 Salmonella serotypes have been identified, and more than half of them belong to Salmonella enteric subsp. enterica, which accounts for the majority of Salmonella infections in humans. Pathogenic Salmonella ingested in food survives passage through the gastric acid barrier, invades the mucosa of the small and large intestines, and produces toxins. Invasion of epithelial cells stimulates the release of proinflammatory cytokines, which induce an inflammatory reaction. The InvA gene is located in Salmonella pathogenicity island I (SPI-1), which encodes structural components, chaperones, and secreted effectors necessary for invasion.
Principle of analysis
The method is based on the isolation of total DNA from pre-enrichment culture samples, further amplification of specific regions of Salmonella spp. DNA (InvA genes) and Salmonella enteritidis specific gene using specific oligonucleotide primers, and the synthesis of complementary DNA chains using the enzyme Hot Start Taq-DNA polymerase. Fluorescent detection of the analysis results is performed both in real-time (real-time mode) or end-point assay using two fluorescence detection channels (FAM525 nm and ROX580 nm), see table below:
Kit contents
Storage and stability
The test kit reagents are stable for 12 months at room temperature in a dry place. Protected from direct sunlight.
Sample preparation
Foods, feeds, and environmental samples should be processed within 24 hours of sampling following ISO 6579:2002 procedures. Briefly, a total of 25 g or 25 mL of sample should be homogenized with 225 mL of Buffered Peptone Water (BPW), stomached for 45 s, and followed by incubation at 370C for 15–20 h. After pre-enrichment, 0.1–1.0 mL of samples should be processed for nucleic acid extractions using commercially available DNA extraction kits.
Reagent preparation
Master Mix preparation: Add 0.5 mL of distilled water directly to the lyophilized master mix, wait 5 minutes, mix well, and dispense 5 μL into PCR testing tubes. Positive control preparation: Add 0.5 mL of distilled water directly to the lyophilized positive control, wait 5 minutes, mix well, and use directly 5 μL for PCR or 100 μL for nucleic acid extraction (to control nucleic acid extraction efficiency). Reagents and controls must be kept on a chilled stand. After use, reagents and controls can be frozen at minus 200C for storage. Several freezing-thawing cycles are allowed.
Assay procedure
1. Select the required amount of PCR tubes or wells in the PCR plate.
2. Using pipette tips with an aerosol filter, dispense Master mix 5 μL, add 5 μL of DNA samples, positive control, and use water as negative control sample.
3. Place testing tubes in the fluorescence PCR instrument and set the negative/positive control and unknown sample parameters for the PCR reaction according to the instrument's operating instructions. Fluorescence is measured at 600C on the FAM, Cy5 and ROX channels. The PCR parameters are shown in table below:
4. Start run.
Result records and their interpretation is shown in the table below:
Example of positive control amplification below:
Product specification:
Salmonella is one of the most common food-borne pathogens and causes an estimated 2–4 million cases of salmonellosis annually. Various serotypes of Salmonella have been associated with raw meats, poultry, eggs, milk and dairy products, fish, shrimp, etc. To date, over 2500 Salmonella serotypes have been identified, and more than half of them belong to Salmonella enteric subsp. enterica, which accounts for the majority of Salmonella infections in humans. Pathogenic Salmonella ingested in food survives passage through the gastric acid barrier, invades the mucosa of the small and large intestines, and produces toxins. Invasion of epithelial cells stimulates the release of proinflammatory cytokines, which induce an inflammatory reaction. The InvA gene is located in Salmonella pathogenicity island I (SPI-1), which encodes structural components, chaperones, and secreted effectors necessary for invasion.
Principle of analysis
The method is based on the isolation of total DNA from pre-enrichment culture samples, further amplification of specific regions of Salmonella spp. DNA (InvA genes) and Salmonella enteritidis specific gene using specific oligonucleotide primers, and the synthesis of complementary DNA chains using the enzyme Hot Start Taq-DNA polymerase. Fluorescent detection of the analysis results is performed both in real-time (real-time mode) or end-point assay using two fluorescence detection channels (FAM525 nm and ROX580 nm), see table below:
| Marker | Detection channel |
| Salmonella spp. | FAM |
| Salmonellar enteritidis | Cy5 |
| Internal control, (IC) | ROX |
Kit contents
| Component name | Quantity |
| Salmonella Master Mix | 1 vial |
| Salmonella Positive Control | 1 tube |
Storage and stability
The test kit reagents are stable for 12 months at room temperature in a dry place. Protected from direct sunlight.
Sample preparation
Foods, feeds, and environmental samples should be processed within 24 hours of sampling following ISO 6579:2002 procedures. Briefly, a total of 25 g or 25 mL of sample should be homogenized with 225 mL of Buffered Peptone Water (BPW), stomached for 45 s, and followed by incubation at 370C for 15–20 h. After pre-enrichment, 0.1–1.0 mL of samples should be processed for nucleic acid extractions using commercially available DNA extraction kits.
Reagent preparation
Master Mix preparation: Add 0.5 mL of distilled water directly to the lyophilized master mix, wait 5 minutes, mix well, and dispense 5 μL into PCR testing tubes. Positive control preparation: Add 0.5 mL of distilled water directly to the lyophilized positive control, wait 5 minutes, mix well, and use directly 5 μL for PCR or 100 μL for nucleic acid extraction (to control nucleic acid extraction efficiency). Reagents and controls must be kept on a chilled stand. After use, reagents and controls can be frozen at minus 200C for storage. Several freezing-thawing cycles are allowed.
Assay procedure
1. Select the required amount of PCR tubes or wells in the PCR plate.
2. Using pipette tips with an aerosol filter, dispense Master mix 5 μL, add 5 μL of DNA samples, positive control, and use water as negative control sample.
3. Place testing tubes in the fluorescence PCR instrument and set the negative/positive control and unknown sample parameters for the PCR reaction according to the instrument's operating instructions. Fluorescence is measured at 600C on the FAM, Cy5 and ROX channels. The PCR parameters are shown in table below:
| Step | Temp | Time | Repeats | Data collections |
| 1 | 95 C | 5 min | 1 | No |
| 2 | 95 C | 5 sec | 45 | No |
| 60 C | 15 sec | Yes |
4. Start run.
Result records and their interpretation is shown in the table below:
| Marker | Fluorescent channels | Salmonella enteritidis Positive | Salmonella Negative |
| Salm. | FAM | Ct≤ 45 | Undetectable |
| CY5 | Ct≤ 45 | Undetectable | |
| IC | ROX | Ct≤ 45 | Ct≤ 35 |
Example of positive control amplification below:
Product specification:
| Detection Method | Primer-probe |
| Format | Lyophilized in Glass vials |
| Reaction Speed | Fast |
| Disease | Salmonellosis |
| Label or Dye | |
| Target | FAM |
| Internal control | ROX |
| Target Organism Class | Salmonella spp |
| Shipping Condition | Room temperature |
| For Use With (Application) | Pathogen detection |
| Concentration | 2x |
| Validation | Field strains: S.infantis S.mbandaka S.enteritidis Reference DNA: S. enteritidis (BNCC356120) |
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Certificate of Analysis
Salmonellosis is a food-borne infectious disease occurring worldwide that is caused by the Gram-negative zoonotic pathogen Salmonella spp. and recognized as a major microbial hazard in human and animal food, including pet food, animal feed, raw materials, and ingredients. The test kit «Salmonella enteritidis qPCR kit» is designed for multiplexed detection of the specific to pathogenic Salmonella DNA (InvA gene) and genotyping for Salmonella enteritidis species in food and environmental samples, according to an FDA-validated protocol, and the Internal Control (16 S) gene as sample processing control by real-time polymerase chain reaction (PCR) with fluorescent detection of the analysis results.
