Sheep Species ID qPCR Kit

According to the European Commission Directive 2002/86/EC, food ingredients have to be declared. Species authenticity is highly relevant to consumers for economic, medical, cultural, and religious reasons. Examples like fraudulent substitution of expensive species with cheaper meats, inclusion of meat in vegetarian products, and allergens in food products highlight the importance of this issue. Additionally, meat species must be disclosed on the label with both commercial and scientific denominations to verify the meat's origin and traceability and to analyze the quality control of handling and cleaning processes of production lines. Methods for species detection based on the identification of proteins are not reliable for application in highly processed and heated products due to protein deterioration. DNA is more stable than proteins during processing. Unlike DNA-based methods, traditional protein-based detection methods are unreliable for highly processed and heated products. DNA is more stable, and modern PCR techniques enable the identification of fragmented DNA from the species present in a sample, ensuring accurate and highly sensitive detection with a lower detection limit. Real-time PCR techniques are suitable for amplifying small fragments of DNA with high sensitivity and specificity.

The kit includes an Internal Control (IC). The IC is used to evaluate PCR inhibitors in the sample or to evaluate problems that occurred during PCR preparation/amplification. The kit includes a Master Mix for the target and IC, primers and TaqMan probes, labelled with non-fluorescent quenchers. The kit contains Uracil-DNA Glycosylase (UDG), preventing DNA contamination with PCR products. The kit includes a positive control, which allows an evaluation of the primers and probes used to detect the targets, and a negative control (nuclease-free water) to confirm the integrity of the kit reagents.

The kit is compatible with all thermocyclers working in FAM and ROX channels. The detection kit must not be used for diagnostic procedures. For food use only.

The procedure includes the following main steps:

1. Sample processing
2. DNA extraction
3. PCR set-up and run
4. Results and interpretation

In accordance with ISO 9001, each lot of the kit is tested against predetermined specifications to ensure consistent product quality.

Molecular biology procedures, such as RNA extractions and PCR amplification, require qualified staff to prevent the risk of erroneous results, especially due to sample contamination or degradation of the nucleic acids contained in the samples. It is strongly recommended to have dedicated areas, materials and equipment for the RNA extraction, preparation of the PCR and post-PCR procedures. Workflow in the laboratory must proceed in a unidirectional manner, beginning in the Extraction Area and moving to the Amplification and Detection Area.

The user should always pay attention to the following:

• Read all the instructions provided before running the assay.
• Do not mix reagents from different batches.
• Wear proper PPE, including disposable gloves and laboratory coats.
• Store and extract positive material separately from all other reagents.

The kit should be stored between -20 °C and 5 °C and protected from exposure to light. Repeated thawing and freezing (> 5) should be avoided, as this may reduce the sensitivity. Consider freezing in aliquots to maintain the performance of the assay.

The kit contains reagents for 100 reactions.

• Microcentrifuge
• Laminar air flow cabinets/PCR cabinets
• Disposable powder-free gloves
• Micropipettes and nuclease-free filter tips
• Real-time PCR instrument
• Tubes/strips/multiwell plates and accessories specific for each instrument
• DNA extraction kit

Sample preparation

Depending on the specific characteristics of the sample, procedures like homogenization and grinding may be necessary before DNA extraction.

DNA extraction

Use a kit or suitable protocol for DNA extraction, such as Complex Extraction Kit (LC-6500), Extraction Rapid (LC-6504, LC-6505) or Extraction Column (LC-6506, LC-6507).

PCR preparation: PCR mix

Always wear gloves for all PCR procedures.

1. Thaw the kit solutions. Mix thoroughly (do not vortex the Enzymes tube) and centrifuge briefly.
2. Prepare the reactions, as described in the table below.
3. Mix the prepared Mix by inverting the tube and centrifuge briefly.
4. Dispense 15 µL aliquots of prepared Mix into the plate wells or PCR tubes.
5. For the negative control, pipette 5 µL of the Negative Control tube (clear cap); pipette 5 µL of DNA sample per well; and for the positive control, pipette 5 µL of the Positive Control tube (red cap). Each PCR tube/well should have a final PCR volume of 20 µL.
6. Centrifuge briefly the plate wells or PCR tubes.
7. Place the reactions into the real-time PCR instrument.

Note: Prepare the PCR reaction for each sample or, in the alternative, prepare a Master Mix for the total number of reactions plus 10% (e.g. for 10 samples, prepare a volume for 11). In this case, prepare the Mix in a 1.5 mL sterile, nuclease-free tube. Include 2 PCR reactions for the Positive and Negative controls.

PCR preparation: program set up

Prepare the real-time PCR instrument according to the following temperature/time program:

For analysis of PCR results, select fluorescence display options. Samples with positive Ct values are considered positive.

Important: Please also check amplification curves, not only Ct values. Samples should be inspected both in logarithmic and linear scale view and compared with the negative control. Adjust the threshold, if necessary. Sample results should be assessed after the positive and negative controls have been examined and determined to be valid. If the results of controls are not valid, the sample results cannot be interpreted.

Interpretation: controls

To validate the assay, the controls must have the following results:

Note that if the controls do not match these results, the experiment must be repeated.

Interpretation: samples

Interpretation of sample results is summarized in the following table:

(1) When both sheep and IC detection are negative, this means the presence of PCR inhibitors in the sample. Dilute the sample or perform another DNA extraction.

(2) High DNA concentration of the target in the sample can lead to a reduced or absent fluorescence signal of the IC.

1. 100% exclusivity, determined using non-target DNA species from animal species suitable to occur in the same food products (Tables 1, 2).
2. 100% inclusivity, determined in food samples obtained from different commercial sources and from External Quality Control (Table 3).

The limit of detection (LoD) of the kit is 10 pg of target DNA.

The method's detection limit can detect 0.02% of ovine (Ovis aries) DNA in 50 ng of total DNA.

No false negatives were obtained, either in the defined PCR conditions or in the deviations used for robustness, increasing the stringency.

Table 1: Food samples tested for validation of the kit

Table 2: List of species used for exclusivity of the kit

Table 3: Analytical sensitivity of the kit

Analytical sensitivity: 100%.