Walnut-derived component nucleic acid detection kit (one-tube PCR-fluorescent probe method) instruction manual

Walnut-derived component nucleic acid detection kit (one-tube PCR-fluorescence probe method)

â—† Product Description

The allergen identification reagent in the species identification series can amplify a specific nucleic acid fragment of an allergen component in a food, and judge the result by a real-time amplification curve. The detection limit of the allergen walnut component in the food raw material or processed food, the detection limit is 0. 0 1% .

â—† Product composition (96 test)

• Applicable instrument

Real-time fluorescence PCR instrument such as ABI 7500, CFX 96, Mx 3005P, LineGene9600.

â—† Self-supplied supplies and instruments

1 ice box; 2 pipettes (0.5-10μL, 10-100μL, 100-1000μL) and matching sterilization tips; 3 centrifuges; 4 vortex mixer; 5 metal bath.

â—† Notes

1. This reagent has high detection sensitivity. In order to prevent pollution, the experiment is to be partitioned.

1) First zone: sample preparation zone.

2) The second zone: the template addition zone.

3) Zone 3: Amplification and product analysis zone.

★ It is best to physically isolate the partitions to avoid contamination caused by human factors.

2. Work clothes and latex gloves are worn during the experiment, and tools are used independently in different areas. Gloves and lab coats need to be replaced.

3. Strictly follow the operation steps, reagent preparation and sample loading steps, please operate in strict accordance with the instructions on the ice box.

4. The components in the reaction solution are sensitive to light and should be stored away from light . The reagent should be completely thawed before use, but repeated freezing and thawing should be avoided. It is recommended to centrifuge for 30 seconds before use, and store the reaction solution in an appropriate volume according to the frequency of detection.

5. After the reaction is completed, the expansion tube should be placed in a sealed bag and discarded. On the same day, the lid is opened and the aerosol is easily contaminated. It is forbidden to open the lid.

6. Do not mix different batches of reagents used within the validity period.

â—† Sample processing

Refer to "SN/T 1961.6-2013 Export Food Allergen Ingredients Part 6: Real-time Fluorescence PCR Method for Detection of Walnut Ingredients" or other standard processing samples, and prepare samples for later use.

The collection and preparation of samples is an important step in the identification of allergen components. Measures to prevent cross-contamination are carried out in accordance with the provisions of GB/T 27403.

For detailed steps, please follow the standard operation or check the food safety software.

â—† Experimental operation

1. Template preparation (sample preparation area)

It is recommended to use the supporting animal genomic DNA extraction kit. The specific process is detailed in the product manual.

2. Add a template (template addition area, placed on the ice box)

Cut the PCR tube containing the reaction number, and place it at room temperature to be thawed. After centrifugation for 30 seconds, uncover the sealing film. Add 5 μL of template to each reaction solution in the order of NG, sample template to be tested. , PG-Hua Taoyuan-P. After the matching PCR tube cap was covered, the mixture was vortexed for 30 s, centrifuged for 1 min, and the PCR amplification reaction was immediately performed.

3. Amplification reaction (amplification and product analysis area)

Using a real-time PCR instrument, the fluorophore was selected for FAM and the quencher group was selected for TAMRA.

Set up the amplification reaction according to the following conditions:

4. Baseline and threshold settings

The baseline adjustment takes 3-15 cycles of fluorescence signal and the threshold line should exceed the highest point of the negative control amplification curve.

â—† Result judgment

When the Ct value of the test sample is ≤35, it is determined that the sample to be tested is positive, and the allergen walnut component is detected.

When the Ct value of the test sample is ≥40, it is determined that the sample to be tested is negative, and the allergen walnut component is not detected.

The test sample 35 < Ct value < 40 is repeated once. If the Ct value is still <40.0 after re-amplification, it is judged that the sample to be tested is positive, and the allergen walnut component is detected. If the Ct value is ≥40 after re-amplification, it is judged that the sample to be tested is negative, and the allergen walnut component is not detected.

• The NG reaction is a smooth straight line, and the PG reaction is an "S" type amplification curve. The test result is valid, otherwise it is invalid. If the duplicate test results are still invalid, please contact technical support.
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