Species identification series

Eukaryotic 18S gene nucleic acid detection kit (PCR-fluorescence probe method)

Please save at -20 °C, valid for 12 months

â—† Product Description

The species identification series can be used to amplify specific nucleic acid fragments of animal-derived components in foods, feeds, etc., through real-time amplification

Line judgment result. This product is used for the detection of eukaryotes with a detection limit of 0.1% .

â—† Product composition ( 48 test)

â—† Applicable instruments

A PCR instrument such as a fluorescent PCR instrument (ABI 7500, CFX 96, Mx 3005P, LineGene 9600).

â—† Self-supplied supplies and instruments

1 Sterilize 1.5mL or 2.0mL centrifuge tube; 2 sterilize 0.2mL PCR tube or octa tube; 3 ice box; 4 pipette (1-10μL, 10-100μL, 100-1000μL)

And matching sterilization tips; 5 centrifuge; 6 vortex mixer; 7 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: reagent preparation zone.

2) Second zone: sample preparation area.

3) Zone 3: Amplification and product analysis zone.

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

2. Wear work clothes and latex gloves during the experiment, use pipettes, and treat all waste generated in the test in time.

3. Strictly follow the operation steps, reagent preparation and sample loading steps should be carried out on ice in strict accordance with the instructions.

4. The components in the reaction solution are sensitive to light and should be stored away from light. Resolve the reagent completely before use, but avoid repeated freezing and thawing. It is recommended to centrifuge before use. 30

Seconds, and the reaction solution was stored 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 and use different batches of reagents and use them within the validity period.

â—† Sample processing

Samples prepared by reference to "SN/T 3730-2013 Methods for Identification of Commonly Used Animal Breeds in Food and Feed" or other standards are prepared for use. The collection and preparation of samples is an important step in the identification of animals. To prevent cross-contamination, disposable consumables should be used. The mortar should be dried at 160 °C.

2 h, other utensils that are not suitable for dry roasting or high pressure treatment should be soaked in 1% sodium hypochlorite solution.

Sampling should take muscle tissue as much as possible. For the same batch of samples, multiple points should be collected and combined, and then homogenized as one sample to extract DNA.

The sampling process should be completed quickly. The sample taken is quickly placed in a tissue agitator to homogenize into a braid, fully mixed, and 0.1 g of the well-mixed sample is taken and homogenized by liquid nitrogen or homogenizer.

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

â—† Experimental operation

The reagents were completely thawed and the components were centrifuged for 30 s.

1. Reagent preparation (reagent preparation area, placed in an ice box):

If there are N samples to be tested, refer to the table below, calculate the amount of reaction solution according to N+2 (N samples to be tested + 1 negative control + 1 positive control), vortex and mix, centrifuge for 30s, divide Installed in a 0.2 mL PCR tube.

2. Template preparation (sample preparation area)

It is recommended to use a reagent-based animal-derived DNA extraction series. The specific process is detailed in the product manual.

3. Add a template (sample preparation area, placed in an ice box)

Add 5 μL of template to the PCR tube containing the reaction solution in step 1, in the order of NG-P, sample template to be tested, and PG-18S-P. The mixture was vortexed for 30 s, centrifuged for 1 min, and immediately subjected to PCR amplification.

• Amplification reaction (amplification and product detection zone)

Using a real-time PCR instrument, the fluorophore was selected for FAM and the quenching group was selected for TAMRA. Set up the amplification reaction according to the following conditions:

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

â—† Result judgment

The sample has no Ct value, the curve is straight or slightly oblique, and there is no “S” type amplification curve, which can report negative samples, no eukaryotes or lower than detection limit;

The test sample Ct ≤ 35, the curve is an "S" type amplification curve, can directly report the sample positive, contains eukaryotes; test sample Ct> 35, can directly report the sample negative, does not contain eukaryotes or the content is below the detection limit .

• 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.

Positive control

Negative control

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