Bovine source component nucleic acid detection kit (constant temperature fluorescence method) instruction manual

Bovine-derived component nucleic acid detection kit (constant temperature fluorescence method)

â—† Product Description

The species identification series is based on a unique thermostated fluorescence detection technology that can amplify specific nucleic acid fragments of animal-derived components in food, feed and other samples. The instrument monitors the fluorescence signal changes during the amplification process in real time and automatically interprets the results. This product is used for the detection of bovine-derived ingredients with a detection limit of 0.5% .

â—† Product composition (48 test)

â—† Applicable instruments

Dhelix 1610, Dhelix 3210, ESE Tube Scanner, Genie II, Deaou-308c and other thermostatic fluorescence detectors, ABI 7500, LightCycler 480, CFX 96 and other fluorescent PCR instruments.

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 (0.5-10μL, 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) The third zone: the template addition zone.

4) Zone 4: 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

Samples prepared by SB/T 10923-2012 Determination of Animal-derived Components in Meat and Meat Products by Real-Time Fluorescence PCR Method or other standards are prepared for use.

1 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 dry-baked at 160 °C for 2 h. Other vessels that should not be dry-baked or high-pressure treated should be used. Soak in % sodium hypochlorite solution. 2 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. 3 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 and calculate the amount of each component according to N+2 (N samples to be tested + 1 negative control + 1 positive control), and place the reaction solution in 0.6 ml or In a 1.5 ml centrifuge tube, vortex and mix, centrifuge for 30 seconds, dispense into 0.2 ml PCR tubes, and add 1 drop of CI (about 20 μl) to each tube.

2. Template preparation (sample preparation area)

It is recommended to use the reagents to support the DNA extraction series of animal-derived components. For details, please refer to the product manual.

3. Add a template (template add area, placed in the ice box)

In the PCR tube containing the reaction solution in step 1, 2 μL of the template was added, and the order was NG-I, the sample template to be tested, and PG-cow source-I. The mixture was vortexed for 30 s, centrifuged for 1 min, and the amplification reaction was immediately performed.

4. Amplification reaction (amplification and product detection zone)

1 The constant temperature instrument was reacted at 63 ° C for 45 min.

2 If a real-time PCR instrument is used, the fluorescent group is selected as FAM, the quenching group is selected as None, 63 ° C for 15 s, 63 ° C for 45 s as a cycle, and fluorescence signal is collected at 63 ° C for 45 s, 45 cycles.

For other instruments, please refer to the instrument manual for setting.

â—† Result judgment

1 The instrument automatically determines the result. If “positive” is displayed, the sample contains bovine-derived components; if “negative” is displayed, the sample does not contain bovine-derived components or the content is below the detection limit.

2 On the fluorescence quantitative PCR machine, the results were determined based on the presence or absence of the "S" type amplification curve. If there is an "S" type amplification curve, the sample contains bovine-derived components; if there is no "S"-type amplification curve, the sample does not contain bovine-derived components or the content is below the detection limit.

• The result of the NG reaction tube showed "negative", and the result of the PG reaction tube showed "positive". The test result was valid, otherwise it was invalid. If the duplicate test results are still invalid, please contact technical support.
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