1.LAMP OTG Reagent is in dry powder form, containing Bst DNA polymerase 4.0, dNTP, etc. Before dissolution, the product can be stored for a long time at -20 ℃ and transported at room temperature without any degradation in performance. Before use, dissolve each bottle in 0.75ml of LAMP Buffer and use immediately. The remaining reagents can be stored at -20 ℃ for 1 month and stored for a long time below -60 ℃.
2 OTG dye has been pre added to the EP tube cover in the form of dry powder, which firmly binds to the tube cover. It can only be dissolved after the reaction is completed and the EP tube is inverted. After dissolution, it will immediately terminate the LAMP reaction and form a bright green visible light with the LAMP reaction product. During the preparation of the reaction solution and the reaction process, it is essential not to mix the dye on the EP tube cover with the reaction solution, otherwise it may cause the LAMP reaction to terminate.
3,。ASFV LAMP Primer is in the form of dry powder, which can be stored for a long time at -20 ℃ and transported at room temperature without any degradation in performance. After each bottle is dissolved in 0.2ml of ddH2O before use, it can be used immediately. The remaining reagents can be stored at -20 ℃ for 1 month and stored for a long time below -60 ℃.
4.The positive standard is in dry powder form, and 100% is added before first use μ Dissolve ddH2O in a vortex for 10 seconds and store at -20 ℃. The concentration of the dissolved product is 105 copies/ μ l. Use 1 for each experiment μ L is sufficient.
Preparation reaction system
Add the following reagents to the 0.2ml EP reaction tube provided in this kit
5.Ingredient dosage
Dissolved LAMP OTG Reagent 15 μ L
Dissolved ASFV LAMP Primer 4 μ L
Template DNA 1 μ L
After all the reagents are added, lightly tap the bottom of the EP tube and cover it with the EP tube cover containing OTG dye (after covering the tube cover, it is important not to mix and invert it vigorously to prevent the OTG dye on the tube cover from dissolving. Once the OTG dye is mixed in the reaction solution, it will terminate the LAMP reaction).
After the reaction system is prepared, the reaction is carried out at 60 ℃ for 25 minutes.
Observation results: When observing the results, try not to share them with the preparation reaction space as much as possible to prevent contamination of the operating platform. Invert the reaction EP tube and gently shake it with your wrist. Soak the reaction solution in the OTG dye on the EP tube cover and let it stand for 30 seconds. Place the EP reaction tube upright and gently shake the reaction solution to the bottom of the EP tube. At this point, the amplified sample will turn bright green visible to the naked eye, while the negative tube that has not undergone amplification will turn dark orange. (It is prohibited to open the reaction tube cover after the LAMP reaction is completed, otherwise it may cause aerosol pollution). In case of aerosol pollution, please use our company's DNA contamination remover (product number: MT3001) to remove it
Environmental cleaning.
6.Result interpretation
Conditions for the establishment of result interpretation
When the negative water control turns dark orange and the positive standard turns bright green, the result interpretation is effective.
Positive result interpretation
The positive sample is a bright green color.
Interpretation of negative results
The negative sample is dark orange.
The stable detection sensitivity of this reagent kit is 5 copies.