The detection of LAMP TaqMan can be performed using a standard quantitative PCR instrument (or a constant temperature fluorescence device), with the probe reporting a FAM as the functional group.
The LAMP TaqMan amplification procedure using a quantitative PCR instrument is as follows:
Step 1: 60 ℃ for 10s
Step 2: Collect the signal at 60 ℃ for 50 seconds and cycle 25 times
The total reaction time is 25 minutes.
Usage:
Dissolve LAMP TaqMan freeze-dried products. Carefully open the aluminum cap of the penicillin bottle, add 1mL of LAMP TaqMan Buffer 2D to each bottle of freeze-dried products, and place it on a vortex mixer for 10 seconds to dissolve the freeze-dried products (or use a 1mL suction head to blow and mix evenly). The dissolved product can be immediately used or stored at -20 ℃. The dissolved reagent is labeled as LAMP TaqMan Mix.
Dissolve the positive standard, the positive standard is in dry powder form, and add 100% before first use μ L of ddH2O was dissolved in a vortex for 10 seconds and stored at -20 ℃. The concentration of the dissolved product is 105 copies/ μ l. Use 1 for each experiment μ L is sufficient.
Configuration reaction system
Dissolved LAMP TaqMan Mix 20 μ L
Detection template DNA 1 μ L
After the reaction system is prepared, it is thoroughly mixed and briefly centrifuged, and then placed on a fluorescence quantitative PCR instrument for reaction.
Result determination:
Conditions for the establishment of result interpretation
The negative water control does not peak, and the positive standard is effective in interpreting results under peak conditions of 10-14 minutes.
Positive result interpretation
If there is an amplification curve, it is determined to be positive. When the peak starts after 25 minutes but is not obvious, the number of copies is usually less than 2. A review experiment is required, and if the peak still appears, it indicates a positive result. Otherwise, it indicates negative.
Interpretation of negative results
The horizontal line indicates a negative sample.