Rapid reaction kit for predicting dosage of traserine, and detection method and application thereof
A dose prediction, rapid response technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of high test cost, difficult primer design, and low throughput of multiple gene amplification. question
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Embodiment 1
[0045] Embodiment 1, the preparation of kit
[0046] The rapid reaction kit of the present invention designs specific amplification primers and sequencing primers for CYP2C19 (681G>A) and CYP2C19 (636G>A) for amplification and pyrosequencing detection. Designing primers based on rapid amplification technology is one of the keys of the present invention. In order to ensure the amplification speed and detection sensitivity, the amplification length should be controlled at 60-120bp as much as possible. Gene polymorphism sequence is subject to the public sequence in Genebank.
[0047] (1) The primer sequences of this embodiment are as follows:
[0048]
[0049] (2) The detection kit of the present embodiment comprises the following components:
[0050]
[0051]
[0052] (3) The single serving configuration system of the detection kit PCR reaction solution of the present embodiment is as follows:
[0053] The final concentrations of each component of the PCR reaction so...
Embodiment 2
[0057] Embodiment 2, pyrophosphate detection
[0058] The instruments adopted in the present invention are as follows: amplification instrument, pyrosequencer (Wuhan First Biotechnology Co., Ltd.).
[0059] (1) Reagent preparation (reagent preparation room)
[0060] Take out the reagents in advance, vortex the PCR reaction solution for 15 seconds, and centrifuge at low speed for later use. Determine the number of reactions N, N = number of samples to be tested (n) + number of quality control products (1) + blank control. It is recommended to conduct positive control and blank control analysis for each PCR experiment at the same time. Then the reaction solution was dispensed into PCR reaction tubes at 16 μL / tube.
[0061] (2) Sample testing (sample preparation room)
[0062] Add EDTA anticoagulated whole blood, positive control and blank control into the PCR reaction tube according to the sample volume of 4 μL, close the tube cap tightly, centrifuge at low speed for 15 seco...
Embodiment 3
[0089] Example 3, Amplification efficiency under different EDTA anticoagulated whole blood volumes
[0090] Blood Direct PCR Master Mix (2×), containing blood resistant HemoTaq TM DNA polymerase and anti-inhibitor, showing super resistance to various PCR inhibitors such as heme in whole blood. EDTA anticoagulated whole blood was added to account for 5%, 10%, 20%, 30%, 40%, and 50% of the volume of the PCR reaction solution, respectively. Tested for maximum spiked whole blood sample volume. For test results, see figure 1 , the test showed that 20% volume EDTA anticoagulated whole blood had no effect on the amplification efficiency.
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