High-sensitivity EBV DNA quantitative detection kit based on ddPCR and using method thereof
A quantitative detection and kit technology, applied in the field of DNA detection, can solve the problems of poor detection effect and low sensitivity
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Embodiment 1
[0044] The highly sensitive EBV DNA quantitative detection kit based on ddPCR (Droplet digital PCR) provided in the embodiment of the present invention includes DNA extraction buffer, PCR primers and probes, PCR reaction solution, and EBV negative and positive controls.
[0045] Wherein, the DNA extraction buffer includes proteinase K buffer, buffer1 (buffer), buffer2, buffer3 and buffer4; the PCR reaction solution includes 5×PCR reaction buffer, and the concentration is 0.8-1.2mmol / L deoxyribonucleoside three Phosphoric acid, H-Taq DNA polymerase (Thermusaquaticus, H-thermostable DNA polymerase) at a concentration of 1-7U / μl; PCR primers and probes include a concentration of 700nmol / L-1.0μmol / L for amplifying the target The upstream and downstream primers of the polynucleotide and the probe used to detect the target polynucleotide at a concentration of 0.2mol / L-0.5μmol / L; the EBV negative control is negative artificial serum, and the EBV positive control is B958 cell culture s...
Embodiment 2
[0055] The highly sensitive EBV DNA quantitative detection kit based on ddPCR provided in the embodiment of the present invention can be applied to detect the content of EBV DNA in plasma and serum samples. The specific steps of the detection operation are as follows:
[0056] 1. Preparation of samples to be tested
[0057] 1. Plasma sample preparation
[0058] Centrifuge EDTA anticoagulated whole blood for 3 minutes within 2 hours at a rotational speed of 2000r / min, take the separated supernatant, and centrifuge the supernatant at a rotational speed of 20000g / min for 10min to remove broken cells For fragments, collect the supernatant to obtain a plasma sample. If the plasma sample is not used temporarily, store the plasma sample at -20°C for future use.
[0059] 2. Serum sample preparation
[0060] Let fresh EDTA anticoagulated whole blood stand at room temperature for 30 minutes, then centrifuge at a speed of 20,000 g / min for 3 minutes, and take the separated supernatant, ...
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