Method for fast and accurate quantitative determination of circulating DNA in blood
A quantitative, rapid technique
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Embodiment 1
[0019] A method for fast and accurate quantitative determination of blood circulating DNA, the specific steps are as follows:
[0020] (1) Treatment of plasma or serum: Take peripheral venous blood and place it in an EDTA anticoagulant tube to stand at room temperature or take from coagulated whole blood, centrifuge at 1800g for 4 minutes, take the upper layer of plasma, add the same volume of dilution reagent, mix well, Stir at 92°C for 6 minutes, then centrifuge at 15,000g for 8 minutes, and take the supernatant as a template for PCR;
[0021] (2) configure the quantitative PCR reaction system: quickly revive hot-start DNA polymerase, SuperGreen dye and 2x mixture, the 2x mixture includes 2x reaction buffer, magnesium chloride and deoxynucleoside triphosphate with a final concentration of 3mM, the triphosphate Deoxynucleosides include dATP, dCTP, dGTP and dTTP, and the final concentrations of dATP, dCTP, dGTP and dTTP are all 0.35mM;
[0022] (3) Quantitative PCR reaction: ...
Embodiment 2
[0024] A method for fast and accurate quantitative determination of blood circulating DNA, the specific steps are as follows:
[0025] (1) Treatment of plasma or serum: Take peripheral venous blood and place it in an EDTA anticoagulant tube to stand at room temperature or take from coagulated whole blood, centrifuge at 2000g for 5 minutes, take the upper layer of plasma, add the same volume of dilution reagent, mix well, Stir at 95°C for 8 minutes, then centrifuge at 16,000g for 10 minutes, and take the supernatant as a template for PCR;
[0026] (2) configure the quantitative PCR reaction system: quickly revive hot-start DNA polymerase, SuperGreen dye and 2x mixture, the 2x mixture includes 2x reaction buffer, magnesium chloride and deoxynucleoside triphosphate with a final concentration of 3mM, the triphosphate Deoxynucleosides include dATP, dCTP, dGTP and dTTP, and the final concentrations of dATP, dCTP, dGTP and dTTP are all 0.4mM;
[0027] (3) Quantitative PCR reaction: ...
Embodiment 3
[0029] A method for fast and accurate quantitative determination of blood circulating DNA, the specific steps are as follows:
[0030] (1) Treatment of plasma or serum: Take peripheral venous blood and place it in an EDTA anticoagulant tube to stand at room temperature or take from coagulated whole blood, centrifuge at 2200g for 6 minutes, take the upper layer of plasma, add the same volume of dilution reagent, mix well, Stir at 97°C for 10 minutes, then centrifuge at 17,000g for 12 minutes, and take the supernatant as a template for PCR;
[0031] (2) configure the quantitative PCR reaction system: quickly revive hot-start DNA polymerase, SuperGreen dye and 2x mixture, the 2x mixture includes 2x reaction buffer, magnesium chloride and deoxynucleoside triphosphate with a final concentration of 3mM, the triphosphate Deoxynucleosides include dATP, dCTP, dGTP and dTTP, and the final concentrations of dATP, dCTP, dGTP and dTTP are all 0.45mM;
[0032] (3) Quantitative PCR reaction...
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