Method for fast and accurate quantitative determination of circulating DNA in blood

A quantitative, rapid technique

Inactive Publication Date: 2016-01-06
宋现让
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, directly using plasma or serum for amplification, the proteins contained in serum or plasma will seriously interfere with PCR, making the amplification efficiency inconsistent among samples
In addition, the quantitative PCR technology using SYBRGreen as the dye has low fluorescent dye signal intensity and low sensitivity, and it is not easy to detect low copy number templates

Method used

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  • Method for fast and accurate quantitative determination of circulating DNA in blood
  • Method for fast and accurate quantitative determination of circulating DNA in blood
  • Method for fast and accurate quantitative determination of circulating DNA in blood

Examples

Experimental program
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Effect test

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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Abstract

The invention discloses a method for fast and accurate quantitative determination of circulating DNA in blood. The method comprises the following specific steps that processing of plasma or serum is performed: peripheral venous blood is taken and arranged in an EDTA anticoagulant tube for standing at room temperature, or self-clotting whole blood is taken to undergo centrifugation under 1800-2200 g of centrifugal force for 4 to 6 minutes, upper-layer plasma is taken, the same volume of diluted reagent is added, even mixing is performed, stirring is performed at the temperature of 92-97 DEG C for 6 to 10 minutes, then centrifugation is performed again under 15000-17000 g of centrifugal force for 8 to 12 minutes, and supernate is taken as a template for PCR; a quantitative PCR reaction system is configured; quantitative PCR reaction is performed. The quantitative PCR reaction can be performed by directly using the processed plasma or serum, PCR reaction is not influenced, the amplification efficiency is consistent with the amplification efficiency of purified DNA, DNA extraction steps and experimental errors are decreased, a new fluorescent dye SuperGreen which is higher in concentration, high in fluorescent signal value and response sensitivity and capable of accurately detecting plasma free DNA as low as the nanogram level can be adopted, the reagent price is low, the using cost of a DNA extraction kit is reduced, and operation is easy and convenient.

Description

technical field [0001] The invention relates to the technical field of DNA determination, in particular to a fast and accurate method for quantitatively measuring DNA in blood circulation. Background technique [0002] Circulating DNA (ctDNA), also known as free DNA or cell-free DNA, mainly comes from cell apoptosis and death. It can be used as the basis for the detection of tumors, autoimmune diseases and fetal genetics. The current detection methods all need to extract DNA from serum or plasma, and then amplify a gene in the genome to calculate the concentration of ctDNA or the number of genome copies. Due to the large differences in ctDNA content and fragment integrity between individuals and different pathological states, even if the operator's factors are not considered, the differences in extraction methods and reagents used will result in huge differences in extraction efficiency. The error of ctDNA determination results based on this will be even greater. However,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851
Inventor 宋现让谢丽
Owner 宋现让
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