Method for quantitatively determining blood circulation DNA (Deoxyribonucleic Acid)

Abstract

The invention discloses a method for quantitatively determining blood circulation DNA (Deoxyribonucleic Acid). The method comprises the following steps of: obtaining 1ml of peripheral venous blood of the same tumor patient and placing the peripheral venous blood into an EDTA (Ethylene Diamine Tetraacetic Acid) anti-freezing pipe; standing for 4 hours at 10-20 DEG C; centrifuging 2000g of peripheral venous blood for 5 minutes; taking 100-200 microliters of upper-layer blood plasma; adding a buffering solution with the same volume and sufficiently and uniformly mixing; standing at 95 DEG C for 5-10 minutes; centrifuging 16000g of peripheral venous blood for 10 minutes; and taking a liquid supernatant as a template to be directly used for quantitative PCR (Polymerase Chain Reaction) amplification. The method disclosed by the invention has the beneficial effects that 1. blood plasma or blood serum can be directly used for a quantitative PCR reaction through special technical process treatment and the PCR reaction is not influenced; ratios of amplification efficiencies to the purified DNA are consistent, and extraction steps and experimental errors of the DNA are reduced; and 2. an adopted novel fluorescent dye Super Green has slight inhibition effect in a PCR amplification process and high concentration can be used; and a fluorescence signal value is high and the reaction sensitivity is high, and blood plasma free DNA which is at a nanogram level can be accurately detected.

Description

technical field [0001] The invention relates to the technical field of blood circulation DNA measurement, in particular to a method for quantitatively measuring blood circulation DNA. Background technique [0002] Circulating DNA (ctDNA), also known as cell-free DNA or cell-free DNA, is mainly derived 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. How...

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

[0003] In addition, the quantitative PCR technology using SYBR Green as a dye has low signal intensity and low sensitivity of fluorescent dyes, and it is not easy to detect low copy number templates

Images