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Blood protection solution for preserving cfDNAs (cell-free DNAs) in whole blood at room temperature

A protective liquid and blood technology, which is applied in the field of blood protective liquid for cfDNA preservation in whole blood at room temperature, can solve the problems of low storage temperature, high economic cost, and short storage time, and achieve the goals of avoiding pollution, prolonging storage time, and preventing rupture Effect

Inactive Publication Date: 2019-05-10
SUZHOU GENEPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the existing problems of free DNA in peripheral blood such as short storage time, easy degradation, easy pollution, low storage temperature, and high economic cost, the present invention provides a protective agent for free DNA in peripheral blood, which can effectively inhibit cell metabolism , inhibit nuclease activity, prevent blood cell rupture, protect free DNA in blood

Method used

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  • Blood protection solution for preserving cfDNAs (cell-free DNAs) in whole blood at room temperature
  • Blood protection solution for preserving cfDNAs (cell-free DNAs) in whole blood at room temperature
  • Blood protection solution for preserving cfDNAs (cell-free DNAs) in whole blood at room temperature

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042]Example 1, Preparation of Blood Protection Solution for Preserving cfDNA in Whole Blood at Normal Temperature

[0043] The blood protection solution for cfDNA in whole blood consists of 10-100g / L 1,3-dimethylol-5,5-dimethylhydantoin, 0.1-1mL / L Proclin300, 10-100g / L EDTA salt , 1-10g / L ammonium sulfate, 1-10g / L ATA, glycerol and buffer of 1-10mL / L consist of tris-hydrochloric acid with a concentration of 10mM (pH value 8.0) solution.

[0044] The concentration is 10mM tris-hydrochloric acid solution, the solute is 10mM tris-salt (Tris), and the solvent is water.

[0045] The specific formula is as follows:

[0046] cfDNA blood protection solution formula 1: 50g / L 1,3-dimethylol-5,5-dimethylhydantoin, 0.2mL / L Proclin300, 20g / L EDTA-K2, 1g / L sulfuric acid Ammonium, 1g / L ATA, 5mL / L of glycerol and a buffer solution, the buffer solution is a tris-hydrochloric acid solution with a concentration of 10mM (pH value 8.0);

[0047] cfDNA blood protection solution formula two: 5...

Embodiment 2

[0048] Embodiment 2, detection of blood protection solution room temperature stability

[0049] The blood sample and cfDNA blood protection solution were mixed thoroughly at a ratio of 5:1 to prevent coagulation due to insufficient contact.

[0050] Prepare anticoagulant according to formula one group in embodiment 1, and carry out comparative test with common EDTA blood collection tube, concrete steps are as follows:

[0051] 1. Use EDTA blood collection tubes and vacuum blood collection tubes added with the blood protection solution provided in Example 1 to collect the same amount of peripheral blood respectively. The total volume of the blood collection tubes is 5mL; the volume ratio of blood protection solution to peripheral blood is 1:5, vacuum Accuracy error ≤ 10%;

[0052] 2. The collected blood is placed at room temperature;

[0053] 3. Separate the plasma on days 0, 1, 4, 7, 9, 12, 14, and 15; the step of separating the plasma is to put the blood into a centrifuge t...

Embodiment 3

[0066] Embodiment 3, transport stability of blood protection solution

[0067] According to the vacuum blood collection tubes and common EDTA blood collection tubes prepared by formula 2 in Example 1, the same amount of peripheral blood was respectively taken to carry out the simulated transportation stability comparison test:

[0068] 1. The collected blood is placed at room temperature, and the blood is gently inverted and mixed twice a day to simulate the transportation environment, so as to observe the changes of blood cells and plasma DNA in the transportation environment.

[0069] 2. On days 0, 1, 4, 7, 9, 12, 14, and 15, separate the plasma in EDTA blood collection tubes and vacuum blood collection tubes added with blood protection solution;

[0070] 3. Extract the DNA in the separated plasma;

[0071] 4. Perform q-PCR verification of the housekeeping gene β-actin on the obtained DNA, and judge the test result according to the Ct value displayed by the fluorescent PCR ...

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Abstract

The invention discloses a blood protection solution for preserving cfDNAs (cell-free DNAs) in whole blood at room temperature. The blood protection solution comprises anticoagulants, preservatives, nuclease inhibitors, metabolic inhibitors, cell stabilizers and membrane stabilizers. The blood protection solution has the advantages that by a blood cfDNA preservative, the nuclease activity can be inhibited, and the cfDNAs in the blood can be protected; white blood cells are stabilized, breakage of the white blood cells is prevented, and the cfDNAs are prevented from being contaminated by cell genomes; peripheral blood can be stored at the room temperature for 14 days and transported at the room temperature for at least 14 days, during which no obvious change occurs, so that the storage timeof blood samples at the room temperature is effectively prolonged, great assistance is brought to long-distance transport of the blood samples, the detection cost of the cfDNAs is saved, and application and promotion of cfDNA-related detection techniques are facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a blood protection solution for preserving cfDNA in whole blood at room temperature. Background technique [0002] Circulating cell free DNA (cfDNA) is a cell-free extracellular DNA that exists in human blood. As a marker of cell death, in recent years, cell-free DNA in peripheral blood has been used more and more clinically: in non-invasive prenatal diagnosis, the detection of fetal cell-free DNA in peripheral blood of pregnant women is an important tool for non-invasive prenatal diagnosis. The diagnosis of pre-genetic diseases provides an important research method; at the same time, more and more studies have shown that cell-free DNA in peripheral blood is most likely to be a liquid biopsy marker for early diagnosis of tumors. Compared with the United States, although its tumor incidence rate is 1.6 times that of China, its 5-year survival rate is 1.8 times that of China. One of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 饶品彬李娜张佩琢
Owner SUZHOU GENEPHARMA
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