Stabilizer for free DNA in blood and application thereof
By developing a blood-free DNA stabilizer, the problem of instability of free DNA in blood samples at room temperature was solved, enabling long-term stable and low-cost DNA detection at room temperature, thus improving detection accuracy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INOZAN (JIANGSU) BIOTECHNOLOGY CO LTD
- Filing Date
- 2024-10-28
- Publication Date
- 2026-04-28
AI Technical Summary
Existing technologies struggle to stabilize free DNA in blood at room temperature for extended periods, limiting detection accuracy. Furthermore, cryopreservation is costly, and imported equipment is expensive.
A blood-free DNA stabilizer is prepared, comprising a buffer, an aldehyde fixative, an anticoagulant, a cell stabilizer, and a nuclease inhibitor. This stabilizes the pH, inhibits nuclease activity, prevents blood clotting and cell disruption, and maintains the stability of the free DNA in the blood sample at room temperature after mixing.
It maintains the stability of free DNA at room temperature for up to 7 days, inhibits the release of genomic DNA from blood cells, reduces sample storage and transportation costs, and improves detection accuracy.
Smart Images
Figure CN121931097A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of blood testing reagents, and in particular to a stabilizer for cell-free DNA in blood and its applications. Background Technology
[0002] Cell-free deoxyribonucleic acid (cfDNA) is a component of double-stranded DNA in the blood. Released into the bloodstream through apoptosis or necrosis, it is typically a double-stranded fragment of 130-150 base pairs in length and can be isolated from plasma. It is an important biomarker reflecting human physiological indicators. Currently, the detection of cell-free DNA is widely used in clinical fields such as cancer screening, diagnosis of single-gene diseases, and non-invasive prenatal diagnosis.
[0003] Traditional extraction methods involve collecting whole blood using anticoagulant blood collection tubes and then collecting plasma for cell-free DNA extraction. However, the instability of cell-free DNA and the complex composition of whole blood significantly limit the integrity and accuracy of the extracted DNA. Anticoagulants, such as EDTA, only chelate with calcium ions in the blood to prevent clotting; they cannot completely inhibit the degradation of cell-free DNA by nucleases in the blood. Furthermore, white blood cells gradually break down, releasing genomic DNA that contaminates the cell-free DNA sample, leading to false results. While cryopreservation after collecting blood using anticoagulant blood collection tubes can alleviate cell-free DNA degradation and cell breakage to some extent, it only delays the process by about a day, and it also results in high transportation costs for samples requiring long-distance transport.
[0004] While imported free DNA blood collection tubes from Streck can remain unchanged for 7 days at room temperature, they are expensive. Summary of the Invention
[0005] The purpose of this invention is to provide a low-cost stabilizer for cell-free DNA in blood, which allows cell-free DNA to be stored in whole blood samples at room temperature for a longer period of time, thereby improving the accuracy of cell-free DNA detection.
[0006] To achieve the objectives of this invention, the technical solution is as follows:
[0007] A stabilizer for cell-free DNA in blood, wherein: each 100 ml of the cell-free DNA stabilizer comprises 0.5-5 g of buffer, 2-20 g of aldehyde fixative, 15-20 g of anticoagulant, 10-50 g of cell stabilizer, 2.5-12.5 g of nuclease inhibitor, and ultrapure water to a final volume of 100 ml; the pH of the cell-free DNA stabilizer is 6.8-7.8; the cell stabilizer is selected from one or more of polysucrose-50, polysucrose-70, polysucrose-400, polyethylene glycol-4000, polyethylene glycol-6000, polyethylene glycol-8000, and betaine.
[0008] The function of cell stabilizers is to reduce blood cell sedimentation, prevent platelet aggregation, and reduce mechanical stress damage to cells. Preferably, the cell stabilizer is selected from polysucrose-70, and each 100 ml of blood-free DNA stabilizer contains 10 g of polysucrose-70.
[0009] The buffer is selected from one or more of the following: tris(hydroxymethyl)aminomethane salt, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate; and / or, the aldehyde fixative is selected from one or more of the following: formaldehyde, paraformaldehyde, and glutaraldehyde; and / or, the anticoagulant is selected from one or more of the following: disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, and sodium citrate; and / or, the nuclease inhibitor is selected from one or more of the following: sodium citrate and potassium citrate. The buffer's function is to maintain a stable pH in the blood sample, preventing atmospheric components and cell metabolites from affecting the sample's pH and causing degradation of free DNA. The aldehyde fixative's function is to fix cell morphology by forming a covalent structure with the amino groups of proteins in the sample through aldehyde groups, while simultaneously inhibiting bacterial growth. The anticoagulant's function is to chelate metal ions, prevent blood clotting, maintain cell stability, and inhibit nuclease activity. The cell stabilizer's function is to reduce blood cell sedimentation, prevent platelet aggregation, and reduce mechanical stress damage to cells. Nuclease inhibitors work by inhibiting the activity of nucleases in blood samples, thus preventing the degradation of free DNA.
[0010] Preferably, the buffer is selected from tris(hydroxymethyl)aminomethane hydrochloride, and 1 g of tris(hydroxymethyl)aminomethane hydrochloride is contained in every 100 ml of blood-free DNA stabilizer.
[0011] Preferably, the aldehyde fixative is selected from paraformaldehyde, and each 100 ml of blood-free DNA stabilizer contains 5 g of paraformaldehyde.
[0012] Preferably, the anticoagulant is selected from dipotassium ethylenediaminetetraacetate, and each 100 ml of blood-free DNA stabilizer contains 1.5 g of paraformaldehyde.
[0013] Preferably, the nuclease inhibitor is selected from sodium citrate, and 2.5g of sodium citrate is contained in every 100ml of blood-free DNA stabilizer.
[0014] Preferably, the blood-free DNA stabilizer has a pH of 7.4.
[0015] The stabilizer provided by this invention can be added to whole blood samples after blood is drawn or added to vacuum blood collection tubes in advance. After the blood is fully mixed with the stabilizer, the free DNA in the sample is kept stable for 7 days at room temperature (15~24℃).
[0016] Furthermore, the volume ratio of the stabilizer to the whole blood sample in this invention is 1:10 to 1:20.
[0017] Furthermore, the stabilizer is used as a stabilizer for cell-free DNA in blood samples during collection, transportation, or storage.
[0018] The significant advantages of this invention compared to existing technologies are:
[0019] This invention utilizes a variety of compounds to formulate a low-toxicity, low-cost blood-free DNA stabilizer that can maintain the stability of free DNA for 7 days at room temperature, inhibit the release of blood cell genomes, effectively reduce the storage and transportation costs of blood samples, and improve the detection accuracy of blood-free DNA. Attached Figure Description
[0020] Figure 1 These are photographs of the blood from the positive control group and the experimental group after centrifugation in Example 1 of this invention.
[0021] Figure 2 This is a diagram showing the β-Actin gene DNA content in each blood collection tube in Example 1 of the present invention.
[0022] Figure 3 This is a graph showing the λDNA content in each blood collection tube in Example 1 of the present invention. Detailed Implementation
[0023] To better illustrate the beneficial effects and technical solutions of the present invention, specific embodiments are now provided for further explanation. The present invention includes, but is not limited to, the following embodiments and combinations thereof.
[0024] Example 1
[0025] Prepare 10 ml of stabilizer using the following method:
[0026] Using ultrapure water, prepare a solution with the following components: 1% Tris-HCl (tris-hydroxymethylaminomethane hydrochloride), 5% paraformaldehyde, 1.5% dipotassium ethylenediaminetetraacetate, 10% sucrose-70, and 2.5% sodium citrate. Adjust the pH of this stabilizer to 7.4.
[0027] Blood samples were drawn and λDNA fragments were added:
[0028] Fresh blood was drawn from one volunteer and divided into three groups (negative control group, positive control group, and experimental group), with three 2ml parallel samples in each group. For both the negative and positive control groups, 0.2ml of 1.5% dipotassium ethylenediaminetetraacetate solution (prepared using PBS as a solvent) was added at a volume ratio of 1:10 to the blood to represent the effect of a common dipotassium ethylenediaminetetraacetate anticoagulant on cell-free DNA. For the experimental group, 0.2ml of stabilizer was added at a volume ratio of 1:10. All three groups of samples were thoroughly mixed by inverting and then allowed to stand.
[0029] 100 ng of a 147 bp λDNA fragment derived from the λ phage genome was added to all three groups of samples. The gene sequence is shown in Table 1. Its size matches that of cell-free DNA in blood and is used to simulate cell-free DNA in blood samples. Since the genome of blood cells does not contain this sequence, even if the cell genome is broken and released extracellularly in the blood, subsequent detection of λDNA fragment concentration changes can show the degradation of cell-free DNA in blood samples over time, reducing false positives in the test results.
[0030] Table 1
[0031]
[0032] Experimental results verify:
[0033] Negative control group: Free DNA was extracted immediately after treatment. Positive control group and experimental group: Free DNA was extracted after standing at room temperature for 7 days.
[0034] The methods for extracting cell-free DNA are as follows:
[0035] For plasma separation, the sample was centrifuged at 1000×g for 10 minutes at room temperature.
[0036] Extraction was performed using 500 μl of plasma from the VAHTS Serum / Plasma Circulating DNA Kit (magnetic bead method) from Nanjing Novizan Biotechnology Co., Ltd., following the manufacturer's instructions. The extracted cell-free DNA was dissolved and stored using Elution Buffer.
[0037] Cell-free DNA was detected and analyzed by quantitative real-time PCR (qPCR). The detection genes were β-Actin and λDNA. Primers are shown in Table 2, and reaction systems are shown in Tables 3 and 4.
[0038] Table 2. Reaction primer sequences
[0039]
[0040] Table 3 Amounts of each component in the reaction system
[0041]
[0042] Table 4 qPCR reaction program conditions
[0043]
[0044] After the reaction, the data were analyzed using the software included with the Boge fluorescent PCR detection system. The results are as follows: Figure 2 and Figure 3 As shown.
[0045] like Figure 1 As shown in the figure, the test tube on the left is the positive control group, and the one on the right is the experimental group. After centrifugation for 7 days, hemolysis was visible to the naked eye in the plasma of the positive control group, while the plasma of the experimental group was clear and bright yellow, indicating no obvious hemolysis and stable red blood cells.
[0046] like Figure 2 As shown, the β-Actin detection results were as follows: In the positive control group, the β-Actin level increased to approximately 30 times that of the freshly extracted level after 7 days of standing at room temperature. This indicates that the nucleated cells in the blood, such as leukocytes, in the positive control group were broken down and released a large amount of genomic DNA during storage at room temperature, which interfered with the detection of cell-free DNA in the plasma. However, the β-Actin gene detection results in the experimental group after 7 days of standing were not different from those in the freshly extracted negative control group, indicating that the stabilizer had a good stabilizing effect on blood cells and did not cause the release or interference of genomic DNA.
[0047] like Figure 3 As shown, the λDNA detection results showed that the λDNA in the positive control group decreased by nearly 70% after standing at room temperature for 7 days, indicating that the free DNA in the blood sample was severely degraded under normal temperature conditions. In contrast, the λDNA in the experimental group decreased less, indicating that the stabilizer effectively prevented the degradation of free DNA in the blood sample.
[0048] Since cell-free DNA in blood is typically a short-chain DNA of 130-150 bp, and β-Actin is one of the housekeeping genes in the human genome, detecting its fragment concentration is commonly used as a method for detecting cell-free DNA in blood, serving as a reference for the concentration of extracted cell-free DNA. However, it is not suitable for assessing the degradation of cell-free DNA in blood over extended storage time. This is because, as the sample is stored for a longer period, some blood cells break down and release their own genomic DNA into the sample, causing false detection results for cell-free DNA. Therefore, in this experiment, to verify the protective effect of the stabilizer against cell-free DNA degradation, external λDNA was added and its content was tested. Testing the β-Actin gene confirmed that the stabilizer provided by this invention has a good stabilizing effect on cells in blood, without causing the release or interference of genomic DNA.
[0049] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application.
Claims
1. A stabilizer for cell-free DNA in blood, characterized in that: The blood-free DNA stabilizer per 100 ml comprises 0.5-5 g buffer, 2-20 g aldehyde fixative, 15-20 g anticoagulant, 10-50 g cell stabilizer, 2.5-12.5 g nuclease inhibitor, and ultrapure water to a final volume of 100 ml; the pH of the blood-free DNA stabilizer is 6.8-7.8; the cell stabilizer is selected from one or more of the following: polysucrose-50, polysucrose-70, polysucrose-400, polyethylene glycol-4000, polyethylene glycol-6000, polyethylene glycol-8000, and betaine.
2. The stabilizer for cell-free DNA in blood according to claim 1, characterized in that: The cell stabilizer is selected from polysucrose-70, and each 10 ml of blood-free DNA stabilizer contains 10 g of polysucrose-70.
3. The stabilizer for cell-free DNA in blood according to claim 1, characterized in that: The buffer is selected from one or more of the following: tris(hydroxymethyl)aminomethane salt, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate. And / or, the aldehyde fixative is selected from one or more of formaldehyde, paraformaldehyde, and glutaraldehyde; And / or, the anticoagulant is selected from one or more of disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, and sodium citrate; And / or, the nuclease inhibitor is selected from one or more of sodium citrate and potassium citrate.
4. The stabilizer for cell-free DNA in blood according to claim 3, characterized in that: The buffer solution is selected from tris(hydroxymethyl)aminomethane hydrochloride, and 1 g of tris(hydroxymethyl)aminomethane hydrochloride is contained in every 100 mL of blood-free DNA stabilizer.
5. The stabilizer for cell-free DNA in blood according to claim 3, characterized in that: The aldehyde fixative is selected from paraformaldehyde, and each 100 ml of blood-free DNA stabilizer contains 5 g of paraformaldehyde.
6. The stabilizer for cell-free DNA in blood according to claim 3, characterized in that: The anticoagulant is selected from dipotassium ethylenediaminetetraacetate, and each 100 ml of blood-free DNA stabilizer contains 1.5 g of paraformaldehyde.
7. The stabilizer for cell-free DNA in blood according to claim 3, characterized in that: The nuclease inhibitor is selected from sodium citrate, and 2.5g of sodium citrate is contained in every 100ml of blood-free DNA stabilizer.
8. The stabilizer for cell-free DNA in blood according to claim 1, characterized in that: The blood-free DNA stabilizer has a pH of 7.
4.
9. The stabilizer for cell-free DNA in blood according to any one of the preceding claims, characterized in that: The volume ratio of the blood-free DNA stabilizer to the blood sample is 1:10 to 1:
20.
10. The stabilizer for cell-free DNA in blood according to any one of the preceding claims, characterized in that: The stabilizer is used as a stabilizer for free DNA in blood samples during collection, transportation, or storage.