Disposable free DNA preservation tube

Abstract

The invention discloses a disposable free DNA preservation tube, and relates to the field of biological material preservation devices.The disposable free DNA preservation tube comprises a cover, a rubber plug, a collection tube and a label, the rubber plug is arranged in the cover and can be tightly plugged in a tube opening of the collection tube, and it is guaranteed that the interior of the collection tube is in a vacuum environment; the preservation pipe contains a preservation agent, and the preservation agent is composed of a chelating agent, a buffering agent and a protective agent; according to the disposable free DNA storage tube, through optimization in multiple aspects of long-acting stabilization of free DNA, inhibition of a pollution source, simplification of an operation process, improvement of detection sensitivity, reduction of cost, popularization and application and the like, the integrity and the concentration of the free DNA are kept for a long time under the condition of normal temperature, and dependence on cold chain transportation is reduced; meanwhile, the risk of genomic DNA and microbial contamination is reduced by preventing blood cell lysis and nuclease activity; the method is suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to a biological material preservation technology, in particular to a disposable free DNA preservation tube. Background Art

[0002] Circulating free DNA (cfDNA) refers to DNA fragments that exist outside cells and are present in bodily fluids such as blood, urine, and cerebrospinal fluid. cfDNA has a wide range of applications, primarily in oncology for early cancer detection, disease monitoring, and treatment guidance. By detecting circulating tumor DNA (ctDNA) in the blood, accurate cancer diagnosis and personalized treatment can be achieved. In prenatal diagnosis, non-invasive prenatal testing (NIPT) is performed by analyzing fetal cfDNA in maternal plasma to detect fetal chromosomal abnormalities and certain genetic diseases. In addition, cfDNA can be used for organ transplant monitoring, by detecting donor cfDNA in the recipient's blood to monitor immune rejection reactions and the health of the transplanted organ. At the same time, cfDNA also shows potential in other disease detection, such as for assessing the level of damage and disease progression in diseases such as trauma, burns, sepsis, myocardial infarction, and stroke.

[0003] Existing technologies have many problems in the preservation and detection of cell-free DNA (cfDNA) in the blood: chemical additives (such as EDTA and heparin) in existing preservation tubes inhibit molecular detection reactions such as PCR and NGS library construction, have poor compatibility, and free DNA will continue to slowly degrade after more than 14 days; physical adsorption-type preservation tubes have low DNA recovery rates, especially for short-fragment free DNA (such as <100bp), with low binding efficiency, and high operational complexity, requiring steps such as centrifugation and liquid transfer, which increases the risk of contamination; high-end preservation tubes are expensive and rely on imports, limiting their use in primary care or large-scale screening; traditional anticoagulant tubes (such as EDTA tubes) can only inhibit coagulation in the short term and cannot inhibit nucleases in the long term, and the high cost of low-temperature transportation makes cfDNA easily degraded; ordinary anticoagulant tubes cannot prevent blood cell lysis, leading to genomic DNA (gDNA) contamination, interfering with the purity and detection sensitivity of free DNA. Summary of the Invention

[0004] The purpose of the present invention is to provide a disposable free DNA storage tube to solve the above-mentioned shortcomings in the prior art.

[0005] To achieve the above-mentioned object, the present invention provides the following technical solution: a disposable free DNA storage tube, comprising a lid, a rubber stopper, a collection tube, and a label; the rubber stopper is placed in the lid and can be tightly plugged into the tube mouth of the collection tube to ensure a vacuum environment in the collection tube; the storage tube contains a preservative, which is composed of a chelating agent, a buffer, and a protective agent.

[0006] Furthermore, the chelating agent is EDTA.2K.2H2O or EDTA.3K.2H2O.

[0007] Furthermore, the mass percentage concentration of the EDTA.2K.2H2O is 3%-16%, and the mass percentage concentration of the EDTA.3K.2H2O is 3%-16%.

[0008] Furthermore, the buffer is glycine.

[0009] Furthermore, the mass percentage concentration of the glycine is 4%-11%.

[0010] Furthermore, the protective agent is imidazolidinyl urine, and the mass percentage concentration of the imidazolidinyl urine is 1.5%-13%.

[0011] Compared with the existing technology, the disposable free DNA preservation tube provided by the present invention achieves long-term maintenance of the integrity and concentration of free DNA at room temperature conditions through optimization in many aspects, such as long-term stabilization of free DNA, suppression of pollution sources, simplification of operating procedures, improvement of detection sensitivity, reduction of costs and popularization of applications, thereby reducing dependence on cold chain transportation; at the same time, by preventing blood cell lysis and nuclease activity, the risk of genomic DNA and microbial contamination is reduced; in addition, it realizes an integrated design of "blood collection and preservation", reducing manual operation steps and contamination probability; by providing high-purity free DNA samples, it supports the efficient detection of low-frequency mutations (such as tumor ctDNA); finally, by breaking the monopoly of imported technology, it provides a cost-effective solution, thereby promoting large-scale clinical screening and scientific research applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0013] Figure 1 A schematic diagram of the genetic test results 7 days after blood collection using a K3EDTA blood collection tube provided in an embodiment of the present invention;

[0014] Figure 2Schematic diagram of cfDNA test results after 7 days of static storage (A) or transportation (B) provided in an embodiment of the present invention;

[0015] Figure 3 Schematic diagram comparing the cfDNA results with proteinase K cleavage (red curve) and without proteinase K cleavage (blue curve) in the cfDNA extraction step provided in an embodiment of the present invention;

[0016] Figure 4 The cfDNA amplification curve and C in blood provided by the embodiment of the present invention T Schematic diagram of value results;

[0017] Figure 5 The cfDNA amplification curve and C in blood after adding the mutant EGFR fragment provided in the embodiment of the present invention T Schematic diagram of value results;

[0018] Figure 6 A schematic diagram showing the stable storage performance of a disposable cell-free DNA storage tube provided by an embodiment of the present invention;

[0019] Figure 7 A schematic diagram of the cfDNA extraction results of samples of different volumes provided in an embodiment of the present invention;

[0020] Figure 8 Schematic diagram of cfDNA extraction results for samples of different volumes and different processing methods provided in an embodiment of the present invention;

[0021] Figure 9 A schematic diagram of the overall structure of a disposable cell-free DNA storage tube provided in an embodiment of the present invention;

[0022] Figure 10 A schematic diagram of the longitudinal cross-section of a disposable cell-free DNA storage tube provided in an embodiment of the present invention;

[0023] Figure 11 An exploded view of a disposable cell-free DNA storage tube provided in an embodiment of the present invention.

[0024] Description of reference numerals:

[0025] 1. Lid; 2. Rubber stopper; 3. Collection tube; 4. Label. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] Comparative Example 1:

[0028] K3EDTA blood collection tube is a vacuum blood collection tube containing tripotassium ethylenediaminetetraacetic acid (K3EDTA) as an anticoagulant. It is mainly used to collect, preserve and process blood samples to prevent blood coagulation and ensure the accuracy and reliability of test results. It can be used to extract nucleic acid substances such as DNA and RNA.

[0029] See also Figure 1 , fresh blood was collected and cfDNA was quickly extracted as a standard ( Figure 1 The blood collected by K3EDTA blood collection tube was first stored for 7 days and then cfDNA was extracted. As a result, a large amount of cell-derived genomic DNA contamination was detected ( Figure 1 (The red curve is in the middle.) This indicates that a large amount of genomic DNA is released into the blood sample during long-term storage in K3EDTA blood collection tubes. This cell-derived genomic DNA contamination significantly reduces the sensitivity and accuracy of cfDNA analysis. Therefore, K3EDTA blood collection tubes are not suitable for the storage and transportation of blood related to cfDNA research.

[0030] Example 1:

[0031] See also Figures 9 to 11 A disposable cell-free DNA storage tube comprises a cap 1, a rubber stopper 2, a collection tube 3, and a label 4. The rubber stopper 2 is placed inside the cap 1 and can be tightly plugged into the mouth of the collection tube 3 to ensure a vacuum environment inside the collection tube 3. The storage tube contains a preservative, which comprises a chelating agent, a buffer, and a protective agent. The chelating agent is EDTA.2K.2H2O or EDTA.3K.2H2O; the mass percentage concentration of EDTA.2K.2H2O is 3%-16%, and the mass percentage concentration of EDTA.3K.2H2O is 3%-16%. The buffer is glycine; the mass percentage concentration of glycine is 4%-11%. The protective agent is imidazolidinyl urea, and the mass percentage concentration of imidazolidinyl urea is 1.5%-13%.

[0032] In a specific embodiment, the collection tube 3 is a cylindrical vacuum blood collection tube made of glass or plastic. The outside of the tube 3 is marked with a scale indicating the blood collection volume. The tube cap, consisting of a lid 1 and a rubber stopper 2, provides a good seal, ensuring a vacuum state within the tube and preventing the ingress of air, moisture, etc. A label 4 can be used to record information related to the collected sample.

[0033] The collection tube 3 is the main body that holds the blood sample and preservative solution. It has a certain strength and transparency to facilitate observation of the sample's status. The preservative inside the collection tube 3 can inhibit the activity of nucleases in the blood, stabilize nucleated blood cells, and prevent the release of genomic DNA.

[0034] The preservative in the collection tube 3 preserves the cell-free DNA. On the one hand, the preservative inhibits the activity of nucleases in the blood, preventing nuclease degradation of the cell-free DNA. On the other hand, the preservative stabilizes nucleated blood cells, preventing cell rupture and the release of genomic DNA, thereby reducing interference and contamination with the cell-free DNA and ensuring the integrity and purity of the cell-free DNA in the sample.

[0035] Chelating agents (EDTA, 2K, 2H2O or EDTA, 3K, 2H2O), EDTA (ethylenediaminetetraacetic acid) and its salts can form stable complexes with metal ions. During the storage of free DNA, the activity of many enzymes (such as DNase, which can cause DNA degradation) depends on metal ions (primarily magnesium and calcium ions). EDTA chelates these metal ions, depriving them of the metal ion "helpers" they need for activity, effectively inhibiting DNase activity, preventing enzymatic degradation of free DNA and safeguarding its integrity.

[0036] The buffer (glycine) acts as a buffer solution. It maintains the pH of the storage solution relatively stable. The structure and chemical properties of free DNA are very sensitive to pH. When the pH is too high or too low, it may cause cleavage of DNA's phosphate bonds or other changes in its chemical structure. Glycine can stabilize the pH at around 5.2. This pH environment helps inhibit DNA hydrolysis, reduce chemical damage to DNA molecules during storage, and ensure the chemical stability and integrity of DNA.

[0037] A protective agent (imidazolidinyl urethane) is a moisturizing and protective agent. It provides a relatively stable and moist environment for free DNA, preventing DNA denaturation due to drying. It also protects DNA molecules from external physical and chemical interference to a certain extent. For example, during storage, there may be slight temperature fluctuations, light exposure, and other environmental factors. Imidazolidinyl urethane helps maintain the three-dimensional structure of DNA molecules and reduce the risk of DNA breakage and degradation.

[0038] Instructions for use of the disposable cell-free DNA preservation tube: Use a blood collection needle to collect venous blood directly into the disposable cell-free DNA preservation tube. The blood collection volume is generally 1ml-10ml as needed.

[0039] After blood enters the disposable cell-free DNA storage tube, it mixes thoroughly with the preservative inside the tube, which begins to stabilize and protect the cell-free DNA. At 6-37°C, cell-free DNA and genomic DNA can be stably stored for 14 days, and circulating tumor cells can be stably stored for 7 days at 15-30°C.

[0040] Disposable cell-free DNA storage tubes containing blood samples can be transported at room temperature. After arriving at the testing laboratory, they can be directly used for subsequent cell-free DNA extraction and analysis experiments, such as second-generation sequencing, digital PCR, etc.

[0041] This disposable cell-free DNA preservation tube can directly draw whole blood, enabling rapid and convenient collection of blood samples; it effectively protects the free DNA, genomic DNA, and circulating tumor cells in the blood, keeping them stable within the specified time and temperature range; it does not require a cold chain and can be transported and stored at room temperature, greatly reducing the difficulty and cost of sample transportation and management; it can provide high-quality cell-free DNA samples for a variety of clinical medical research and diagnostic tests, including liquid biopsy, oncology research, and non-invasive prenatal testing.

[0042] Example 2:

[0043] See also Figure 2 This embodiment provides a technical solution based on the first embodiment: anti-genomic DNA contamination performance test.

[0044] Blood was collected using EDTA blood collection tubes, disposable cfDNA storage tubes (cfDNA BCT) and other similar blood collection tubes (Competitor product). After collecting blood in EDTA blood collection tubes, cfDNA was quickly extracted as a standard. The blood collected in the disposable cfDNA storage tubes and the blood collected in other similar blood collection tubes were stored separately ( Figure 2 A) or transport ( Figure 2 B) 7 days, cfDNA was extracted after 7 days.

[0045] See also Figure 2 A. The yield of cfDNA extracted using disposable cfDNA storage tubes reached over 90% (red curve), essentially identical to that of a standard (blue curve). However, the 170bp peak of cfDNA extracted using other similar tubes shifted to 200bp (green curve). Furthermore, no cell-derived genomic DNA contamination was observed in blood stored for 7 days in disposable cfDNA storage tubes.

[0046] See also Figure 2 B, with Figure 2 Consistent with the conclusion of A, blood stored in disposable cfDNA storage tubes was extracted after 7 days of transportation (red curve) and compared with the standard on day 0 (blue curve), and no genomic DNA contamination was found.

[0047] Disposable cfDNA collection tubes are stored or transported for at least seven days after blood collection, minimizing contamination with cell-derived genomic DNA. The lower the DNA contamination, the more sensitive and accurate downstream cfDNA research can be. While other similar blood collection tubes also offer protection against genomic DNA contamination, chemical reactions in these tubes can cause cross-linking of nucleic acids with other biomolecules, leading to an increase in the 170bp peak and a shift to 200bp.

[0048] Example 3:

[0049] See also Figure 3 This embodiment provides a technical solution based on the first embodiment: simplifying the effect of cfDNA extraction steps on cfDNA.

[0050] See also Figure 3 A. Comparison of cfDNA extraction after 7 days of blood collection using a comparable blood collection tube with and without proteinase K cleavage (blue curve). Proteinase K cleavage is essential for cfDNA extraction using comparable products; otherwise, no cfDNA is isolated. Furthermore, the cfDNA peak extracted using comparable products shifts from 170bp to 200bp.

[0051] See also Figure 3 B, Comparison of cfDNA extraction after 7 days of blood collection using disposable cfDNA storage tubes with and without proteinase K cleavage (red curve). There was no significant difference in cfDNA extraction between blood collected using disposable cfDNA storage tubes with and without proteinase K cleavage.

[0052] When using other similar blood collection tubes to extract blood, the nucleic acid in the sample will cross-link with the protein, which will affect the detection of cfDNA downstream. When extracting cfDNA using a disposable cfDNA storage tube, proteinase K treatment is not required, and this step can be skipped to simplify the extraction process. However, blood collected by other similar blood collection tubes must be lysed with proteinase K, otherwise cfDNA cannot be separated ( Figure 3 (as shown in A).

[0053] Example 4:

[0054] See also Figure 4 This embodiment provides a technical solution based on the first embodiment: cfDNA collection capacity detection of disposable cfDNA storage tubes.

[0055] Fresh blood samples were collected using EDTA blood collection tubes and disposable cfDNA storage tubes, respectively, and stored at room temperature for 7 days before cfDNA extraction. Immediately after collecting blood samples using EDTA blood collection tubes, cfDNA was extracted from plasma as a standard (Day 0), and then the level of β-globulin cfDNA was detected by fluorescent quantitative PCR. Regardless of static storage or transportation, and whether or not proteinase K cleavage was performed, the amplification curves of samples and standards using disposable cfDNA storage tubes were ( Figure 4 A) and CT value ( Figure 4 B) are consistent. However, for blood stored in EDTA tubes for 7 days, the CT value of β-globulin fluorescence quantitative detection results was significantly lower due to contamination by cell-derived genomic DNA.

[0056] This is another evidence that similar blood collection tube competitors have cross-linked cfDNA with other biological molecules. In contrast, cfDNA isolated and extracted using disposable cfDNA storage tubes does not require proteinase K cleavage treatment, and the peak is located at the normal approximately 170bp ( Figure 3 B), which shows that disposable cfDNA storage tubes can effectively avoid cfDNA cross-linking. Fluorescence quantitative PCR detection of β-globin encoding gene levels in samples is also an effective method to test the preservation ability of blood cfDNA. Use disposable cfDNA storage tubes to collect blood samples, store them statically or transport them for 7 days, and then extract cfDNA, and then perform fluorescence quantitative PCR detection. At the same time, EDTA blood collection tubes are used to collect blood and the samples immediately extracted are used as standards. The samples using disposable cfDNA storage tubes have the same CT value as the standards ( Figure 4 ), indicating that the disposable cfDNA storage tube has high endogenous cfDNA extraction capacity and high-quality blood preservation capacity

[0057] Embodiment 5:

[0058] See also Figure 5 This embodiment provides a technical solution based on the first embodiment: cfDNA extraction efficiency detection of disposable cfDNA storage tubes: artificial nucleic acid addition and recovery experiment.

[0059] In order to further verify the extraction efficiency of the disposable non-invasive cfDNA storage tube, a "Spike in & recover" experiment was designed to verify it.

[0060] Multiple tubes of fresh blood samples from the same individual were collected using disposable cfDNA storage tubes. A 0.01ng / uL concentration of a synthetic DNA fragment containing the c.2573T>G (L858R) EGFR mutation (approximately 170bp) was added as a marker. CFDNA was extracted from one tube immediately after blood collection as a standard, and cfDNA was extracted from the other tube after being stored at room temperature for 7 days as a test sample. Fluorescence quantitative PCR was then performed on both tubes. Amplification curve ( Figure 5 A) and CT value ( Figure 5 B) showed that there was basically no significant difference in the results of the test product and the standard product: the CT value showed that the DNA extraction rate in the test product was as high as 99%, confirming that the disposable cfDNA preservation tube has a stable and efficient cfDNA preservation ability, and the results of the test product and the standard product are basically consistent.

[0061] Disposable cfDNA storage tubes prevent cfDNA degradation, cross-linking, and genomic nucleic acid contamination, providing high-quality cfDNA for downstream applications.

[0062] Example 6:

[0063] See also Figure 6 This embodiment provides a technical solution based on the first embodiment: testing the cfDNA preservation performance of disposable cfDNA storage tubes relative to Streck BCT.

[0064] Streck BCT is a collection tube for stabilizing blood samples, primarily used to preserve and transport blood samples, ensuring their integrity prior to analysis. It is used to stabilize cfDNA and circulating tumor cells (CTCs), preserving cfDNA for up to 14 days at 6°C to 37°C and CTCs for up to 7 days at 15°C to 30°C.

[0065] Fresh blood samples were collected using Streck BCT and disposable cfDNA storage tubes, respectively. cfDNA was extracted after storage at room temperature for 22 hours, 3 days, and 7 days. The figure shows that the disposable cfDNA storage tube has better cfDNA preservation performance than Streck BCT.

[0066] Embodiment seven:

[0067] See also Figures 7 and 8 This embodiment provides a technical solution based on the first embodiment: the effect of different volumes of blood samples in disposable cfDNA storage tubes on cfDNA extraction.

[0068] See also Figure 71ml, 3ml, and 5ml fresh blood samples were collected using disposable cfDNA storage tubes, and cfDNA was extracted. Different cfDNA extraction kits (Kit 1 and Kit 2) were used to extract cfDNA from 1ml, 3ml, and 5ml fresh blood samples. Disposable cfDNA storage tubes provide efficient capture and enrichment capabilities and are more suitable for cfDNA extraction from large samples larger than 5ml.

[0069] See also Figure 8 The study compared cfDNA concentrations (pg / μL) in samples of different volumes (1mL, 3mL, and 5mL) using products and disposable cfDNA tubes from different suppliers (Supplier 1 and Supplier 2), using KAPA and 1:4KAPA treatments. The results showed that the disposable cfDNA tubes achieved significantly higher DNA concentrations than the products from the other two suppliers at all tested volumes, regardless of whether they were treated with KAPA or 1:4KAPA. This suggests that the disposable cfDNA tubes are superior in preventing inhibitors and genomic DNA from affecting downstream steps, enabling more efficient storage and extraction of cfDNA.

[0070] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A disposable free DNA storage tube, characterized in that: The storage tube comprises a cover (1), a rubber stopper (2), a collection tube (3) and a label (4); the rubber stopper (2) is placed in the cover (1) and can be tightly plugged into the tube mouth of the collection tube (3) to ensure that the collection tube (3) is in a vacuum environment; the storage tube contains a preservative, which is composed of a chelating agent, a buffer and a protective agent.

2. The disposable free DNA storage tube according to claim 1, characterized in that: The chelating agent is EDTA.2K.2H2O or EDTA.3K.2H2O.

3. The disposable free DNA storage tube according to claim 2, characterized in that: The mass percentage concentration of the EDTA.2K.2H2O is 3%-16%, and the mass percentage concentration of the EDTA.3K.2H2O is 3%-16%.

4. The disposable free DNA storage tube according to claim 1, characterized in that: The buffer is glycine.

5. The disposable free DNA storage tube according to claim 4, characterized in that: The mass percentage concentration of the glycine is 4%-11%.

6. The disposable free DNA storage tube according to claim 1, characterized in that: The protective agent is imidazolidinyl urine, and the mass percentage concentration of the imidazolidinyl urine is 1.5%-13%.

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