A nucleic acid preservation card, its preparation method and usage method

Abstract

This invention discloses a nucleic acid preservation card, its preparation method, and its usage method, belonging to the technical field of sample collection devices. The nucleic acid preservation card includes a card body, a cover plate, a sample dispersion pad, a nucleic acid preservation pad, and a drying block. The nucleic acid preservation pad is made of chemically modified cellulose. The card body contains a sample preservation chamber and a drying chamber. The sample dispersion pad and the nucleic acid preservation pad are placed in the sample preservation chamber, and the drying block is placed in the drying chamber. The sample preservation chamber and the drying chamber are connected by a gas channel. This invention effectively solves the edge effect problem of traditional nucleic acid preservation cards through chemical modification, ensuring the uniformity of nucleic acid preservation throughout the sample area. Simultaneously, the integrated active drying system can complete sample drying in a short time, greatly improving the convenience and operability of the product in practical applications.

Description

Technical Field

[0001] This invention belongs to the technical field of sample collection devices, specifically relating to a nucleic acid preservation card, its preparation method, and its usage method. Background Technology

[0002] Nucleic acid testing plays a crucial role in many fields, including biomedical research, disease diagnosis, and epidemic prevention and control. Proper preservation of nucleic acid samples throughout the entire process, from collection to final testing, is of paramount importance. Nucleic acid preservation cards were developed in this context.

[0003] In the early days, traditional methods of preserving nucleic acid samples faced numerous challenges. Nucleic acids in samples such as blood, saliva, and tissue fluid are highly susceptible to degradation by nucleases, oxidants, and ultraviolet light under natural conditions. Furthermore, the growth of bacteria and other microorganisms accelerates nucleic acid degradation, making it difficult to preserve samples stably for extended periods. Especially during field sampling, testing in remote areas, and large-scale screening, traditional methods rely on cold chain transportation and low-temperature storage, which are demanding in terms of equipment, expensive, and inconvenient to operate, severely limiting the scope of nucleic acid testing.

[0004] With the continuous advancement of technology, researchers have developed nucleic acid preservation cards through extensive research and experimentation. These cards are typically made of specially formulated filter paper, whose fiber matrix is ​​soaked in a patented formula of powerful denaturing agents and chelating agents, containing special chemical substances. This ingenious design allows nucleic acids to be stably fixed, protected from damage by nucleases, oxidants, and ultraviolet light, and effectively inhibits the growth of bacteria and other microorganisms, thus maintaining the integrity of the nucleic acids. However, some existing nucleic acid preservation cards typically use ordinary cellulose soaked in a corresponding solution to preserve nucleic acids. In actual use, when the fibers come into contact with the filter paper, the solution undergoes a process of reconstitution and re-drying. Especially in the central area where the sample is added, the solution concentration is significantly reduced due to the edge effect, thus affecting the nucleic acid preservation effect in that area. This invention chemically modifies the filter paper used in the nucleic acid preservation card, coupling it with a protein denaturing agent through covalent bonds. Filter paper treated by this method effectively avoids the edge effect of the solution during sample addition, resulting in more uniform nucleic acid samples during testing. Meanwhile, this invention also solves the drying problem of blood collection cards, nucleic acid preservation cards, etc. This process typically requires natural drying at room temperature for more than 4 hours, which greatly increases the complexity of the operation and thus affects the user experience of nucleic acid preservation cards. This invention effectively solves this problem by integrating a drying module into the collection card. Summary of the Invention

[0005] Based on the problems existing in the background technology, the present invention provides a new nucleic acid preservation card technology, which can solve the problem of uneven preservation caused by edge effect, and can also achieve rapid drying, improve the convenience of operation and the stability of preservation effect.

[0006] This invention is implemented through the following technical solutions:

[0007] The first aspect of this invention discloses a nucleic acid preservation card, comprising a card body, a cover plate, a sample dispersion pad, a nucleic acid preservation pad, and a drying block;

[0008] The nucleic acid preservation pad is made of chemically modified cellulose filter paper with a thickness of 0.4–4 mm;

[0009] The card body is provided with a sample preservation chamber and a drying chamber. The sample dispersion pad and nucleic acid preservation pad are placed in the sample preservation chamber, and the drying block is placed in the drying chamber.

[0010] The sample preservation chamber and the drying chamber are connected by a gas channel.

[0011] Furthermore, the preparation method of the nucleic acid preservation pad includes the following steps:

[0012] a) Prepare a 0.1M MES solution and adjust the pH to 5-6 with HCl;

[0013] b) Add guanidine acetate to a concentration of 1 g / 100 ml, and heat to 60–80 °C to dissolve it completely;

[0014] c) Add N-hydroxysuccinimide (NHS) to a final concentration of 100 mmol / L;

[0015] d) Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to a final concentration of 1 mol / L, and react for 60–90 min;

[0016] e) Adjust the pH to 6-7 using dilute sodium hydroxide;

[0017] f) Immerse the cellulose filter paper in the solution from step e overnight;

[0018] g) Remove the filter paper and squeeze it until it is semi-dry;

[0019] h) Spraying with a solution containing additives;

[0020] i) Dry at 80±10℃.

[0021] Further, in step h), the solution containing the adjuvant contains 2%–5% EDTA-2Na, 5%–10% SDS, 1%–5% vitamin C, and 5%–10% glutathione, and the spraying volume is 30–50 μL / cm. 2.

[0022] Furthermore, the drying block is prepared by combining cellulose with at least one desiccant selected from calcium chloride, magnesium chloride, and calcium oxide; the drying block and drying chamber can be in multiple sets, and the gas channel can be multiple.

[0023] Furthermore, the sample dispersion pad is a hydrophilic porous material, specifically any one of cellulose acetate, cellulose nitrate, glass fiber, and polyester fiber, with an average pore size of 1–50 μm and a thickness of 0.1–0.5 mm.

[0024] Furthermore, the cover plate is provided with a sample dispensing hole, which is located above the sample dispersion pad.

[0025] Furthermore, it also includes a sealing sticker that covers the sample application hole of the cover plate; the sealing sticker can be reused and forms a seal.

[0026] The second aspect of this invention discloses a method for using a nucleic acid preservation card, comprising the following steps:

[0027] Add the nucleic acid sample to the sample well of the nucleic acid preservation card;

[0028] After the sample is absorbed by the nucleic acid preservation pad, the sample is actively dried by the drying block through the gas channel;

[0029] Seal and store the item in a sealed container after applying the sealing tape.

[0030] Furthermore, the amount of nucleic acid sample added to each sample addition unit shall not exceed 500 μL, and the drying time shall not exceed 4 hours.

[0031] Furthermore, the method also includes a nucleic acid extraction step, specifically:

[0032] Remove the top cover and take out the nucleic acid preservation pad containing the sample;

[0033] Use a special punch to cut eight circular pieces with a diameter of 5-8mm from the filter paper.

[0034] Place the prepared discs into centrifuge tubes and add nucleic acid extraction reagents for nucleic acid extraction.

[0035] The beneficial effects of this invention are:

[0036] 1. This invention fundamentally solves the edge effect problem existing in the prior art by chemically modifying the cellulose carrier and using an NHS and EDC crosslinking system to covalently couple the protein denaturant to the cellulose. Compared with the traditional simple soaking process, covalent coupling ensures the stable immobilization of the protein denaturant on the cellulose carrier, avoiding uneven concentration distribution caused by reagent redissolution and re-drying during sample loading. This improvement makes the nucleic acid preservation effect uniform throughout the entire sample area, significantly improving the reliability and reproducibility of nucleic acid preservation.

[0037] 2. This invention integrates an active drying system. By incorporating a drying chamber, drying block, and gas channel within the card body, it achieves active drying of the loaded sample. Compared to traditional nucleic acid preservation cards that require more than 4 hours of natural air drying, this invention can complete sample drying in a much shorter time, achieving a drying efficiency of over 95%. This active drying mechanism not only significantly shortens operation time and improves work efficiency but also reduces the risk of sample contamination or degradation during the drying process, greatly enhancing the product's convenience and operability in practical applications. Attached Figure Description

[0038] The accompanying drawings are provided to further explain the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0039] Figure 1 This is a schematic diagram showing the exploded structure of the nucleic acid preservation card of the present invention;

[0040] In the diagram: 1. Card body; 2. Cover plate; 3. Sealing sticker; 4. Sample dispensing hole; 5. Sample dispersion pad; 6. Nucleic acid preservation pad; 7. Drying block; 8. Drying chamber; 9. Gas channel; 10. Sample preservation chamber. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0042] Example 1

[0043] A nucleic acid preservation card, such as Figure 1 As shown, it includes card body 1, cover plate 2, sample dispersion pad 5, nucleic acid preservation pad 6, and drying block 7;

[0044] The nucleic acid preservation pad 6 is made of chemically modified cellulose filter paper with a thickness of 1 mm;

[0045] The card body 1 is provided with a sample preservation chamber 10 and a drying chamber 8. The sample dispersion pad 5 and the nucleic acid preservation pad 6 are arranged in the sample preservation chamber 10, and the drying block 7 is arranged in the drying chamber 8.

[0046] The sample preservation chamber 10 and the drying chamber 8 are connected by a gas channel 9.

[0047] The drying block 7 is prepared by combining cellulose with at least one desiccant selected from calcium chloride, magnesium chloride, and calcium oxide; the drying block 7 and the drying chamber 8 can be in multiple sets, and the gas channel 9 can be in multiple lines.

[0048] The sample dispersion pad was a nitrocellulose membrane with an average pore size of 25 μm and a thickness of 0.3 mm.

[0049] The cover plate 2 is provided with a sample dispensing hole 4, which is located above the sample dispersion pad 5.

[0050] It also includes a sealing sticker 3, which covers the sample application hole 4 of the cover plate 2; the sealing sticker 3 can be reused and forms a seal.

[0051] The preparation method of the nucleic acid preservation pad 6 includes the following steps:

[0052] a) Prepare a 0.1M MES solution and adjust the pH to 5.5 with HCl;

[0053] b) Add guanidine acetate to a concentration of 1 g / 100 ml, and heat to 70 °C to dissolve completely;

[0054] c) Add N-hydroxysuccinimide to a final concentration of 100 mmol / L;

[0055] d) Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to a final concentration of 1 mol / L, and react for 60–90 min;

[0056] e) Adjust the pH to 6.5 using dilute sodium hydroxide;

[0057] f) Immerse the cellulose filter paper in the solution from step e overnight;

[0058] g) Remove the filter paper and squeeze it until it is semi-dry;

[0059] h) Spray a solution containing adjuvants, wherein the solution contains 3.5% EDTA-2Na, 7.5% SDS, 3% Vitamin C, and 7.5% glutathione, at a spraying volume of 40 μL / cm. 2 ;

[0060] i) Dry at 80±10℃.

[0061] Experimental Example 1

[0062] The nucleic acid preservation ability in plasma was tested using a nucleic acid preservation card prepared from the nucleic acid preservation filter paper of Example 1.

[0063] Sample preparation:

[0064] A: Using fresh whole blood samples from healthy individuals, SARS-CoV-2 genomic nucleic acid was added to prepare SARS-CoV-2 nucleic acid-positive whole blood samples. 500 μL of this sample was added dropwise to a blood collection card treated with a patented method. After air-drying at room temperature for 4 hours, the sample was sealed in an aluminum foil bag and placed in a 37°C incubator. Testing was conducted one week later. For testing, a 6 mm diameter disc was taken from both the central and peripheral regions using a punch, placed in a 1.5 mL conical centrifuge tube, and 500 μL of nucleic acid extraction reagent was added. The tube was vortexed thoroughly for approximately 1 minute. Subsequent nucleic acid extraction and PCR testing were performed, and the CT values ​​were recorded as A1 and A2. This was repeated three times, and the results were recorded as: A1-1, A1-2, A1-3; A2-1, A2-2, A2-3.

[0065] B: Using fresh whole blood samples from healthy individuals, SARS-CoV-2 genomic nucleic acid was added to prepare SARS-CoV-2 nucleic acid-positive whole blood samples. 500 μL of this sample was added dropwise to a blood collection card prepared from blank, untreated filter paper. After drying at room temperature for 4 hours, the sample was sealed in an aluminum foil bag and placed in a 37°C incubator. Testing was conducted one week later. For testing, a 6 mm diameter disc was taken from both the central and peripheral regions using a punch, placed in a 1.5 mL conical centrifuge tube, and 500 μL of nucleic acid extraction reagent was added. The tube was vortexed thoroughly for approximately 1 minute. Subsequent nucleic acid extraction and PCR testing were performed, and the CT values ​​were recorded as B1 and B2. This was repeated three times, and the results were recorded as: B1-1, B1-2, B1-3; B2-1, B2-2, B2-3.

[0066] The nucleic acid test results are shown in Table 1 (A0 and B0 are the test results before the product was dried and stored at 37 degrees Celsius).

[0067] Table 1: Nucleic Acid Test Results

[0068] Group CtORF1ab CtN Group CtORF1ab CtN A0 25.17 20.06 B0 25.35 21.24 A1-1 25.12 20.11 B1-1 37.16 35.61 A1-2 25.36 20.25 B1-2 35.24 33.60 A1-3 25.31 20.30 B1-3 37.62 33.56 A2-1 25.82 20.19 B2-1 34.35 32.85 A2-2 25.91 20.28 B2-2 35.63 33.26 A2-3 25.07 20.64 B2-3 37.28 32.07

[0069] Table 1 shows that the nucleic acid preservation effect of the detection card prepared in Example 1 of this invention is significantly better than that of the untreated detection card, and no edge effect of the sample occurs. The detection results of the central sample and the edge sample are basically consistent.

[0070] Experimental Example 2

[0071] The drying ability was tested using nucleic acid preservation cards prepared with nucleic acid preservation filter paper from Example 1.

[0072] Sample preparation:

[0073] A: Use pure water as a simulated sample and add it dropwise to the nucleic acid preservation card at a volume of 500μL. After sealing for 72 hours, measure the weight and calculate the overall mass difference between the sample dispersion pad 5 and the nucleic acid preservation pad 6 before and after sample addition, and calculate the drying efficiency.

[0074] Table 2. Drying Efficiency of Nucleic Acid Preservation Cards

[0075]

[0076]

[0077] Finally, it should be noted that the above embodiments are merely illustrative of several implementations of the present invention and are not intended to limit the scope of the invention. For those skilled in the art, any modifications, equivalent substitutions, or improvements made without departing from the concept of the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A nucleic acid preservation card, comprising a card body (1), a cover plate (2), a sample dispersion pad (5), a nucleic acid preservation pad (6), and a drying block (7), characterized in that: The nucleic acid preservation pad (6) is made of chemically modified cellulose and has a thickness of 0.4~4 mm; The card body (1) is provided with a sample preservation chamber (10) and a drying chamber (8). The sample dispersion pad (5) and the nucleic acid preservation pad (6) are placed in the sample preservation chamber (10), and the drying block (7) is placed in the drying chamber (8). The sample preservation chamber (10) and the drying chamber (8) are connected by a gas channel (9); The preparation method of the nucleic acid preservation pad (6) includes the following steps: a) Prepare a 0.1M MES solution and adjust the pH to 5-6 with HCl; b) Add guanidine acetate to a concentration of 1 g / 100 ml, and heat to 60-80°C to dissolve it completely; c) Add N-hydroxysuccinimide to a final concentration of 100 mmol / L; d) Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to a final concentration of 1 mol / L, and react for 60-90 min; e) Adjust the pH to 6-7 using dilute sodium hydroxide; f) Immerse the cellulose filter paper in the solution from step e overnight; g) Remove the filter paper and squeeze it until it is semi-dry; h) Spraying with a solution containing additives; i) Dry at 80±10℃.

2. The nucleic acid preservation card according to claim 1, characterized in that, The solution containing the adjuvant in step h) contains 2%~5% EDTA-2Na, 5%~10% SDS, 1%~5% Vitamin C, and 5%~10% glutathione, and the spraying volume is 30~50 μL / cm. 2 .

3. The nucleic acid preservation card according to claim 1, characterized in that, The drying block (7) is prepared by combining cellulose with at least one of the following desiccants: calcium chloride, magnesium chloride, and calcium oxide; the drying block (7) and the drying chamber (8) can be in multiple sets, and the gas channel (9) can be in multiple lines.

4. The nucleic acid preservation card according to claim 1, characterized in that, The sample dispersion pad is a hydrophilic porous material with an average pore size of 1~50μm and a thickness of 0.1~0.5mm.

5. The nucleic acid preservation card according to claim 1, characterized in that, The cover plate (2) is provided with a sample addition hole (4), which is located above the sample dispersion pad (5).

6. The nucleic acid preservation card according to claim 1, characterized in that, It also includes a sealing sticker (3) that covers the sample feeding hole (4) of the cover plate (2); the sealing sticker (3) can be reused and forms a seal.

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