A device and method for long-term storage of DNA based on filter paper adsorption

Abstract

The application discloses a DNA long-term storage device and method based on filter paper adsorption and belongs to the technical field of clinical pathological detection. The device comprises a filter paper carrier, a storage isolation component and an operation auxiliary tool. The filter paper carrier is A4 size high-purity cellulose filter paper, is divided into a square matrix, each square corresponds to one DNA sample and is labeled with a unique sample number, and a batch information filling area is arranged at the top. The storage isolation component comprises a single-layer plastic membrane and an archive folder. The operation auxiliary tool comprises a disposable sterile blade, a micro pipette and an elution tube. The matching method comprises a storage method and an elution method. Fast sample drying is realized by relying on the strong water absorption of the filter paper, pollution isolation is realized by wrapping the double-layer plastic membrane, and long-term storage can be realized in a low-temperature environment of-20 DEG C or-80 DEG C. The application has the advantages of significant improvement of space utilization, simple operation, low cost, stable storage effect, realization of sample accurate traceability and adaptation to the large-scale and low-cost archiving demand of the pathology department.

Description

Technical Field

[0001] This invention relates to the field of clinical pathology testing technology, and in particular to a long-term DNA storage device and method based on filter paper adsorption. Background Technology

[0002] In the field of clinical pathology testing, long-term DNA preservation is a routine procedure to ensure sample retesting, secondary applications, and scientific research retrospection. Current technologies mostly use centrifuge tube cryopreservation, which involves storing extracted DNA samples in a -20°C or -80°C freezer. The preservation period is usually 3 years or more.

[0003] This traditional technical solution has significant drawbacks, as follows: 1. Large space occupation: Each DNA sample requires an independent centrifuge tube. As the storage period increases, the volume requirement of the low-temperature freezer far exceeds the available area of ​​the molecular laboratory, which puts great pressure on site and equipment resources and is not conducive to large-scale sample archiving. 2. Cumbersome operation process: Frequent opening and closing of the low-temperature freezer is required during freezing, retrieval and retesting, which not only increases the operating cost, but also easily leads to sample contamination and affects the accuracy of subsequent test results; 3. High cost: Although existing commercial nucleic acid preservation cards (such as FTA cards) can achieve room temperature preservation, they are expensive and difficult to meet the needs of pathology departments for large-scale, low-cost long-term archiving, especially unsuitable for primary healthcare institutions. 4. Poor preservation stability: Traditional cryopreservation methods are highly dependent on the cold chain. Once the temperature control is interrupted, DNA degradation is very likely to occur, which will affect the accuracy of subsequent tests and cannot guarantee the long-term effective use of the sample.

[0004] Therefore, this invention proposes a long-term DNA storage solution that is space-efficient, easy to operate, low-cost, and has stable preservation effects, in order to solve the above-mentioned problems existing in the prior art. Summary of the Invention

[0005] To address the shortcomings of existing DNA long-term storage technologies, such as large space occupation, cumbersome operation, high cost, and poor preservation stability, this invention provides a DNA long-term storage device and method based on filter paper adsorption, which achieves efficient, low-cost, and stable long-term storage of DNA samples, while simplifying the operation process, improving space utilization, and adapting to the large-scale archiving needs of clinical pathology departments.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A long-term DNA storage device based on filter paper adsorption includes a filter paper carrier, a storage and isolation component, and operating aids. The filter paper carrier is a high-purity cellulose filter paper. The filter paper carrier is divided into an m×n square matrix by an oil-based water-resistant pen. The spacing between the squares is set to avoid smudging and contamination after the sample is added. Each square corresponds to one DNA sample. A unique sample number is marked on the edge of the square. A batch information filling area is set on the top of the filter paper carrier. The storage and isolation component includes a plastic diaphragm of the same size as the filter paper carrier and an archive folder. The plastic diaphragm is used to wrap the upper and lower layers of the filter paper carrier, and the archive folder is used to hold the wrapped filter paper carrier and stack them in batches in an orderly manner. A label area is pasted on the outside of the archive folder for marking archive information. The operational aids include a disposable sterile blade, a micropipette, and an elution tube. The disposable sterile blade is used to cut the target grid filter paper, the micropipette is used to add the DNA sample, and the elution tube is used for eluting the DNA sample.

[0007] Furthermore, the high-purity cellulose filter paper is selected from Whatman Grade 1 high-purity cellulose filter paper.

[0008] Furthermore, the batch information filling area on the top of the filter paper carrier includes the sample batch, storage date, and operator information.

[0009] A method for long-term DNA storage based on filter paper adsorption includes the following steps: S1: Sample addition and adsorption: Adjust the concentration of the extracted DNA sample to ≥10ng / μL, and use a micropipette to add 50-100μL of DNA sample to the corresponding squares on the filter paper carrier. The sample is quickly adsorbed by relying on the strong water absorption of the filter paper carrier, ensuring that the sample does not spread over a large area. S2: Sample drying: Place the filter paper carrier with the DNA sample added to it in a room temperature environment to dry naturally for 3-5 minutes until there is no liquid residue in the sample area; S3: Wrapping and Storage: Take two single-layer plastic diaphragms of the same size as the filter paper carrier. Place the dried filter paper carrier in the center of one of the plastic diaphragms, and then cover the filter paper carrier with the other plastic diaphragm to complete the double-layer diaphragm wrapping. Place the wrapped filter paper carrier into the archive folder, stack them in batches in an orderly manner, label the relevant information in the label area on the outside of the archive folder, and then store them in a low-temperature environment of -20℃ or -80℃ for standardized archiving.

[0010] Furthermore, in step 1, the amount of DNA sample added is 80 μL, and in step 2, the air drying time at room temperature is 4 minutes.

[0011] A DNA elution method based on filter paper adsorption is used to elute DNA samples. The specific steps are as follows: Take out the corresponding packaged filter paper carrier and single-layer plastic septum from the file folder, peel off the upper single-layer plastic septum, cut the grid filter paper corresponding to the target sample with a disposable sterile blade, put the cut grid filter paper into an elution tube containing 100-200μL of distilled water, soak at room temperature for 5 minutes and gently shake, and the DNA liquid that can be directly used for subsequent molecular gene detection can be obtained by elution.

[0012] Furthermore, the amount of distilled water used in the elution tube is 150 μL, and it is gently shaken 3 times during the room temperature soaking process.

[0013] The beneficial effects of this invention are as follows: 1. Significantly improved space utilization: A single A4-sized filter paper carrier can stably preserve approximately 60 DNA samples, occupying less than 1 / 50 of the space required for traditional centrifuge tube cryopreservation. This significantly saves on the volume of low-temperature freezers and laboratory space resources, effectively solving the space pressure problem of traditional cryopreservation methods and meeting the needs of large-scale sample archiving.

[0014] 2. Simple and quick operation: The adsorption, drying and preservation process of samples does not require complicated equipment and the operation steps are simple and easy to understand; when retesting, only the corresponding square filter paper needs to be cut to complete the elution, without the need to frequently open and close the low temperature freezer. This not only simplifies the operation process, but also reduces the risk of sample contamination and significantly improves the daily work efficiency of the pathology department.

[0015] 3. Stable and reliable preservation effect: DNA samples preserved by the method of this invention show that the retest results are completely consistent with the initial test in common pathological tests such as HPV genotyping and BRAFV600E mutation detection, with no decrease in specificity or accuracy; and the integrity of DNA can be guaranteed in both room temperature and low temperature environments, avoiding the strong dependence of traditional cryopreservation on the cold chain and reducing the risk of DNA degradation.

[0016] 4. Cost-effective and easy to promote: Using ordinary high-purity cellulose filter paper instead of expensive special nucleic acid preservation cards greatly reduces the storage cost per sample, making it inexpensive; the device has a simple structure, is easy to operate, and is suitable for the large-scale sample archiving needs of pathology departments, making it especially suitable for promotion and application in primary healthcare institutions.

[0017] 5. Convenient and standardized archiving management: The unique sample number is marked on the grid of the filter paper carrier, which, together with the batch information filling area at the top and the label on the outside of the archive folder, enables accurate traceability of the sample; the stacking and storage of archive folders and the double-layer membrane isolation between filter papers conform to the laboratory's routine management habits, facilitates the classification, retrieval and archiving of samples, and improves the standardization of sample management. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the DNA long-term storage method based on filter paper adsorption according to the present invention; Figure 2 This is a schematic diagram of the DNA elution method based on filter paper adsorption according to the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0021] A long-term DNA storage device based on filter paper adsorption includes a filter paper carrier 1, a storage and isolation component, and operating aids. These components work together to achieve safe and orderly storage of DNA samples. 1. Filter Paper Carrier 1: High-purity cellulose filter paper is selected, preferably Whatman Grade 1 high-purity cellulose filter paper, in A4 size for easy routine laboratory operations and storage. Divided into a 6×10 grid matrix using an oil-based water-resistant pen, the grid spacing is carefully controlled to effectively prevent cross-contamination caused by sample spreading after addition. Each square corresponds to one DNA sample, and a unique sample number is marked on the edge of each square to ensure accurate sample identification. A batch information area is provided at the top of Filter Paper Carrier 1 for labeling sample batch, storage date, operator, etc., facilitating sample traceability and management.

[0022] 2. Storage and Isolation Components: These include a single-layer plastic diaphragm 2 of the same size as the filter paper carrier 1 and a file folder 3. The single-layer plastic diaphragm 2 is used to double-wrap the filter paper carrier 1, covering both the top and bottom of the filter paper carrier 1 with a single-layer plastic diaphragm 2, which can completely isolate external contamination and avoid cross-contamination between different filter paper carriers 1. The file folder 3 is used to hold the wrapped filter paper carriers 1, allowing them to be stacked in an orderly manner by batch, saving storage space. The outer label area of ​​the file folder 3 is used to mark key information such as batch number, storage date, and storage conditions, facilitating quick sample retrieval.

[0023] 3. Operational aids: including disposable sterile blades 4, micropipettes, and elution tubes. Disposable sterile blades 4 are used to cut the target grid filter paper during retesting to avoid cross-contamination; micropipettes are used to accurately add DNA samples and control the amount added; elution tubes are used for eluting DNA samples, providing a carrier for subsequent testing.

[0024] A method for long-term DNA storage based on filter paper adsorption, using the above-mentioned storage device, includes the following specific storage steps: Step 1: Sample Addition and Adsorption: Adjust the concentration of the extracted DNA sample to ≥10 ng / μL to ensure that the sample concentration meets the requirements for long-term storage. Using a micropipette, slowly add 50-100 μL of DNA sample to the corresponding square of filter paper carrier 1. Utilizing the strong absorbency of the high-purity cellulose filter paper, rapid sample adsorption is achieved while ensuring that the sample does not spread over a large area, avoiding cross-contamination between samples in different squares. Preferably, an 80 μL DNA sample addition volume yields better adsorption results.

[0025] Step 2: Sample Drying: Place the filter paper carrier 1 with the added DNA sample on the lab bench at room temperature to air dry for 3-5 minutes, until no liquid residue remains on the sample area. This ensures the DNA sample is stably adsorbed onto the filter paper and avoids sample degradation or contamination caused by liquid residue. Preferably, the drying time is 4 minutes, balancing drying efficiency and sample stability.

[0026] Step 3: Wrapping and Storage: Take two single-layer plastic diaphragms 2 of the same size as the filter paper carrier 1. Place the dried filter paper carrier 1 in the center of one of the single-layer plastic diaphragms 2, and then cover the filter paper carrier 1 with the other single-layer plastic diaphragm 2 to complete the double-layer diaphragm wrapping, achieving complete isolation of the filter paper carrier 1. Place the wrapped filter paper carrier 1 into the file folder 3, stacking them in an orderly manner according to batch. Label the batch, storage date, storage conditions (-20℃ or -80℃) and other information on the outer label area of ​​the file folder 3. Then place the file folder 3 in the corresponding low-temperature environment for standardized archiving, completing the long-term storage of the DNA sample.

[0027] A DNA elution method based on filter paper adsorption, when retesting or conducting secondary experiments on stored DNA samples, employs the following elution steps: Take the corresponding batch of wrapped filter paper and single-layer plastic diaphragm 2 from the file folder 3; gently peel off the upper single-layer plastic diaphragm 2; precisely cut the square filter paper corresponding to the target sample using a disposable sterile blade 4, avoiding contact with other squares to prevent contamination; place the cut square filter paper into an elution tube containing 100-200 μL of distilled water, soak at room temperature for 5 minutes with gentle shaking to fully elute the DNA adsorbed on the filter paper into the distilled water, thus obtaining DNA liquid that can be directly used for subsequent experiments such as molecular gene detection. Preferably, the volume of distilled water in the elution tube is 150 μL, and gentle shaking 3 times during room temperature soaking ensures more thorough elution.

[0028] Example 1: Low-temperature preservation of HPV-positive DNA samples at -20℃ 1. Preparation of filter paper carrier 1: Select A4 size Whatman Grade 1 high-purity cellulose filter paper, and divide it into a 6×10 square matrix evenly with an oil-based water-resistant pen. The spacing between the squares should be reasonably controlled to avoid smudging and contamination after the sample is added. Mark the sample number S01-S60 on the edge of each square, and mark the batch information area at the top of the filter paper as HPV001 and the operator as XXX.

[0029] 2. Sample addition and adsorption: 60 HPV-positive cervical exfoliated cell DNA samples were collected, and the concentration of all samples was uniformly adjusted to 50 ng / μL. 80 μL of each sample was taken using a micropipette and slowly added to the corresponding numbered square. The strong absorbency of the filter paper enabled rapid adsorption, ensuring that the sample did not spread over a large area.

[0030] 3. Sample drying: Place the filter paper after dripping in the laboratory table and let it air dry at room temperature for 4 minutes to confirm that there is no liquid residue in the sample area.

[0031] 4. Wrapping and Storage: Take two single-layer plastic diaphragms 2 of the same size as the filter paper. Place the dried filter paper in the center of one of the diaphragms, and then cover the filter paper with the other diaphragm to complete the double-layer plastic diaphragm wrapping. Place the wrapped filter paper into the file folder 3, stack them in an orderly manner according to batch, and label the outside of the folder with batch HPV001, storage date, and storage conditions -20℃. Place the file folder 3 in a -20℃ low-temperature freezer for standardized archiving and retest after 12 months.

[0032] 5. Sample elution and detection: During the retest, the corresponding filter paper and septum were taken out from the file folder 3, the upper plastic septum 2 was peeled off, and the grid filter paper corresponding to the sample to be tested was cut with a disposable sterile blade 4. The sample was placed in an elution tube containing 150 μL of distilled water, soaked at room temperature for 5 minutes and gently shaken 3 times to complete the DNA elution. The elution solution was used for HPV genotyping detection. The detection results were completely consistent with the initial detection, with no missed detections, misjudgments, or cross-contamination, proving that the preservation effect of this invention is stable and reliable.

[0033] General Implementation Steps During routine DNA archiving in the pathology department, the operator prepares corresponding filter paper carriers 1 according to the sample batch, controls the grid spacing, and adds the extracted and adjusted DNA sample to the corresponding grid as required. Relying on the strong water absorption of the filter paper, it is allowed to dry naturally at room temperature for 3-5 minutes. After drying, two single-layer plastic diaphragms 2 of the same size as the filter paper are taken and wrapped with a single filter paper in the manner of "bottom layer padding + top layer covering". The wrapped filter papers are then placed in the archive folder 3 in an orderly manner according to the batch, and the archive is standardized in a low-temperature environment of -20℃ or -80℃ according to the preservation requirements.

[0034] When retesting or secondary experiments are required, quickly locate the corresponding batch of packaged filter paper according to the label on the outside of the file folder 3. After peeling off the upper plastic diaphragm 2, cut the target square filter paper with a disposable sterile blade 4 and proceed with the elution method described above to obtain usable DNA liquid. The used filter paper is disposed of in accordance with medical waste regulations. The single-layer plastic diaphragm 2 and the file folder 3 can be reused after sterilization, further reducing usage costs.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A long-term DNA storage device based on filter paper adsorption, characterized in that: Includes filter paper carrier (1), storage and isolation components, and operation aids; The filter paper carrier (1) is a high-purity cellulose filter paper. The filter paper carrier (1) is divided into an m×n square matrix by an oil-based water-resistant pen. The spacing between the squares is set to avoid the sample from spreading and contaminating after being dropped. Each square corresponds to one DNA sample. The edge of the square is marked with a unique sample number. A batch information filling area is set on the top of the filter paper carrier (1). The storage and isolation assembly includes a plastic diaphragm (2) of the same size as the filter paper carrier (1) and an archive folder (3). The plastic diaphragm (2) is used to wrap the upper and lower layers of the filter paper carrier (1). The archive folder (3) is used to hold the wrapped filter paper carrier (1) and stack it in batches in an orderly manner. The outer side of the archive folder (3) has a label area for marking archive information. The operation aids include a disposable sterile blade (4), a micropipette and an elution tube. The disposable sterile blade (4) is used to cut the target grid filter paper, the micropipette is used to add DNA samples, and the elution tube is used for eluting DNA samples.

2. The DNA long-term storage device based on filter paper adsorption according to claim 1, characterized in that, The high-purity cellulose filter paper used is Whatman Grade 1 high-purity cellulose filter paper.

3. The DNA long-term storage device based on filter paper adsorption according to claim 1, characterized in that, The batch information filling area at the top of the filter paper carrier (1) includes the sample batch, storage date and operator information.

4. A method for long-term DNA storage based on filter paper adsorption, characterized in that, Using the storage device according to any one of claims 1-3, Includes the following steps: S1: Sample addition and adsorption: Adjust the concentration of the extracted DNA sample to ≥10ng / μL, and use a micropipette to add 50-100μL of DNA sample to the corresponding square of the filter paper carrier (1). Relying on the strong water absorption of the filter paper carrier (1), the sample is quickly adsorbed, ensuring that the sample does not spread over a large area. S2: Sample drying: Place the filter paper carrier (1) with the DNA sample added into the filter paper carrier at room temperature and let it dry naturally for 3-5 minutes until there is no liquid residue in the sample area; S3: Wrapping and Storage: Take two single-layer plastic diaphragms (2) of the same size as the filter paper carrier (1), place the dried filter paper carrier (1) in the center of one of the plastic diaphragms (2), and then cover the filter paper carrier (1) with the other plastic diaphragm (2) to complete the double-layer diaphragm wrapping; insert the wrapped filter paper carrier (1) into the file folder (3), stack them in batches in an orderly manner, mark the relevant information in the label area on the outside of the file folder (3), and then store them in a low temperature environment of -20℃ or -80℃ for standardized archiving.

5. The method for long-term DNA storage based on filter paper adsorption according to claim 4, characterized in that, In step 1, the amount of DNA sample added was 80 μL, and in step 2, the air drying time at room temperature was 4 minutes.

6. A DNA elution method based on filter paper adsorption, characterized in that, The specific steps for eluting DNA samples stored by the method of claim 4 or 5 are as follows: Take out the corresponding packaged filter paper carrier (1) and single-layer plastic diaphragm (2) from the file folder (3), peel off the upper single-layer plastic diaphragm (2), cut the grid filter paper corresponding to the target sample with a disposable sterile blade (4), put the cut grid filter paper into an elution tube containing 100-200 μL of distilled water, soak at room temperature for 5 minutes and shake gently to obtain DNA liquid that can be directly used for subsequent molecular gene detection.

7. The DNA elution method based on filter paper adsorption according to claim 6, characterized in that, The amount of distilled water used in the elution tube is 150 μL, and it is gently shaken 3 times during the room temperature soaking process.

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