Biological sample pretreatment method and glass slide
By using slides with adhesion and transparent layers combined with microscope labeling and dewaxation treatment, the accuracy of nucleic acid sample extraction in formalin-fixed paraffin-embedded sample sections was solved, and efficient target cell enrichment and high-quality nucleic acid extraction were achieved, improving the accuracy of detection.
Patent Information
- Application Number
- CN202510461363.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-14
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, when extracting nucleic acid samples from specific cells from formalin-fixed paraffin-embedded sample sections, there are problems such as poor accuracy, easy to lead to false negative results, relying on manual experience, and easy to cause cross-contamination.
Using slides including an adhesive layer and a transparent layer, the target area was marked by a microscope, and the target area was cut and separated by an adhesive layer. The target area was treated with a dewax agent and lysis buffer to obtain a high-purity target nucleic acid sample.
The target area of sample sections is achieved more accurately, reducing tissue residues and cross-contamination, improving DNA yield and detection accuracy, especially for samples with low tumor cell content, and improving the frequency of tumor mutation detection.
Smart Images
Figure CN120253380A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological sample processing, and particularly to a method for preprocessing biological samples and a glass slide. Background Art
[0002] Formalin-fixed paraffin-embedded (FFPE) sample sections are a commonly used sample type in clinical pathological diagnosis, with the advantages of simple operation and easy preservation. Currently, tumor gene targeted sequencing based on FFPE sections has become an important means for accurate tumor diagnosis, providing an important basis for subsequent treatment decisions and prognosis evaluation.
[0003] Currently, high-throughput sequencing technology (NGS, Next-Generation Sequencing) can usually reliably detect mutations with an allele frequency above 5%. This means that when the proportion of tumor cells is less than 10%, false negative results may occur; in FFPE sections, tumor tissue usually coexists with a large amount of normal tissue, such as tumor-infiltrating lymphocytes (TILs), nerves, blood vessels, and fibroblasts. These normal cells dilute the proportion of tumor cells, increasing the difficulty of detecting tumor cells. Especially for samples with a low content of tumor cells, the detection sensitivity of tumor-specific mutations is greatly reduced.
[0004] To solve this problem, clinically, tumor enrichment is often performed on FFPE sections of tumor tissue, and the tumor tissue is extracted for selective detection to avoid the influence of surrounding normal tissue and obtain more accurate detection results. The blade scraping method is the most commonly used tumor enrichment technique in clinical practice. It is simple to operate and has a low cost. Its steps include using a HE-stained (Hematoxylin-Eosin Staining) section marked with the tumor area as a reference, and using a surgical blade to scrape the tumor area from an unstained glass slide to physically separate tumor cells from normal tissue. However, this method depends on the experience of the operator, and can only roughly scrape the tumor area in the section by visually referring to the marked area of the HE-stained section, unable to accurately separate tumor from normal tissue, and the enrichment effect of tumor cells is not ideal. In addition, wax debris scattered during the scraping process may cause cross-contamination between samples, resulting in false positive detection results; static electricity effects may also occur during the scraping process, and some samples adhere to the blade and glass slide, resulting in sample loss. Due to the above factors, for small-volume samples such as biopsy or puncture samples, the accuracy of nucleic acid samples extracted using the blade scraping method is poor, and there is a risk of sequencing failure due to too low total DNA extraction. Summary of the Invention
[0005] The present invention provides a method for preprocessing biological samples and a glass slide, which solves the technical problem of poor accuracy in extracting nucleic acid samples of specific cells from sample sections.
[0006] In a first aspect of the present invention, a method for preprocessing biological samples is provided, including:
[0007] Adhering a first sample section to a first glass slide; the first glass slide includes an adhesion layer and a transparent layer, and the surface of the adhesion layer includes a first surface for detachably bonding with the surface of the transparent layer and a second surface for adhering the sample section.
[0008] Placing a second glass slide carrying a stained section with a marked target area below the first glass slide, and making the projection of the stained section and the first sample section coincide in the normal direction.
[0009] Cutting the adhesion layer according to the marks on the stained section to obtain a target adhesion part with the target area of the first sample section adhered thereto.
[0010] Performing impurity removal treatment on the target adhesion part to obtain a target nucleic acid sample.
[0011] Optionally, before placing the second glass slide carrying the stained section with the marked target area below the first glass slide and making the projection of the stained section and the first sample section coincide in the normal direction, it further includes:
[0012] Fixing a second sample section on the second glass slide.
[0013] Performing staining treatment on the second sample section, observing the range of the target area where the target cells are located by a microscope and making marks to obtain a stained section with the marked target area.
[0014] Optionally, the step of cutting the adhesion layer according to the marks on the stained section to obtain a target adhesion part with the target area of the first sample section adhered thereto includes:
[0015] Cutting the adhesion layer along the boundary between the target area and the non-target area according to the marks on the stained section to obtain a target adhesion part with the target area of the first sample section adhered thereto and a non-target adhesion part with the non-target area of the first sample section adhered thereto.
[0016] Separating and collecting the target adhesion part from the transparent layer.
[0017] Optionally, the first sample section is a formalin-fixed paraffin-embedded sample section.
[0018] The adhesion layer is made of a material that can be completely dissolved by a dewaxing agent at a preset temperature.
[0019] Optionally, the impurity removal treatment of the target adhesion part to obtain the target nucleic acid sample includes:
[0020] Mix the target adhesion part with a dewaxing agent, and successively perform shaking treatment, first centrifugation treatment, first heat preservation treatment, second centrifugation treatment, and second heat preservation treatment to obtain a first mixed solution;
[0021] Remove the supernatant of the first mixed solution and retain the precipitate;
[0022] After adding a dewaxing agent to the precipitate, perform a third heat preservation treatment to obtain a second mixed solution;
[0023] After the second mixed solution returns to room temperature, add a lysis buffer and a protease and perform a fourth heat preservation treatment to obtain a third mixed solution;
[0024] Extract the bottom aqueous phase of the third mixed solution and perform purification treatment to obtain the target nucleic acid sample.
[0025] The second aspect of the present invention provides a glass slide, including an adhesion layer and a transparent layer;
[0026] The surface of the adhesion layer includes a first surface for detachably bonding with the surface of the transparent layer and a second surface for adhering to a sample section.
[0027] Optionally, the thickness of the adhesion layer is 30 μm to 40 μm.
[0028] Optionally, the light transmittance of the adhesion layer is greater than or equal to 50%.
[0029] Optionally, the overall thickness of the glass slide is less than 1.2 mm.
[0030] Optionally, the adhesion layer is made of a material that can be completely dissolved by a dewaxing agent at a preset temperature.
[0031] From the above technical solutions, it can be seen that the present invention has the following advantages:
[0032] Compared with the traditional blade scraping method, the biological sample pretreatment method provided by the present invention can more accurately separate the target area and non-target area of the sample section pair with simple and inexpensive instruments, and can avoid partial tissue remaining on the glass slide caused by the traditional blade scraping method, reduce the residue on the blade, improve the DNA yield, and have a good target cell enrichment effect; it can also avoid the risk of sample contamination caused by wax chips falling out during the operation of the traditional blade scraping method; the above factors comprehensively improve the accuracy of extracting the nucleic acid sample of target cells from the sample section. Description of the Drawings
[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0034] Figure 1 It is a real - shot picture before and after the first glass slide adheres to the first sample section provided in Embodiment 1 of the present invention;
[0035] Figure 2 It is a real - shot picture of the process of separating the target adhesion part from the transparent layer provided in Embodiment 1 of the present invention;
[0036] Figure 3 It is the electrophoresis band picture of the gel electrophoresis detection in Experimental Example 2 of the present invention. Detailed implementation manners
[0037] The embodiments of the present invention provide a biological sample pretreatment method and a glass slide, which are used to solve the technical problem of poor accuracy in extracting nucleic acid samples of specific cells from sample sections.
[0038] To make the object, features, and advantages of the present invention more obvious and understandable, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the embodiments described below are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.
[0039] It should be noted that in addition to the blade scraping method, laser capture microdissection (LCM) is also clinically used to enrich tumors in FFPE sections of tumor tissues; laser capture microdissection is a high - precision tumor enrichment method that can accurately cut tumor tissues and separate them from the surrounding normal tissues; however, the LCM technology requires the use of professional laser cutting equipment, which is expensive and has high maintenance costs, and the operation process requires high professionalism and takes a long time, and cannot meet the needs of large - scale sample processing.
[0040] The raw materials and reagents involved in the embodiments of the present invention are all commercially available.
[0041] Embodiment 1
[0042] Embodiment 1 of the present invention provides a biological sample pretreatment method, including steps 101 to 113:
[0043] Step 101: Prepare the first sample section and the second sample section: After fixing the tissue sample with formalin, perform operations such as dehydration, wax infiltration, and paraffin embedding to obtain a formalin-fixed paraffin-embedded block. Then, use a microtome to continuously cut thin sections from the formalin-fixed paraffin-embedded block with a thickness of 5 μm, and take two adjacent sample sections as the first sample section and the second sample section.
[0044] It should be noted that the first sample section and the second sample section targeted in the method provided by the present invention are both sample sections obtained by means of continuous sectioning to obtain sections containing the target area, such as formalin-fixed paraffin-embedded sample sections and frozen sections. Preferably, they are formalin-fixed paraffin-embedded sample sections with a thickness preferably ranging from 5 μm to 10 μm; the target area is the area where the target cells are located, and the target cells refer to the cells of the cell type to be enriched; the original biological samples used to make the first sample section and the second sample section can be common samples that need to undergo gene detection or have other requirements for extracting nucleic acids from specific cells, including tissue samples, cell samples, etc.
[0045] The first sample section and the second sample section are two sample sections continuously cut from the same sample. The morphological differences between the first sample section and the second sample section are small and negligible, and the areas where the target cells are located are basically the same; the preparation of the first sample section and the second sample section can be completed in the same manner as that for making a conventional observation section specimen.
[0046] Step 102: Adhere the second sample section to the second glass slide: Use a commercially available glass slide with the brand "DAKEWE,LTD.", a length of 75 mm × a width of 25 mm × a thickness of 1.1 mm as the second glass slide. Float the second sample section on the heated distilled water in a spreading machine to fully stretch the second sample section, and then it can be adhered to the second glass slide in an unfolded state.
[0047] In the present invention, the step of adhering the second sample section to the second glass slide can be completed in the same manner as that for making a conventional observation section specimen, and the specific steps can refer to Step 102 of this embodiment; the second glass slide can be any glass slide suitable for optical microscope observation, preferably a glass slide; the present invention does not particularly limit the shape and size of the second glass slide, and preferably, it is based on the shape and size of a well-known microscope slide for making observation specimens. For example, the glass slide used in this embodiment can be selected according to the size of the sample section.
[0048] Step 103: Prepare the first glass slide: Use a commercially available glass slide with the brand "DAKEWE, LTD.", having a length of 75 mm × width of 25 mm × thickness of 1.1 mm as the transparent layer of the first glass slide. Cut a 40 mm × 25 mm commercially available Parafilm sealing film of the Amcor brand and adhere it to one side of the transparent layer to obtain the first glass slide.
[0049] It should be noted that in the present invention, the first glass slide includes an adhesive layer and a transparent layer. The surface of the adhesive layer includes a first surface for detachably bonding with the surface of the transparent layer and a second surface for adhering the sample section; the transparent layer is made of a transparent material capable of bonding with the adhesive layer, such as glass, resin, etc. A commercially available glass slide can be directly used as the transparent layer of the first glass slide. When there is a transparency requirement for the glass slide in optical microscope observation, it is preferably glass; the bonding between the adhesive layer and the transparent layer is detachable, and the adhesive layer can be a flexible film layer so that it can be completely torn off from the surface of the transparent layer; since in the subsequent steps, the adhesive layer needs to be completely removed and only the nucleic acid adhering to the cells above is retained, the adhesive layer should be made of a material that can be completely dissolved in a solution that does not damage the nucleic acid or causes less damage to the nucleic acid.
[0050] In order to ensure that after the second glass slide is placed below the first glass slide in the subsequent steps, the projections of the stained section and the first sample section in the normal direction coincide, the light transmittance of the adhesive layer is preferably greater than or equal to 50%, the thickness of the adhesive layer is preferably 30 μm to 40 μm, and the overall thickness of the first glass slide is preferably less than 1.2 mm; the present invention does not particularly limit the shape, length, and width dimensions of the transparent layer of the first glass slide, and preferably uses the shape and length and width dimensions based on the well-known microscope glass slide for making observation specimens, such as a rectangular shape with a length of about 75 mm and a width of about 25 mm, or can be selected according to the size of the sample section; the adhesive layer is bonded to at least one surface of the transparent layer surface, and there is no particular limitation on the area and shape of the adhesive layer covering the transparent layer surface, and it can be selected according to the size of the transparent layer or the sample section.
[0051] In this embodiment, the main components of the Parafilm sealing film are paraffin and polyethylene, which can be dissolved by organic solvents at high temperatures and can be completely dissolved in the dewaxing agent added in the subsequent steps at 90 °C, and can be dissolved and removed together with the paraffin; the thickness of the Parafilm sealing film is 127 μm, which is translucent in the unstretched state, and it has good ductility at 15 °C to 25 °C. When preparing the first glass slide, the operator can stretch the Parafilm sealing film to 3 to 4 times its original length, making its thickness and light transmittance better, and helping to improve the tightness of the Parafilm sealing film adhered to the transparent layer; since the Parafilm sealing film has good viscosity at 55 °C to 65 °C and does not change its physical properties, after the Parafilm sealing film is coated on the surface of the transparent layer, it can be placed in a spreading machine at the corresponding temperature and incubated for 5 to 10 seconds to increase the viscosity of the sealing film and firmly adhere to the transparent layer.
[0052] Step 104: Adhere the first sample section to the first glass slide: Float the first sample section on the heated distilled water in the spreading machine to fully stretch the first sample section, and then place it on the second surface of the adhesion layer of the first glass slide in the unfolded state, and place the whole in the spreading machine at 56 °C and incubate until the paraffin becomes transparent (melts), so that the first sample section is adhered and fixed on the first glass slide.
[0053] This step can refer to Figure 1 , Figure 1 which are the actual photos before and after the first glass slide adheres to the first sample section in this embodiment; in this step, the temperature of the spreading machine can be set to 55 °C to 65 °C; in the present invention, if an adhesion layer of other materials is used, the step of adhering the first sample section to the adhesion layer can be adjusted according to the physical properties of the adhesion layer based on the method of making a conventional observation section specimen.
[0054] Step 105: Perform HE staining on the second sample section, observe the range of the target area where the target cells are located on the stained sample section under a microscope, and use a marker pen to mark it to obtain a stained section with the target area marked.
[0055] The stained section with the target area marked obtained in this step can be referred to Figure 2In Figure ①, the lower slide glass in Figure ① is the second slide glass. The purple area on the second slide glass is the stained tissue cells in the stained section, and the blue area is the marked target area. In the present invention, the method for staining the second sample section can be any staining method that enables the operator to clearly identify the target area under the microscope, such as HE staining, Papanicolaou staining, etc. After staining, the second slide glass should be sealed. The operator can use a microscope to identify and clearly mark the target area (such as the area where tumor cells are located) in the stained section on the cover glass with common marking tools such as a marker pen.
[0056] Step 106: Place the second slide glass carrying the stained section with the marked target area below the first slide glass, and make the projection of the stained section coincide with that of the first sample section in the normal direction.
[0057] This step can be referred to Figure 2 In Figure ②, with the surface of the first slide glass adhered with the first sample section as the top surface and the surface of the second slide glass after sealing as the top surface, after placing the second slide glass below the first slide glass, the top surface of the second slide glass should be closely attached to the bottom surface of the transparent layer of the first slide glass to ensure that the projection contours of the stained section and the first sample section can precisely coincide in the normal direction.
[0058] Step 107: According to the marks on the stained section, cut the adhesion layer of the first slide glass along the boundary between the target area and the non-target area to obtain a target adhesion part with the first sample section adhered to the target area and a non-target adhesion part with the first sample section adhered to the non-target area.
[0059] The first sample section and the adhesion layer can be cut with a cutting tool such as a surgical blade. The cutting tool is preferably vertically cut into the first sample section and the adhesion layer.
[0060] Step 108: Separate and collect the target adhesion part from the transparent layer: As shown in Figures ③ and ④, first use forceps to tear off the non-target adhesion part from the edge of the non-target adhesion part to separate the non-target adhesion part from the transparent layer, and then tear off the target adhesion part and collect it in a centrifuge tube. Figure 2
[0061] It should be noted that using clamping tools such as forceps to clamp the edge of the adhesion layer and tear it off can achieve the separation of the adhesion layer from the transparent layer. In the present invention, only the target adhesion part can be separately separated and collected from the transparent layer without tearing off the non-target adhesion part, or first separate the non-target adhesion part from the edge of the non-target adhesion part from the transparent layer, and then separate and collect the target adhesion part from the transparent layer. The latter is preferably adopted, and the separation accuracy is higher. The target adhesion part can be collected in a centrifuge tube or other containers convenient for centrifugation treatment.
[0062] Step 109: Add 500 μL of xylene dewaxing agent from ComWin Biotech to the centrifuge tube containing the target adhesion part, and perform vortex oscillation for 10 seconds (oscillation treatment), centrifuge until the sample is collected at the bottom of the centrifuge tube (first centrifugation treatment), incubate in a constant temperature metal bath at 90 °C for 3 minutes (first heat preservation treatment), centrifuge at 12,000 rpm for 2 minutes (second centrifugation treatment), and incubate in a constant temperature metal bath at 90 °C for 3 minutes (second heat preservation treatment) to obtain the first mixture.
[0063] In a specific experimental example, when the Parafilm sealing film was added to the dewaxing agent at 56 °C, the dewaxing agent after sufficient mixing was turbid, indicating that the dewaxing agent could not completely dissolve the Parafilm sealing film at this temperature; while when the Parafilm sealing film was added to the dewaxing agent at 90 °C, the dewaxing agent after sufficient mixing was transparent, indicating that the dewaxing agent could fully dissolve the Parafilm sealing film at this temperature.
[0064] Step 110: Thoroughly aspirate the supernatant of the first mixture and retain the precipitate.
[0065] Step 111: After adding 500 μL of xylene dewaxing agent from ComWin Biotech to the precipitate, incubate in a constant temperature metal bath at 90 °C for 3 minutes (third heat preservation treatment) to obtain the second mixture.
[0066] Step 112: After the second mixture returns to room temperature, add lysis buffer 1 and proteinase K from the fixed tissue DNA extraction kit of ComWin Biotech. The amount of lysis buffer 1 is 180 μL, and the amount of proteinase K is 20 μL. Incubate at 56 °C for at least 1 hour until the solids in the mixture are completely dissolved, and then incubate at 90 °C for 1 hour (fourth heat preservation treatment) to obtain the third mixture.
[0067] Step 113: Transfer the bottom aqueous phase of the third mixture to a new centrifuge tube, and use the adsorption column from the fixed tissue DNA extraction kit of ComWin Biotech to purify the bottom aqueous phase to obtain the target nucleic acid sample.
[0068] It can be understood that the target nucleic acid sample refers to the nucleic acid sample of the cells in the target area. The target nucleic acid sample in this embodiment is a DNA sample; after obtaining the target adhesion part, the target nucleic acid sample can be obtained through impurity removal treatment. The impurity removal treatment is used to remove impurities such as the adhesion layer, embedding material, and other cell components (proteins, lipids, etc.) in the target adhesion part. These impurities can be removed step by step separately, or multiple of them can be removed in the same step; when the first sample section is a formalin-fixed paraffin-embedded sample section, the embedding material to be removed is paraffin; when the first sample section is a frozen section, the embedding material to be removed is usually a water-soluble colloidal medium such as OCT or CMC.
[0069] In this embodiment, steps 109 to 113 are for impurity removal treatment of the target adhesion part; steps 109 to 111 are used to remove the Parafilm sealing film adhesion layer, paraffin, and some lipid-soluble impurities in the target adhesion part; the dewaxing agent uses an organic solvent that does not dissolve the sample section but can dissolve paraffin and the adhesion layer material; when aspirating and discarding the supernatant in step 110, care should be taken to avoid aspirating the precipitate, and the precipitate can be used for RNA extraction or DNA extraction.
[0070] In the present invention, adding a lysis buffer and a protease to the second mixture is to remove cell components such as proteins, lipids, and cell membrane components. The protease can be selected from protease K, protease E, etc., and the lysis buffer can be selected from SDS (sodium dodecyl sulfate)-based lysis buffer, chaotropic salt-based lysis buffer, etc.; the temperature and duration of the first heat preservation treatment, the second heat preservation treatment, and the third heat preservation treatment can be set according to the characteristics of the dewaxing agent, the melting point of paraffin, and the experimental requirements of impurity removal treatment, and the temperature and duration of the fourth heat preservation treatment can be set according to the optimal temperature for protease reaction, the lysis rate, and the decrosslinking temperature; the adhesion layer can also be made of other materials that can be completely dissolved by the dewaxing agent at a preset temperature, and the preset temperature can refer to the temperature settings of the first heat preservation treatment, the second heat preservation treatment, and the third heat preservation treatment; the purification treatment is used to further remove residual impurities such as proteins, polysaccharides, lipids, and organic solvents or salts that may interfere with subsequent experiments to ensure obtaining high-quality nucleic acid samples, such as adsorption column purification, magnetic bead method, phenol-chloroform extraction method, etc.
[0071] Compared with the traditional blade scraping method, the biological sample pretreatment method provided by the present invention can more precisely separate the target area and non-target area of the sample section pair with simple and inexpensive instruments, and can avoid partial tissue remaining on the glass slide caused by the traditional blade scraping method, and reduce the residue on the blade, improve the DNA yield, and have a good target cell enrichment effect; it can also avoid the risk of sample contamination caused by wax chips falling out during the operation of the traditional blade scraping method; the above factors comprehensively improve the accuracy of extracting nucleic acid samples of target cells from sample sections; when the method provided by the present invention is used to enrich tumor cells and extract nucleic acid samples for gene detection, the final tumor mutation detection frequency can be increased, and the accuracy of gene detection can be improved.
[0072] Comparative Example 1
[0073] Comparative Example 1 of the present invention provides a blade scraping biological sample pretreatment method, including:
[0074] Step 201, preparing a third sample section: Take a section adjacent to the first sample section or the second sample section in Example 1 as the third sample section; the specific sample section preparation method is the same as step 101 of Example 1.
[0075] Step 202: Adhere the third sample section to the third glass slide: Use commercially available glass slides with the brand "DAKEWE, LTD.", 75 mm in length × 25 mm in width × 1.1 mm in thickness as the third and fourth glass slides; Float the third sample section on the heated distilled water in a spreading machine to fully stretch the third sample section, and then it can be adhered to the third glass slide in the unfolded state.
[0076] Step 203: Refer to the marks on the stained section obtained in step 105 of Example 1, and use a blade to scrape the same area in the third sample section to obtain an intermediate sample, and collect the intermediate sample in a centrifuge tube.
[0077] Step 205: Add 500 μL of xylene dewaxing agent of the brand ComWin Biotech to the centrifuge tube containing the intermediate sample, and perform vortex oscillation for 10 seconds, short centrifugation to collect the sample at the bottom of the centrifuge tube, incubation in a constant temperature metal bath at 90 °C for 3 minutes, centrifugation at 12,000 rpm for 2 minutes, and incubation in a constant temperature metal bath at 90 °C for 3 minutes to obtain a fourth mixture.
[0078] Step 206: Thoroughly aspirate the supernatant of the fourth mixture and retain the precipitate.
[0079] Step 207: After adding 500 μL of xylene dewaxing agent of the brand ComWin Biotech to the precipitate, incubate in a constant temperature metal bath at 90 °C for 3 minutes (the third heat preservation treatment) to obtain a fifth mixture.
[0080] Step 208: After the fifth mixture returns to room temperature, add 180 μL of lysis buffer 1 of the fixed tissue DNA extraction kit of the brand ComWin Biotech and 20 μL of proteinase K of the fixed tissue DNA extraction kit of the brand ComWin Biotech, incubate at 56 °C for at least 1 hour until the solids in the mixture are completely dissolved, and then incubate at 90 °C for 1 hour (the fourth heat preservation treatment) to obtain a sixth mixture.
[0081] Step 209: Transfer the bottom aqueous phase of the sixth mixture to a new centrifuge tube, and use the adsorption column of the fixed tissue DNA extraction kit of the brand ComWin Biotech to purify the bottom aqueous phase to obtain a target nucleic acid sample.
[0082] Comparative Example 2
[0083] The present invention provides a biological sample pretreatment method for Comparative Example 2, including:
[0084] Step 301: Prepare a fourth sample section: Take a section adjacent to the first sample section or the second sample section in Example 1 as the fourth sample section; specifically, the sample section preparation method is the same as step 101 of Example 1.
[0085] Step 302: Add 500 μL of xylene dewaxing agent of the brand ComWin Biotech to the centrifuge tube containing the fourth sample section, and perform vortex oscillation for 10 seconds, followed by brief centrifugation to collect the sample at the bottom of the centrifuge tube, incubation in a constant temperature metal bath at 90 °C for 3 minutes, centrifugation at 12,000 rpm for 2 minutes, and incubation in a constant temperature metal bath at 90 °C for 3 minutes to obtain the seventh mixture.
[0086] Step 306: Thoroughly aspirate and discard the supernatant of the seventh mixture, and retain the precipitate.
[0087] Step 307: After adding 500 μL of xylene dewaxing agent of the brand ComWin Biotech to the precipitate, incubate in a constant temperature metal bath at 90 °C for 3 minutes (the third heat preservation treatment) to obtain the eighth mixture.
[0088] Step 308: After the eighth mixture returns to room temperature, add 180 μL of lysis buffer 1 of the fixed tissue DNA extraction kit of the brand ComWin Biotech and 20 μL of proteinase K of the fixed tissue DNA extraction kit of the brand ComWin Biotech, incubate at 56 °C for at least 1 hour until the solids in the mixture are completely dissolved, and then incubate at 90 °C for 1 hour (the fourth heat preservation treatment) to obtain the ninth mixture.
[0089] Step 309: Transfer the bottom aqueous phase of the ninth mixture to a new centrifuge tube, and use the adsorption column of the fixed tissue DNA extraction kit of the brand ComWin Biotech to purify the bottom aqueous phase to obtain the target nucleic acid sample.
[0090] Experimental Example 1
[0091] Using the biological sample pretreatment methods provided in Example 1 and Comparative Example 1, different original biological samples 1 to 5 were processed to obtain ten target nucleic acid samples. Each original biological sample was a tumor puncture or biopsy tissue; a Qubit fluorometer (Thermo Fisher Scientific) was used to measure the absorbance of the ten target nucleic acid samples. The measurement results can be referred to Table 1:
[0092]
[0093] Table 1 Absorbance measurement result table
[0094] It can be seen from the measurement results in Table 1 that the total amount of DNA in the target nucleic acid samples obtained by using the biological sample pretreatment method provided in Example 1 is higher than that in the target nucleic acid samples obtained by the blade scraping method in Comparative Example 1. The biological sample pretreatment method provided in Example 1 can effectively improve the DNA yield compared with the blade scraping method.
[0095] Experimental Example 2
[0096] Using the biological sample pretreatment methods provided in Example 1 and Comparative Example 2, different original biological samples 1 to 5 were processed to obtain ten target nucleic acid samples; the DNA quality of the ten target nucleic acid samples was evaluated by PCR amplification and gel electrophoresis detection respectively. The amplification target was set as the GAPDH gene, and the primer sequences used in the determination are shown in Table 2:
[0097]
[0098] Table 2 Primer sequence table for PCR amplification
[0099] The reaction composition and reaction conditions of PCR are shown below:
[0100] Using 4 ng of the target nucleic acid sample as the PCR starting template, the forward primer (F primer) and reverse primers 1, 2, 3 (R primer) were each 200 nmol / L, and the Premix Taq premixed solution (Takara) was used for the determination; after the PCR reaction system was configured, the reaction was carried out under the following conditions: maintained at 95°C for 3 minutes, and then 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 40 seconds as one cycle, and 30 cycles were carried out. Finally, it was maintained at 72°C for 5 minutes; then the reaction system was loaded onto an agarose gel and electrophoresed at a constant voltage of 160 V for 19 minutes; finally, the ultraviolet projection function of the gel imager was used to take pictures and observe the electrophoresis bands.
[0101] The electrophoresis bands obtained by gel electrophoresis detection are as Figure 3 shown. The clarity and brightness of the electrophoresis bands corresponding to the samples processed by the method of Comparative Example 1 and the samples processed by the method of Example 1 have little difference. It can be seen that the biological sample pretreatment method provided in Example 1 does not affect the DNA quality of tissue sections.
[0102] Experimental Example 3
[0103] The ten target nucleic acid samples obtained in Experimental Example 1 were respectively fragmented, end-repaired, ligated with adapters, PCR-enriched, etc. using a human tumor multi-gene detection (NGS) small panel kit (Zhiben) to prepare a pre-library; then a DNA probe with a specific sequence was hybridized with the pre-library to specifically capture the library containing the region to be detected, thereby obtaining a final library; finally, a next-generation sequencer (Illumina) was used for next-generation sequencing, and the detection results of next-generation sequencing are shown in Table 3:
[0104]
[0105] Table 3 Table of DNA variant detection results of next-generation sequencing
[0106] As can be seen from the detection results in Table 3, the mutation detection rates of the nucleic acid samples obtained by using the biological sample pretreatment methods provided in Example 1 and Comparative Example 1 are both 100%.
[0107] Experimental Example 4
[0108] Using the biological sample pretreatment methods provided in Example 1 and Comparative Example 1, different original biological samples 6 to 10 were processed to obtain ten target nucleic acid samples. Each original biological sample was a tumor tissue. The ten target nucleic acid samples were respectively subjected to steps such as fragmentation, end repair, adapter ligation, and PCR enrichment using a human tumor multi-gene detection (NGS) small panel kit (Zhiben) to prepare a pre-library. Subsequently, DNA probes with specific sequences were used to hybridize with the pre-library to specifically capture the library containing the region to be detected, thereby obtaining a final library. Finally, a gene sequencer (Illumina) was used for next-generation sequencing. The results of the detection frequencies of DNA mutation sites in the next-generation sequencing can be referred to in Table 4:
[0109]
[0110] Table 4 Results table of the detection frequencies of DNA mutation sites in next-generation sequencing
[0111] The results in the table show that the tumor mutation detection frequency of the target nucleic acid sample obtained by using the biological sample pretreatment method provided in Example 1 is higher than that of the target nucleic acid sample obtained by using the method provided in Comparative Example 1. It can be seen that the tumor enrichment efficiency of the method provided in Example 1 is better than that of the method provided in Comparative Example 1.
[0112] As described above, the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A biological sample pretreatment method, characterized in that, Comprising: Adhere the first sample section to the first glass slide; the first glass slide includes an adhesion layer and a transparent layer, the surface of the adhesion layer includes a first surface for detachably bonding with the surface of the transparent layer and a second surface for adhering the sample section; Place the second glass slide carrying the stained section with the target area marked below the first glass slide, and make the projection of the stained section and the first sample section coincide in the normal direction; Cut the adhesion layer according to the mark on the stained section to obtain a target adhesion part with the target area of the first sample section adhered thereto; Perform impurity removal treatment on the target adhesion part to obtain a target nucleic acid sample.
2. The biological sample pretreatment method according to claim 1, characterized in that, Before placing the second glass slide carrying the stained section with the target area marked below the first glass slide and making the projection of the stained section and the first sample section coincide in the normal direction, further comprising: Fix the second sample section on the second glass slide; Perform staining treatment on the second sample section, observe the range of the target area where the target cells are located by microscope and mark it to obtain a stained section with the target area marked.
3. The biological sample pretreatment method according to claim 1, wherein The cutting the adhesion layer according to the mark on the stained section to obtain a target adhesion part with the target area of the first sample section adhered thereto includes: Cut the adhesion layer along the boundary between the target area and the non-target area according to the mark on the stained section to obtain a target adhesion part with the target area of the first sample section adhered thereto and a non-target adhesion part with the non-target area of the first sample section adhered thereto; Separate and collect the target adhesion part from the transparent layer.
4. The biological sample pretreatment method according to claim 1, characterized in that, The first sample section is a formalin-fixed paraffin-embedded sample section; The adhesion layer is made of a material that can be completely dissolved by a dewaxing agent at a preset temperature.
5. The biological sample pretreatment method according to claim 4, wherein The performing impurity removal treatment on the target adhesion part to obtain a target nucleic acid sample includes: Mix the target adhesion part with a dewaxing agent, and perform shaking treatment, first centrifugation treatment, first heat preservation treatment, second centrifugation treatment and second heat preservation treatment in sequence to obtain a first mixed solution; Remove the supernatant of the first mixed solution and retain the precipitate; After adding a dewaxing agent to the precipitate, perform a third heat preservation treatment to obtain a second mixed solution; After the second mixed solution returns to room temperature, add a lysis buffer and a protease and perform a fourth heat preservation treatment to obtain a third mixed solution; Extract the bottom aqueous phase of the third mixed solution and perform purification treatment to obtain a target nucleic acid sample.
6. A glass slide, characterized in that, Including an adhesion layer and a transparent layer; The surface of the adhesion layer includes a first surface for detachably bonding with the surface of the transparent layer and a second surface for adhering the sample section.
7. The glass slide according to claim 6, characterized in that, The thickness of the adhesion layer is 30μm to 40μm.
8. The glass slide according to claim 6, characterized in that, The light transmittance of the adhesion layer is greater than or equal to 50%.
9. The slide according to claim 6, characterized in that, The overall thickness of the glass slide is less than 1.2mm.
10. The slide according to claim 6, wherein, The adhesion layer is made of a material that can be completely dissolved by a dewaxing agent at a preset temperature.