A method for preparing a nucleic acid detection sample

Abstract

The present application provides a method for preparing a nucleic acid detection sample, comprising the following steps: adding a pH-responsive hydrogel to an inactivated virus preservation solution for mixing, wherein the pH-responsive range of the pH-responsive hydrogel is 5-6, and the pH of the inactivated virus preservation solution is 7.5-8; using the inactivated virus preservation solution for virus sample collection to obtain a sample solution; adding proteinase K and magnetic beads to the sample solution and mixing evenly for enzymatic digestion; adjusting the pH to 5-6, incubating for 10-30 min; centrifuging the container containing the sample solution and fixing the magnetic beads to the bottom of the container by a magnet; removing the supernatant; washing the magnetic beads at least once; and eluting the washed magnetic beads to obtain the nucleic acid detection sample. The above preparation method can obtain a nucleic acid detection sample with a higher nucleic acid content, thereby improving the accuracy of nucleic acid detection.

Description

Technical Field

[0001] This application relates to the field of nucleic acid detection, and particularly to a method for preparing a nucleic acid detection sample. Background Art

[0002] Nucleic acid detection is an important step in molecular biology detection, and the preparation of nucleic acid detection samples is a key link among them. The method for preparing nucleic acid detection samples (taking inactivated virus preservation solution as an example) includes the following steps: sample collection and preservation, sample inactivation, nucleic acid extraction, and finally obtaining a sample that can be used for nucleic acid detection. The accuracy of nucleic acid detection is closely related to the method for preparing nucleic acid detection samples. However, the latter still has some deficiencies that affect the accuracy of nucleic acid detection. Inactivated virus preservation solution generally uses a high concentration of guanidine salts to obtain better inactivation efficiency. However, the virus preservation solution containing a high concentration of guanidine salts may cause damage to the nucleic acid structure during long-term storage.

[0003] CN115274235A discloses a magnetic carbon nanotube with amino magnetic beads coupled on its surface. By using the protonated magnetic carbon nanotube with a positive charge between pH = 1 and 2, it can be electrostatically combined with negatively charged RNA and adsorbed through π-π stacking to achieve nucleic acid extraction. Then, the binding effect can be reduced by adding a reaction buffer to increase the pH value to achieve nucleic acid amplification. However, it requires the use of a relatively acidic environment, while the pH of conventional inactivated virus preservation solution is weakly alkaline. Therefore, it cannot be directly applied to conventional inactivated virus preservation solution to play the role of nucleic acid protection, and the cost of carbon nanotubes is significantly higher than that of conventional magnetic beads.

[0004] Therefore, it is necessary to provide a method for preparing a nucleic acid detection sample. Summary of the Invention

[0005] In order to further improve the accuracy of nucleic acid detection, it is necessary to provide a method for preparing a nucleic acid detection sample.

[0006] This application provides a method for preparing a nucleic acid detection sample, including the following steps: adding a pH-responsive hydrogel to an inactivated virus preservation solution and mixing. The pH-responsive range of the pH-responsive hydrogel is 5 - 6, and the pH of the inactivated virus preservation solution is 7.5 - 8. Using the inactivated virus preservation solution for virus sample collection to obtain a sample solution, adding proteinase K and magnetic beads to the sample solution and mixing evenly for enzymatic hydrolysis, adjusting the pH to 5 - 6, incubating for 10 - 30 min, centrifuging the container containing the sample solution, and fixing the magnetic beads at the bottom of the container with a magnet, removing the supernatant, washing the magnetic beads at least once, and eluting the washed magnetic beads to obtain the nucleic acid detection sample.

[0007] In this application, a pH-responsive hydrogel with a pH response range of 5-6 is added to the inactivated virus preservation solution. The latter is in a hydrophobic state in the inactivated virus preservation solution with a pH of 7.5-8 and will not dissolve in the virus preservation solution, so it presents as a gel state. Under the electrostatic shielding effect provided by high salt, there is no electrostatic adsorption between the hydrogel and nucleic acid. However, the hydrogel can form a water-transporting domain through deprotonation to preserve the viral nucleic acid inside the hydrogel, thus avoiding the degradation of nucleic acid during transportation. After the sample solution is enzymolyzed, by adjusting the pH, the hydrogel is transformed into a hydrophilic state and dissolved in the virus preservation solution. The adsorption of the free hydrogel to nucleic acid is reduced to less than 10% (it can be seen from the data of 0d in the examples and comparative example 1 that this scheme will not cause a significant reduction in the nucleic acid recovery rate), thereby releasing the nucleic acid it preserves. Thus, the magnetic beads can bind to the nucleic acid through non-electrostatic adsorption. Moreover, when the hydrogel dissolves in the virus preservation solution, a steric hindrance effect can be generated to prevent the aggregation of magnetic beads, thereby improving the adsorption rate of nucleic acid. By combining the dynamic protection-release mechanism of the pH-responsive hydrogel with the magnetic bead adsorption mechanism, this application can improve the preservation effect of nucleic acid, is applicable to conventional inactivated virus preservation solutions, and has high universality.

[0008] Furthermore, the pH-responsive hydrogel contains a polymer with an aromatic group. Aromatic groups such as benzene rings and pyridine rings can specifically adsorb nucleic acid molecules through π-π stacking, thereby improving the selective capture ability of the hydrogel for nucleic acid and reducing the adsorption of impurities (such as proteins).

[0009] Furthermore, the pH-responsive hydrogel contains a pyridine-based polymer. For pyridine-based polymers, such as the pyridine group of polyvinylpyridine, it is deprotonated and hydrophobic at pH>7, transforming into a gel state and can adsorb nucleic acid; when pH<6, the pyridine group is protonated and hydrophilic, and the hydrogel dissolves in water and transforms into a free state, and the adsorption to nucleic acid is significantly reduced, thus releasing nucleic acid.

[0010] Furthermore, the pyridine-based polymer contains polyvinylpyrrolidone. Polyvinylpyrrolidone has good biocompatibility and can reduce the non-specific adsorption of the hydrogel to impurities such as viral proteins.

[0011] Furthermore, in the virus preservation solution, the addition amount of the pH-responsive hydrogel is 0.1-5% w / v. Controlling the hydrogel concentration within this range can not only provide sufficient adsorption sites for nucleic acid molecules to adsorb, but also will not affect the movement and adsorption effect of magnetic beads after transforming into a gel state.

[0012] Furthermore, the average particle size of the hydrogel particles is 1-10 μm. If the average particle size of the hydrogel particles is too large, the specific surface area of the hydrogel will be small, affecting the adsorption effect; if the average particle size of the hydrogel particles is too small, the adsorption effect on nucleic acids will be reduced.

[0013] Furthermore, the magnetic beads are silicon-based magnetic beads. Under the conditions of high salt and low pH (5-6), the positively charged salt ions in the solution can bind to the phosphate backbone of nucleic acids to neutralize their negative charges, and at the same time shield the electrostatic repulsion between the silanol groups on the surface of the magnetic beads and nucleic acids. The hydroxyl groups on the surface of the silicon-based magnetic beads can bind to the phosphate groups of nucleic acids through hydrogen bonds.

[0014] Furthermore, the hydrogel is prepared by free radical polymerization. The free radical polymerization process is simple and controllable, and the pH response range of the hydrogel can be regulated by adjusting the monomer ratio, which is suitable for industrialization.

[0015] Furthermore, the inactivated virus preservation solution contains 4-6 mol / L guanidine salt. The high-concentration guanidine salt does not affect the non-electrostatic adsorption of the hydrogel through hydrophobicity or π-π stacking, etc., thus ensuring the adsorption of the hydrogel on nucleic acids in a weakly alkaline environment, and can shield the electrostatic adsorption to avoid the electrostatic adsorption of the hydrogel on nucleic acids in an acidic environment from interfering with the adsorption of magnetic beads.

[0016] Furthermore, the virus preservation solution contains 4-6 mol / L guanidine salt, 0.1-0.3 mol / L Tris-HCl, 5-20 mmol / L EDTA, 0.05-0.5% sodium azide by mass fraction, and 0.01-0.05% TritonX-100 by mass fraction. This application can be applicable to conventional inactivated virus preservation solutions and has universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic flow chart of the preparation method of a nucleic acid sample in an embodiment.

[0018] Figure 2 It is the Cq value test results of the examples and comparative examples.

[0019] Figure 3 It is a graph showing the change trend of the Cq value of the nucleic acid sample in the example with the storage time. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] To facilitate the understanding of the present application, the present application will be described more comprehensively below. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure content of the present application more thorough and comprehensive.

[0021] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one such feature. In the description of the application, the meaning of "a plurality" is at least two, such as two, three, etc., unless otherwise specifically defined. In the description of the present application, the meaning of "several" is at least one, such as one, two, etc., unless otherwise specifically defined.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0023] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0024] In this application, regarding numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0025] In this application, for the percentage content involved, unless otherwise specified, for solid-liquid mixing and solid-solid mixing, it refers to the mass percentage, and for liquid-liquid mixing, it refers to the volume percentage.

[0026] In this application, for the percentage concentration involved, unless otherwise specified, it refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding this component.

[0027] For the temperature parameters in this application, unless otherwise specifically limited, it allows both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0028] The "particles" mentioned in this application, or substances with a defined average particle size distribution, do not necessarily have a spherical shape and may be irregular. They can be primary particles or secondary particles. The average particle size of irregular particles is calculated as the average of their maximum diameter and minimum diameter.

[0029] Example 1: This example provides a method for preparing a nucleic acid detection sample. The process schematic diagram is as shown in Figure 1 the figure.

[0030] Virus preservation solution formula (the same formula is used in other examples and comparative examples, and other commercially available inactivated virus preservation solutions can also be used): 4 mol / L guanidine isothiocyanate, 0.1 mol / L Tris-HCl, 5 mmol / L EDTA, 0.05% sodium azide by mass fraction, 0.03% Triton X-100 by mass fraction, 2% glycerol by mass fraction. The addition amount of BSA is 4 g / L, and the pH of the virus preservation solution is 7.5.

[0031] The preparation method of chitosan / polyvinylpyrrolidone hydrogel (the preparation methods of other examples and comparative examples are the same) is as follows: Prepare a 3% chitosan solution by mass fraction, dissolve polyvinylpyrrolidone in water to make a 25% polyvinylpyrrolidone aqueous solution by mass fraction, dissolve 0.01 g of initiator potassium persulfate and 0.02 g of crosslinking agent N,N-methylenebisacrylamide in 2 mL of water, add 8 mL of polyvinylpyrrolidone aqueous solution and 5 mL of chitosan solution, mix and place in an inert gas environment, react at 60 °C for 4 h to obtain a colloid, and break the colloid by ultrasonic method to obtain a micron-sized hydrogel. Hydrogels with different particle size distributions (such as 1 μm, 2 μm, 4 μm, 8 μm, and 10 μm, etc.) can be obtained by controlling the ultrasonic power and ultrasonic time. The average particle size of the hydrogel in this example is 8.2 μm.

[0032] Use a sampling swab to collect an equal amount of standard influenza A virus, add it to 5 mL of virus preservation solution to obtain a sample solution. 0.05 g of chitosan / polyvinylpyrrolidone hydrogel is pre-added to the virus preservation solution, and it is placed on a shaker and shaken for 30 min to fully inactivate the virus and release nucleic acid, and the hydrogel fully adsorbs free nucleic acid. Place the sample solution at room temperature, take 200 μL of samples at 0 d, 2 d, 4 d, and 8 d (take samples after shaking to avoid sedimentation of the hydrogel. The same applies to other examples and comparative examples. When sampling, cut the tip of the pipette to expand the opening to prevent the hydrogel from blocking the tip) for nucleic acid extraction.

[0033] Add 10 μL of proteinase K and 5 μL of magnetic bead suspension (concentration: 20 mg / mL, silicon-based magnetic beads, Haisen, product number: cz1001, average particle size 1 μm) to the sample solution, adjust the pH to 5, shake and mix well, incubate at 65 °C for 10 min, centrifuge instantaneously, place the test tube on a magnetic rack and adsorb for 3 min, and remove the supernatant.

[0034] After rinsing the magnetic beads with the washing solution, discard the supernatant. The composition of the washing solution is: 0.1 M EDTA, 0.05 M Tris, 3 M guanidine hydrochloride, 50% (v / v) ethanol, and the balance is water. Then, rinse the magnetic beads with 70% ethanol and discard the supernatant.

[0035] Add 100 μL of eluent to the magnetic beads to obtain a nucleic acid detection sample.

[0036] Example 2: This example provides a method for preparing a nucleic acid detection sample.

[0037] Use a sampling swab to collect an equal amount of the standard product of influenza A virus, add it to 5 mL of virus preservation solution to obtain a sample solution. The virus preservation solution is pre-added with 0.05 g of chitosan / polyvinylpyrrolidone hydrogel, and placed on a shaker and shaken for 30 min to fully inactivate the virus and release nucleic acids, and the hydrogel fully adsorbs free nucleic acids. Place the sample solution at room temperature, and take 200 μL samples at 0 d, 2 d, 4 d, and 8 d for nucleic acid extraction.

[0038] Add 10 μL of proteinase K and 5 μL of magnetic bead suspension (concentration: 20 mg / mL, silica-based magnetic beads, Haisen, product number: cz1001, average particle size 1 μm) to the sample solution, adjust the pH to 5, mix well by shaking, incubate at 55 °C for 30 min, centrifuge instantaneously, place the test tube on a magnetic rack and adsorb for 3 min, and remove the supernatant.

[0039] After rinsing the magnetic beads with the washing solution, discard the supernatant. The composition of the washing solution is: 0.1 M EDTA, 0.05 M Tris, 3 M guanidine hydrochloride, 50% (v / v) ethanol, and the balance is water. Then, rinse the magnetic beads with 70% ethanol and discard the supernatant.

[0040] Add 100 μL of eluent to the magnetic beads to obtain a nucleic acid detection sample.

[0041] Example 3: This example provides a method for preparing a nucleic acid detection sample.

[0042] Use a sampling swab to collect an equal amount of the standard product of influenza A virus, add it to 5 mL of virus preservation solution to obtain a sample solution. The virus preservation solution is pre-added with 0.05 g of chitosan / polyvinylpyrrolidone hydrogel, and placed on a shaker and shaken for 30 min to fully inactivate the virus and release nucleic acids, and the hydrogel fully adsorbs free nucleic acids. Place the sample solution at room temperature, and take 200 μL samples at 0 d, 2 d, 4 d, and 8 d for nucleic acid extraction.

[0043] Add 10 μL of proteinase K and 5 μL of magnetic bead suspension (concentration of 20 mg / mL, silica-based magnetic beads, Hisan, product number: cz1001, average particle size 1 μm) to the sample solution and adjust the pH to 6. After vortex mixing, incubate at 65°C for 10 min, centrifuge briefly, place the test tube on a magnetic stand for adsorption for 3 min, and remove the supernatant.

[0044] The magnetic beads were rinsed with a washing solution containing 0.1 M EDTA, 0.05 M Tris, 3 M guanidine hydrochloride, 50% ethanol by volume, and the remainder water, and the magnetic beads were rinsed with 70% ethanol and the supernatant was discarded.

[0045] Add 100 μL of elution solution to the magnetic beads to obtain the nucleic acid detection sample.

[0046] Example 4: This example provides a method for preparing a nucleic acid detection sample.

[0047] An equal amount of type A virus standard was collected using a sampling swab and added to 5 mL of virus preservation solution to prepare a sample solution. The solution was pre-added with 0.01 g of chitosan / polyvinyl pyrrolidone hydrogel. The sample solution was shaken on a shaker for 30 minutes to fully inactivate the virus, release nucleic acids, and allow the hydrogel to fully absorb the free nucleic acids. The sample solution was allowed to stand at room temperature, and 200 μL of the sample was collected at 0, 2, 4, and 8 days for nucleic acid extraction.

[0048] Add 10 μL of proteinase K and 5 μL of magnetic bead suspension (concentration of 20 mg / mL, silica-based magnetic beads, Hisan, product number: cz1001, average particle size 1 μm) to the sample solution and adjust the pH to 5. After vortex mixing, incubate at 65°C for 10 min, centrifuge briefly, place the test tube on a magnetic stand for adsorption for 3 min, and remove the supernatant.

[0049] The magnetic beads were rinsed with a washing solution containing 0.1 M EDTA, 0.05 M Tris, 3 M guanidine hydrochloride, 50% ethanol by volume, and the remainder water, and the magnetic beads were rinsed with 70% ethanol and the supernatant was discarded.

[0050] Add 100 μL of elution solution to the magnetic beads to obtain the nucleic acid detection sample.

[0051] Example 5: This example provides a method for preparing a nucleic acid detection sample.

[0052] Collect an equal amount of standard influenza A virus samples using a sampling swab, and add them to 5 mL of virus preservation solution to obtain a sample solution. The virus preservation solution is pre-added with 0.25 g of chitosan / polyvinylpyrrolidone hydrogel, and placed on a shaker for 30 min to inactivate the virus and release nucleic acids, and the hydrogel fully adsorbs the free nucleic acids. Place the sample solution at room temperature, and take 200 μL samples at 0 d, 2 d, 4 d, and 8 d for nucleic acid extraction.

[0053] Add 10 μL of proteinase K and 5 μL of magnetic bead suspension (concentration: 20 mg / mL, silica-based magnetic beads, Hisian, product number: cz1001, average particle size 1 μm) to the sample solution, and adjust the pH to 5. After shaking and mixing evenly, incubate at 65 °C for 10 min, centrifuge instantaneously, place the test tube on a magnetic stand for adsorption for 3 min, and remove the supernatant.

[0054] Rinse the magnetic beads with the washing solution and discard the supernatant. The composition of the washing solution is: 0.1 M EDTA, 0.05 M Tris, 3 M guanidine hydrochloride, 50% ethanol by volume, and the balance of water. Then rinse the magnetic beads with 70% ethanol and discard the supernatant.

[0055] Add 100 μL of elution solution to the magnetic beads to obtain a nucleic acid detection sample.

[0056] Example 6: This example provides a method for preparing a nucleic acid detection sample.

[0057] Collect an equal amount of standard influenza A virus samples using a sampling swab, and add them to 5 mL of virus preservation solution to obtain a sample solution. The virus preservation solution is pre-added with 0.05 g of chitosan / polyvinylpyrrolidone hydrogel, and placed on a shaker for 30 min to inactivate the virus and release nucleic acids, and the hydrogel fully adsorbs the free nucleic acids. Place the sample solution at room temperature, and take 200 μL samples at 0 d, 2 d, 4 d, and 8 d for nucleic acid extraction.

[0058] Add 10 μL of proteinase K and 5 μL of magnetic bead suspension (concentration: 20 mg / mL, silica-based magnetic beads, Hisian, product number: cz1001, average particle size 0.5 μm) to the sample solution, and adjust the pH to 5. After shaking and mixing evenly, incubate at 65 °C for 10 min, centrifuge instantaneously, place the test tube on a magnetic stand for adsorption for 3 min, and remove the supernatant.

[0059] Rinse the magnetic beads with the washing solution and discard the supernatant. The composition of the washing solution is: 0.1 M EDTA, 0.05 M Tris, 3 M guanidine hydrochloride, 50% ethanol by volume, and the balance of water. Then rinse the magnetic beads with 70% ethanol and discard the supernatant.

[0060] Add 100 μL of elution solution to the magnetic beads to obtain a nucleic acid detection sample.

[0061] Comparative Example 1: This comparative example provides a method for preparing a nucleic acid detection sample.

[0062] Use a sampling swab to collect an equal amount of the standard product of influenza A virus, add it to 5 mL of virus preservation solution to obtain a sample solution, and place it on a shaker for 30 min to fully inactivate the virus and release nucleic acid. Place the sample solution at room temperature, and take 200 μL of samples at 0 d, 2 d, 4 d, and 8 d for nucleic acid extraction.

[0063] Add 10 μL of proteinase K and 5 μL of magnetic bead suspension (concentration: 20 mg / mL, silica-based magnetic beads, Haisian, product number: cz1001, average particle size 1 μm) to the sample solution and adjust the pH to 5. After shaking and mixing evenly, incubate at 65 °C for 10 min, perform instantaneous centrifugation, place the test tube on a magnetic rack for adsorption for 3 min, and remove the supernatant.

[0064] Rinse the magnetic beads with the washing solution and discard the supernatant. The composition of the washing solution is: 0.1 M EDTA, 0.05 M Tris, 3 M guanidine hydrochloride, 50% (v / v) ethanol, and the balance of water. Then rinse the magnetic beads with 70% ethanol and discard the supernatant.

[0065] Add 100 μL of eluent to the magnetic beads to obtain a nucleic acid detection sample.

[0066] Comparative Example 2: This comparative example provides a method for preparing a nucleic acid detection sample.

[0067] Use a sampling swab to collect an equal amount of the standard product of influenza A virus, add it to 5 mL of virus preservation solution to obtain a sample solution. 0.05 g of chitosan / polyvinylpyrrolidone hydrogel is pre-added to the virus preservation solution. Place it on a shaker for 30 min to fully inactivate the virus and release nucleic acid, and the hydrogel fully adsorbs free nucleic acid. Place the sample solution at room temperature, and take 200 μL of samples at 0 d, 2 d, 4 d, and 8 d for nucleic acid extraction.

[0068] Add 10 μL of proteinase K and 5 μL of magnetic bead suspension (concentration: 20 mg / mL, silica-based magnetic beads, Haisian, product number: cz1001, average particle size 1 μm) to the sample solution and maintain the pH at 7.5. After shaking and mixing evenly, incubate at 65 °C for 10 min, perform instantaneous centrifugation, place the test tube on a magnetic rack for adsorption for 3 min, and remove the supernatant.

[0069] Rinse the magnetic beads with the washing solution and discard the supernatant. The composition of the washing solution is: 0.1 M EDTA, 0.05 M Tris, 3 M guanidine hydrochloride, 50% (v / v) ethanol, and the balance of water. Then rinse the magnetic beads with 70% ethanol and discard the supernatant.

[0070] Add 100 μL of eluent to the magnetic beads to obtain the nucleic acid detection sample.

[0071] Detect the above nucleic acid detection sample with an influenza A virus subtype N9 nucleic acid detection kit (PCR-fluorescence method, South China Bio) and compare the changes in its Cq value.

[0072] Table 1: Experimental data of Examples 1-6 and Comparative Examples 1-2.

[0073] Project 0d 2d 4d 8d Example 1 26.74 28.13 28.45 28.70 Example 2 26.85 28.25 28.62 28.95 Example 3 27.10 28.50 28.91 29.35 Example 4 27.50 29.03 29.88 30.51 Example 5 26.55 27.95 28.33 28.47 Example 6 26.32 27.87 28.22 28.67 Comparative Example 1 26.18 30.18 Cq NA Cq NA Comparative Example 2 29.41 Cq NA Cq NA Cq NA

[0074] According to Table 1 and Figure 2 - Figure 3 the data, it can be seen that the data of Examples 1-6 are all better than those of Comparative Examples 1-2. This is because the present application adds a pH-responsive hydrogel with a pH response range of 5-6 to the inactivated virus preservation solution. The latter is in a hydrophobic state in the inactivated virus preservation solution with a pH of 7.5-8 and will not dissolve in the virus preservation solution, so it presents a gel state. Under the electrostatic shielding effect provided by high salt, there is no electrostatic adsorption between the hydrogel and the nucleic acid. However, the hydrogel can store the viral nucleic acid inside the hydrogel through hydrophobic interaction, thus avoiding the degradation of nucleic acid during transportation. After the sample solution is enzymolyzed, by adjusting the pH, the hydrogel is converted into a hydrophilic state and thus dissolves in the virus preservation solution. The adsorption of the free hydrogel on the nucleic acid is reduced to less than 10%, thereby releasing the stored nucleic acid. Therefore, the magnetic beads can bind to the nucleic acid through non-electrostatic adsorption. When the hydrogel dissolves in the virus preservation solution, a steric hindrance effect can be generated to prevent the aggregation of magnetic beads, thereby improving the adsorption rate of nucleic acid. The Cq values of Examples 1-6 and Comparative Example 1 were close at 0 d, indicating that the recovery rate of nucleic acid using this scheme is relatively high, and the hydrogel can efficiently and reversibly adsorb nucleic acid. In Comparative Example 1, lacking the protection of the hydrogel, the nucleic acid degraded rapidly in the guanidine salt environment, and the Cq value increased rapidly. In Comparative Example 2, the pH was too high during magnetic bead adsorption, and it was difficult for the hydrogel to release nucleic acid, resulting in too low a recovery rate.

[0075] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0076] The above-described embodiments only represent several implementation manners of the present application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for preparing a nucleic acid detection sample, characterized in that, It includes the following steps: adding a pH-responsive hydrogel to an inactivated virus preservation solution and mixing them. The pH-responsive range of the pH-responsive hydrogel is 5-6. The pH-responsive hydrogel contains a pyridine polymer. The pH of the inactivated virus preservation solution is 7.5-8. The inactivated virus preservation solution contains 4-6 mol / L of guanidine salt. Using the inactivated virus preservation solution for virus sample collection to obtain a sample solution, adding proteinase K and magnetic beads to the sample solution and mixing evenly for enzymatic digestion, adjusting the pH to 5-6, incubating for 10-30 min, centrifuging the container containing the sample solution and fixing the magnetic beads to the bottom of the container by a magnet. The magnetic beads are silicon-based magnetic beads. Removing the supernatant, washing the magnetic beads at least once, and eluting the washed magnetic beads to obtain the nucleic acid detection sample.

2. The preparation method according to claim 1, characterized in that, The pyridine polymer contains polyvinylpyrrolidone.

3. The preparation method according to claim 1, wherein In the virus preservation solution, the addition amount of the pH-responsive hydrogel is 0.1-5% w / v.

4. The preparation method according to claim 1, wherein The average particle size of the pH-responsive hydrogel is 1-10 μm.

5. The preparation method according to claim 1, characterized in that, The hydrogel is prepared by free radical polymerization.

6. The preparation method according to claim 1, characterized in that, The virus preservation solution contains 4-6 mol / L of guanidine salt, 0.1-0.3 mol / L of Tris-HCl, 5-20 mmol / L of EDTA, 0.05-0.5% by mass of sodium azide, and 0.01-0.05% by mass of Triton X-100.

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