Nucleic acid extraction method and nucleic acid extraction kit

Abstract

The invention relates to the technical field of nucleic acid extraction, in particular to a nucleic acid extraction method and a nucleic acid extraction kit. The nucleic acid extraction kit can remove protein in a sample and further release / purify nucleic acid. According to the nucleic acid extraction method, the protease K is added in the washing liquid of the extraction reagent, protein which is deeply combined can be treated, nucleic acid is further released, high-temperature dissociation is used in the nucleic acid elution process, the protease K is inactivated, and the influence on subsequent amplification is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid extraction, in particular to a nucleic acid extraction method and a nucleic acid extraction kit. Background Art

[0002] Nucleic acid extraction or purification is a common step in molecular biology experiments, used to extract pure nucleic acids (such as DNA or RNA) from cell or tissue samples. The basic principle of this process is to separate nucleic acids from other cellular components using chemical, physical, or biological methods.

[0003] The basic steps of nucleic acid extraction or purification include: Cell disruption: Use physical or chemical methods to break the cell membrane and release the nucleic acids in the cell. Dissolution and dilution: Dissolve the sample in an appropriate buffer to dilute the interference of other components in the cell. Protein elimination: Separate proteins from nucleic acids through the action of proteases or organic solvent extraction. Removal of pollutants: Remove impurities and contaminants in the sample through alcohol precipitation, silica gel columns, centrifugation or magnetic beads. Precipitation or extraction of nucleic acids: Utilize the characteristics of the buffer to precipitate nucleic acids under appropriate pH conditions, or extract and purify nucleic acids through column chromatography or magnetic bead extraction. Washing and drying: Wash the precipitated nucleic acids to remove residual contaminants, and dry the nucleic acids to obtain pure nucleic acid products. Through the above steps, pure nucleic acids extracted from cells or tissues can be obtained for subsequent molecular biology experiments, such as PCR, gene cloning, sequencing, etc. The choice of nucleic acid extraction method depends on the sample type, nucleic acid type and experimental purpose.

[0004] The products of nucleic acid extraction and purification are generally used for further downstream analysis and testing, such as PCR or sequencing. The quality of the extracted products directly affects the reliability of the measured results. Currently, proteinase K is commonly added during the sample cleavage step during nucleic acid extraction or purification. In the process of realizing the present invention, the inventors discovered that the nucleic acids of some samples are specifically bound to proteins. After cleavage, washing, and elution with conventional nucleic acid extraction reagents, the nucleic acids and proteins are still difficult to separate, which has a certain impact on subsequent PCR detection. Summary of the Invention

[0005] In view of this, the present invention provides a nucleic acid extraction method and a nucleic acid extraction kit. The nucleic acid extraction kit of the present invention can remove protein from the sample, further release / purify nucleic acids, and use high temperature dissociation during the nucleic acid elution process to inactivate proteinase K to avoid affecting subsequent amplification.

[0006] In some specific embodiments of the present invention, the methods, components, and kits described herein are further used to release / purify nucleic acids from a lysed sample.

[0007] To achieve the above object, the present application provides the following technical solutions.

[0008] The present application provides a method for extracting nucleic acid, comprising the following steps:

[0009] Step S1, lysing the sample to be tested to obtain a lysed sample;

[0010] Step S2, washing the lysed sample with a washing solution containing proteinase K;

[0011] Step S3, separating to obtain the nucleic acid;

[0012] The concentration of the proteinase K in step S2 includes no less than 0.6 U / mL.

[0013] In some embodiments of the present application, the washing time in step S2 includes 10-600 s.

[0014] In some embodiments of the present application, the washing solution in step S2 further includes a combination of one or more of a buffer, a preservative, a surfactant or a reducing agent;

[0015] The buffer includes one or more of Tris-HCl buffer, citrate buffer, phosphate buffer; or

[0016] The preservative includes one or more of NaN3, P300, P950; or

[0017] The surfactant includes one or more of SDS, CTAB, Triton X-100, Brij35, NP40; or

[0018] The reducing agent includes DTT or β-mercaptoethanol.

[0019] In some embodiments of the present application, the washing solution in step S2 includes 0.1%-40% buffer, 0.1%-5% preservative and 0.6 U-80 U / mL proteinase K in g / mL.

[0020] In some embodiments of the present application, the washing solution in step S2 further includes 1%-25% surfactant or 0.5%-20% reducing agent in g / mL.

[0021] In some embodiments of the present application, the washing solution in step S2 includes 0.1%-2% Tris-HCl, 0.01%-1% P300 and 0.6 U-80 U / mL proteinase K in g / mL.

[0022] In some specific embodiments of the present invention, the separation in step S3 comprises separation using magnetic beads or centrifugal columns.

[0023] In some specific embodiments of the present invention, the extraction method comprises the following steps:

[0024] Step S1: Mix the sample to be tested with the lysis solution, incubate at 10-60°C, and separate to obtain a lysed sample;

[0025] Step S2, washing the lysed sample with the washing solution to obtain a washed sample;

[0026] Step S3: separation to obtain the nucleic acid.

[0027] In some specific embodiments of the present invention, step S1 includes mixing the sample to be tested, proteinase K and lysis buffer, incubating at 10-60° C., and separating to obtain a lysed sample.

[0028] In some specific embodiments of the present invention, the lysate comprises, in g / mL, 10% to 46% of a saline solution, 1% to 25% of a surfactant, 5% to 40% of a buffer solution, and the balance of water.

[0029] Preferably, the saline solution comprises guanidine isothiocyanate, guanidine thiocyanate or sodium chloride.

[0030] In some specific embodiments of the present invention, step S3 further includes a step of removing proteinase K before or after the separation step.

[0031] In some specific embodiments of the present invention, the step of removing proteinase K comprises high temperature inactivation or centrifugal column purification.

[0032] In some specific embodiments of the present invention, the temperature of the high-temperature inactivation ranges from 70° C. to 100° C.; and the time of the high-temperature inactivation is ≥2 min.

[0033] The present invention also provides a combined reagent comprising a lysis solution and a washing solution; the washing solution comprises proteinase K at a concentration of not less than 0.6 U / mL.

[0034] In some specific embodiments of the present invention, when the amount of proteinase K added to the washing solution reaches 80 U / mL, the nucleic acid extraction efficiency of proteinase K will no longer be further improved when the amount of proteinase K is further increased. When cost control is required, the washing solution includes 0.6 U~80 U / mL proteinase K.

[0035] In some embodiments of the present invention, the wash solution further comprises a combination of one or more of a buffer, a preservative, a surfactant, or a reducing agent;

[0036] The buffer comprises one or more of Tris-HCl buffer, citrate buffer, and phosphate buffer; or

[0037] The surfactant includes one or more of SDS, CTAB, Triton X-100, Brij35, and NP40; or

[0038] The preservative includes one or more of NaN3, P300, and P950; or

[0039] The reducing agent includes DTT or β-mercaptoethanol.

[0040] In some specific embodiments of the present invention, the washing solution comprises, in terms of g / mL, 0.1% to 40% buffer, 0.1% to 5% preservative, and 0.6 U to 80 U / mL proteinase K.

[0041] In some specific embodiments of the present invention, the washing solution further comprises 1% to 25% of a surfactant or 0.5% to 20% of a reducing agent, calculated in g / mL.

[0042] In some specific embodiments of the present invention, the washing solution comprises, in terms of g / mL, 0.1%-2% Tris-HCl, 0.01%-1% P300, and 0.6U-80U / mL of proteinase K.

[0043] In some specific embodiments of the present invention, the lysate comprises, in g / mL, 10% to 46% saline solution, 1% to 25% surfactant, 5% to 40% buffer, and the balance water. Preferably, the saline solution comprises guanidine isothiocyanate, guanidine thiocyanate, or sodium chloride.

[0044] In some specific embodiments of the present invention, the nucleic acid extraction kit further comprises an eluent, which comprises, in g / mL, 0.1% to 5% of a preservative, 5% to 40% of a buffer, and the remainder being water.

[0045] In some specific embodiments of the present invention, the pH value of the lysate is comprised between 3.5 and 8.0; the pH value of the washing solution is comprised between 5.0 and 8.0; and the pH value of the eluent is comprised between 5.0 and 9.5.

[0046] In some specific embodiments of the present invention, the pH value of the lysate is 4.0; the pH value of the washing solution is 6.8; and the pH value of the eluent is 8.8.

[0047] The present invention also provides a nucleic acid extraction kit, comprising the combined reagents and an acceptable auxiliary agent or carrier.

[0048] The application also provides application of any of the following in extracting nucleic acid in a biological sample:

[0049] (I) the combination reagent; and / or

[0050] (II) the nucleic acid extraction kit.

[0051] In some specific embodiments of the application, the sample to be tested comprises one or more of a virus, a fungus, a bacterium, mycoplasma or chlamydia.

[0052] Preferably, the virus comprises hepatitis B virus.

[0053] The application provides the following advantages:

[0054] The nucleic acid extraction method of the application adds proteinase K in the washing solution of the extraction reagent, can treat the deeply bound protein, makes the nucleic acid further release, detects the proteinase K in the washing solution, and the target detection Ct value is advanced by 1-2 (the target nucleic acid concentration is increased by about 2-4 times). The detection effect is better than that of the washing solution formula without proteinase K. High temperature dissociation is used in the nucleic acid elution process, the proteinase K is inactivated, the subsequent amplification is avoided, generally needs to be maintained at 70 DEG C or more, and the maintaining time is more than 2 min. DETAILED DESCRIPTION

[0055] The application discloses a nucleic acid extraction method and a nucleic acid extraction kit, and those skilled in the art can refer to the content herein, and appropriately improve process parameters to realize. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as being included in the application. The method and application of the application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the application, to realize and apply the technology of the application.

[0056] At present, common nucleic acid extraction or purification products and patents all add proteinase K in the sample lysis step (Table 1). Some nucleic acids are specially combined with proteins, so that the nucleic acid and the protein are not easy to separate, and therefore have a certain influence on subsequent PCR detection.

[0057] Table 1 Common nucleic acid extraction or purification process of proteinase K

[0058]

[0059] This invention provides a method for removing proteins from samples and further releasing / purifying nucleic acids. A unique feature of this method is the addition of proteinase K to the wash solution in the extraction reagent, which can treat deeply bound proteins and further release nucleic acids. Furthermore, during the nucleic acid elution process, high-temperature dissociation is used to inactivate proteinase K and prevent it from interfering with subsequent amplification. This process typically requires temperatures above 70°C for at least 2 minutes.

[0060] Wash solutions typically include a buffer, preservatives, and proteinase K. Typical buffers include Tris-HCl (0.1%–2%), citrate buffer (5%–40%), or phosphate buffer (5%–40%). Preservatives include NaN₃ (0.01%–1%), P300 (0.01%–1%), and P950 (0.01%–1%). The buffer is used to maintain a stable pH (Tris buffer). EDTA can also be added to chelate metal ions such as Mg²⁺ / Ca²⁺, inhibiting nuclease activity and preventing metal ion-mediated oxidative degradation of nucleic acids.

[0061] Proteinase K must be added to the wash buffer. Adding the same amount to the lysis process will not produce the same effect. Surfactants or reducing agents can be added to the wash buffer. Surfactants such as NP40 (1%-25%) and reducing agents such as DTT (0.5%-20%) do not affect the action of Proteinase K. Proteinase K is used to digest structural proteins within cells (such as cytoskeletal proteins and nuclear matrix) and is particularly suitable for protein-rich samples (such as tissue and blood). Surfactants may act by disrupting the lipid bilayer of the cell membrane / cell wall, releasing nucleic acids; denaturing proteins, and unbinding nucleic acids from proteins (such as histones). Reducing agents may act by breaking protein disulfide bonds, enhancing lysis; and protecting nucleic acids from oxidative damage.

[0062] The lysate used in the extraction process typically contains a certain concentration of salt solution, surfactant, and buffer. Examples of salt solutions include guanidine isothiocyanate (10%-46%), guanidine thiocyanate (10%-46%), and sodium chloride (10%-46%). Buffers include Tris-HCl (5%-40%), Trizma hydrochloride (5%-40%), citrate buffer (1%-8%), or phosphate buffer (1%-8%). Surfactants include SDS (1%-15%), Triton (1%-15%), and Brij35 (1%-35%). Salt solutions can strongly denature proteins, disrupting hydrogen bonds and hydrophobic interactions; inhibit nuclease (DNase / RNase) activity; and promote the binding of nucleic acids to silica membranes / magnetic beads (for subsequent purification).

[0063] The eluent used in the extraction process is generally Tris-HCl buffer (0.1%~2%), with preservatives such as P300 (0.01%~1%) added.

[0064] Magnetic beads or centrifugal columns are also used in the extraction, usually commercial magnetic beads or centrifugal columns.

[0065] Unless otherwise specified, “%” throughout the text refers to mass-volume concentration, which is defined as the ratio of the solute mass (unit: g) to the solution volume (unit: mL), that is, the unit is g / mL.

[0066] Unless otherwise specified, the nucleic acid extraction method and the raw materials and reagents used in the nucleic acid extraction kit provided by the present invention can be purchased from the market.

[0067] The present invention will be further described below in conjunction with the embodiments:

[0068] Example 1 Nucleic acid extraction or purification

[0069] 1. Instruments and reagents

[0070] Real-time fluorescence quantitative PCR instrument (qTOWER Jena Analytical Instruments, Germany) 3 G IVD), nucleic acid extraction or purification reagents (see the formula shown in Table 2), hepatitis B virus nucleic acid detection kit (PCR-fluorescence probe method) (National Medical Device Registration No. 20253400064) (FAM channel is the target channel, ROX channel is the internal standard channel), hereinafter referred to as HBV, hepatitis B virus positive samples, hereinafter referred to as HBV samples.

[0071] 2. Preparation of nucleic acid extraction and purification solution - Formula 1

[0072] (1) Preparation of lysis buffer

[0073] Table 2 Lysis buffer formula

[0074]

[0075] Add about 60% of the total volume of purified water to the container, add the above chemical reagents at one time, and after the reagents are fully dissolved, measure the pH value of the solution and adjust it to 4.0. Finally, add purified water to 1000 mL.

[0076] (2) Preparation of lotion

[0077] Table 3 Wash solution (containing 80 U / mL proteinase K) formula

[0078]

[0079] Add 80% of the required volume of purified water to the container. Then add the required amounts of Trizma hydrochloride, Proteinase K, and P300, and mix thoroughly. Measure the pH of the solution, adjust it to 6.80, and add more purified water to 1000 mL.

[0080] (3) Preparation of eluent

[0081] Table 4 Eluent formula

[0082]

[0083] Add 80% of the required total volume of purified water to the container, then add the required amount of Tris hydrochloride, P300, etc. and mix thoroughly. Measure the pH of the solution and adjust it to pH 8.80. Add more purified water to 1000 mL.

[0084] Preparation of nucleic acid extraction and purification solution - Formula 2

[0085] (1) Preparation of lysis buffer

[0086] Table 5 Lysis buffer formula

[0087]

[0088] Add about 60% of the total volume of purified water to the container, add the above chemical reagents at one time, and after the reagents are fully dissolved, measure the pH value of the solution and adjust it to 4.0. Finally, add purified water to 1000 mL.

[0089] (2) Preparation of lotion

[0090] Table 6 Wash solution (containing 0.8U / mL proteinase K) formula

[0091]

[0092] Add 80% of the required total volume of purified water to the container. Then add the required amounts of Trizma hydrochloride, Proteinase K, and P300, and mix thoroughly. Measure the pH of the solution and adjust it to pH 6.80. Add more purified water to 1000 mL.

[0093] (3) Preparation of eluent

[0094] Table 7 Eluent formula

[0095]

[0096] Add 80% of the required total volume of purified water to the container, then add the required amount of Tris hydrochloride, P300, etc. and mix thoroughly. Measure the pH of the solution and adjust it to pH 8.80. Add more purified water to 1000 mL.

[0097] 3. Nucleic acid extraction or purification steps

[0098] (1) Add 10 μL proteinase K (greater than 800 U / mL, Roche), 600 μL HBV sample, 100 μL magnetic bead suspension (mix the magnetic beads before aspiration, Du Bio MS02H), and 1.2 mL lysis buffer to the reaction vessel, mix well, and incubate in a 40°C incubator for 2 min;

[0099] (2) Magnetic aspiration of the incubated mixed solution for 2 minutes and discard the waste liquid;

[0100] (3) Add 2 mL of washing solution, mix for 30 seconds, and magnetically aspirate for 2 minutes to discard the waste liquid;

[0101] (4) Add 100 μL (according to the requirements of subsequent experiments) of eluent, mix well, and dissociate in a dry-bed thermostat at 80°C for 5 min.

[0102] Example 2 Nucleic acid extraction or purification

[0103] Nucleic acid extraction or purification steps (lysis step without proteinase K):

[0104] (1) Add 600 μL of HBV sample, 100 μL of magnetic bead suspension (mix the magnetic beads before aspiration, use Du Bio MS02H), and 1.2 mL of lysis buffer to the reaction vessel, mix well, and incubate in a 40°C incubator for 2 min;

[0105] (2) Magnetic aspiration of the incubated mixed solution for 2 minutes and discard the waste liquid;

[0106] (3) Add 2 mL of washing solution, mix for 30 seconds, and magnetically aspirate for 2 minutes to discard the waste liquid;

[0107] (4) Add 100 μL (according to the requirements of subsequent experiments) of eluent, mix well, and dissociate in a dry-bed thermostat at 80°C for 5 min.

[0108] The remaining operations are the same as in Example 1.

[0109] Comparative Example 1

[0110] The washing solution in Example 1 does not contain proteinase K, and the remaining operations are the same as in Example 1.

[0111] Table 8 Wash solution (excluding proteinase K) formula

[0112]

[0113] In a container, add 80% of the required total volume of purified water, and then add the required amount of Trizma hydrochloride P300, etc., and mix well. Measure the pH of the solution, adjust the pH of the solution to pH 6.80, and add purified water to 1000 mL.

[0114] Comparative Example 2

[0115] The wash solution in Example Formulation 2 does not contain protease K, and the rest of the operations are the same as in Example 1.

[0116] Table 9 Wash solution (without protease K) formulation

[0117]

[0118] In a container, add 80% of the required total volume of purified water, and then add the required amount of Trizma hydrochloride P300, etc., and mix well. Measure the pH of the solution, adjust the pH of the solution to pH 6.80, and add purified water to 1000 mL.

[0119] Comparative Example 3

[0120] In the wash solution in Example Formulation 1, protease K is replaced by NP-40, and the rest of the operations are the same as in Example 1.

[0121] Table 10 Wash solution (without protease K, with increased NP-40) formulation

[0122]

[0123] In a container, add 80% of the required total volume of purified water, and then add the required amount of Trizma hydrochloride, Nonidet P40, P300, etc., and mix well. Measure the pH of the solution, adjust the pH of the solution to pH 6.80, and add purified water to 1000 mL.

[0124] Effect Example 1 Comparison of detection results of different wash solution formulations

[0125] The wash solutions of Examples and Comparative Examples 1-3, i.e. two formulations containing and not containing protease K, were used to extract hepatitis B virus samples, which contained three different concentrations of samples (about 1000 IU / mL, 50 IU / mL and 10 IU / mL, respectively), and then the amplification products were detected by PCR amplification (50°C for 2 min, 95°C for 2 min, 95°C for 10 s, 60°C for 22 s).

[0126] Table 11 Detection results of different wash solution formulations of Formulation 1

[0127]

[0128] Table 12 Test results of different lotion formulas of formula 2

[0129]

[0130] Judging from the results, when the wash solution contains proteinase K, the target detection Ct value is advanced by 1~2. It is generally believed that the detection Ct is greater than 1, and the difference in concentration value is 1 times. 1~2, the difference in concentration value is 1~4 times. The detection effect is better than the wash solution formula without proteinase K. And formula 1 is significantly better than formula 2, so formula 1 is selected as the formula for subsequent research. According to the formula of the experimental group, a scheme verification without adding proteinase K in the lysis step is also set. From the results, it can be seen that proteinase K added to the wash solution can process deeply bound proteins and further release nucleic acids. When the wash solution contains proteinase K, the target detection Ct value is advanced. Proteinase K in the lysis step cannot achieve the effect of this scheme.

[0131] Effect Example 2 Comparison of test results after adjusting the position of proteinase K

[0132] To further clarify whether the effect of proteinase K can be exerted in other places, the following experiments were performed:

[0133] Control group: Proteinase K was removed from the wash solution of Formula 1 and an equal amount of Proteinase K was added during the lysis step.

[0134] Experimental group: Formula 1 washing solution contains proteinase K.

[0135] The remaining operations were the same as in Example 1, and HBV testing was performed using two sets of conditions. The test results are as follows:

[0136] Table 13 Adjustment of Proteinase K Position Detection Results

[0137]

[0138] Judging from the results, adjusting the proteinase K in the washing solution to the cleavage step did not improve the detection situation, and the target detection Ct value was still 1 to 2 values ​​lower than that of the experimental group.

[0139] Effect Example 3: The influence of different dissociation temperatures on the test results

[0140] The final step in the extraction process is to dissociate the nucleic acids from the magnetic beads for subsequent PCR analysis and to inactivate proteinase K in the wash solution. Therefore, the effects of different dissociation temperatures on test results were investigated. To investigate the impact of dissociation conditions on subsequent detection, the extraction process was performed at different temperatures, including 65°C, 70°C, 75°C, 80°C, and 85°C. The remaining steps were the same as in Example 1. A sample with a low HBV level (approximately 10 IU / mL) was tested, and the test results are shown below.

[0141] Table 14 Effects of different dissociation temperatures on test results

[0142]

[0143] Judging from the test results, when the dissociation temperature is 65°C, the proteinase K in the washing solution cannot be inactivated, resulting in a decrease in the detection Ct or missed detection.

[0144] Effect Example 4: Effect of different proteinase K concentrations on test results

[0145] In order to study the effect of different concentrations of proteinase K on subsequent detection, the concentrations of proteinase K in the washing solution were set to 0, 0.6 U / mL, 80 U / mL, and 100 U / mL, respectively. The remaining steps were the same as in Example 1. The HBV low-value sample was tested, and the test results are as follows.

[0146] Table 15 Effects of different proteinase K concentrations on test results

[0147]

[0148] From the test results, it can be seen that as the concentration of proteinase K in the washing solution increases, the extraction effect improves (the Ct mean exceeds 0.5Ct in advance). When the concentration is increased to 80U / mL, the improvement effect no longer increases (Ct no longer advances).

[0149] Effect Example 4: Effect of using Nonidet P40 to replace proteinase K in the wash solution

[0150] To investigate whether proteinase K could be used as an alternative in the wash solution, the proteinase K in the wash solution was replaced with the surfactant Nonidet P40, which has a similar effect (Comparative Example 3, Table 10). The results of the test for low-HBV samples are as follows.

[0151] Table 16 Effects of changing components in the washing solution on the test results

[0152]

[0153] From the test results, when proteinase K in the washing solution was replaced by Nonidet P40, the test Ct decreased by 1 to 2 units. The surfactant Nonidet P40 could not replace the role of proteinase K in the washing solution.

[0154] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting nucleic acid, characterized in that: The steps include: Step S1, lysing the sample to be tested to obtain a lysed sample; Step S2, washing the lysed sample with a washing solution containing proteinase K; Step S3, separating to obtain the nucleic acid; The concentration of proteinase K in step S2 is not less than 0.6 U / mL.

2. The extraction method according to claim 1, wherein The washing solution in step S2 further comprises a combination of one or more of a buffer, a preservative, a surfactant or a reducing agent; The buffer comprises one or more of Tris-HCl buffer, citrate buffer, and phosphate buffer; or The preservative includes one or more of NaN3, P300, and P950; or The surfactant includes one or more of SDS, CTAB, Triton X-100, Brij35, and NP40; or The reducing agent includes DTT or β-mercaptoethanol.

3. The extraction method according to claim 1 or 2, wherein The separation in step S3 includes separation using magnetic beads or centrifugal columns.

4. The extraction method according to any one of claims 1 to 3, characterized in that Step S3 further includes a step of removing proteinase K before or after the separation step.

5. A combination reagent, characterized in that The invention comprises a lysis solution and a washing solution; the washing solution comprises no less than 0.6 U / mL of proteinase K; preferably, the washing solution comprises 0.6 U to 80 U / mL of proteinase K.

6. The combination reagent according to claim 5, characterized in that The washing solution further comprises a combination of one or more of a buffer, a preservative, a surfactant or a reducing agent; The buffer comprises one or more of Tris-HCl buffer, citrate buffer, and phosphate buffer; or The surfactant includes one or more of SDS, CTAB, Triton X-100, Brij35, and NP40; or The preservative includes one or more of NaN3, P300, and P950; or The reducing agent includes DTT or β-mercaptoethanol.

7. The combined reagent according to claim 5 or 6, characterized in that The pH value of the lysate is 3.5-8.0; the pH value of the washing solution is 5.0-8.0; and the pH value of the eluent is 5.0-9.

5.

8. A nucleic acid extraction kit, characterized in that The method comprises the combined agent according to any one of claims 5 to 7 and an acceptable auxiliary agent or carrier.

9. Use of any of the following in extracting nucleic acids from biological samples: (I) The combination agent according to any one of claims 5 to 7; and / or (II) The nucleic acid extraction kit according to claim 8.

10. The use according to claim 9, characterized in that The biological sample includes one or more of viruses, fungi, bacteria, mycoplasmas or chlamydia.

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