Nucleic acid extraction and purification reagent, kit and use method

Abstract

The invention discloses a nucleic acid extraction and purification reagent, a kit and a use method. Reagents of the kit comprise a cell lysis solution, a digestive enzyme, a binding solution, a rinsing solution and an eluent, wherein the binding solution contains an alcoholic solution and sodium salt. Compared with similar kits on the market, the kit provided by the invention has the advantages that a sodium salt reagent is also added in the binding solution besides an alcoholic solution, meanwhile, the mass ratio of sodium salt is optimized, the DNA extract can be efficiently and selectively adsorbed on a centrifugal column silicon matrix membrane by adding the reagent, the yield and purity of obtained nucleic acid are remarkably improved, and the kit is suitable for large-scale popularization and application. Meanwhile, the component NaCl of the lysate is optimized and reduced, and the steps in the preparation and production process are reduced. The reagent / kit is simple in composition and low in cost, the operation method is simple and convenient, large-scale nucleic acid extraction operation is facilitated, in addition, the kit does not contain toxic and harmful reagents such as methylbenzene, dimethylbenzene, phenol and chloroform, harm to the environment and operators is small, and the modern environmental protection concept is better met.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a nucleic acid extraction and purification reagent, kit, and method of use. Background Technology

[0002] Nucleic acid extraction is fundamental to various molecular biology detection methods. Efficient and complete DNA extraction is essential for PCR amplification, library construction, and sequencing. Besides tissue and organ samples, blood samples are the most commonly used ex vivo samples in molecular biology detection. Currently known DNA extraction methods, such as the phenol-chloroform method, adsorption column method, and magnetic bead method, can be used for blood DNA extraction. However, the phenol-chloroform method, due to the use of toxic organic solvents, suffers from poor nucleic acid quality, cumbersome procedures, and long processing time. Currently, the main nucleic acid extraction methods include silica gel membrane adsorption column method and magnetic bead method. Column extraction can obtain high-purity nucleic acids, but the extraction steps are cumbersome, requiring multiple centrifugations, and the entire process is time-consuming. Magnetic bead extraction, combined with automated extraction instruments, can significantly shorten the extraction time compared to column extraction; however, simultaneous processing of multiple samples poses a risk of cross-contamination, and the cost of extraction instruments and reagents is relatively high.

[0003] The main bottleneck in the widespread use of molecular diagnostics in medical laboratories is the requirement to purify nucleic acids from samples, especially for detection during disease (cancer) treatment surgeries. Therefore, there is an urgent need to develop safe, simple, rapid, and high-quality nucleic acid extraction methods. Summary of the Invention

[0004] The purpose of this invention is to provide a nucleic acid extraction and purification reagent, a kit, and a method for using it.

[0005] In one aspect, the present invention claims protection for a reagent used for nucleic acid extraction and purification.

[0006] The reagents for nucleic acid extraction and purification claimed in this invention include cell lysis buffer, digestive enzyme, binding buffer, washing buffer, and elution buffer; wherein the binding buffer contains an alcohol solution and a sodium salt.

[0007] Furthermore, the alcohol solution is anhydrous ethanol or isopropanol.

[0008] Furthermore, the sodium salt may be at least one of sodium acetate, sodium sulfate, sodium phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0009] In a specific embodiment of the present invention, the binding solution is composed of anhydrous ethanol and sodium acetate, wherein the mass percentage of sodium acetate is 0.6-1.0%; and the pH value of the binding solution is 6.5-8.5.

[0010] Specifically, in the first embodiment of the present invention, the binding solution is composed of anhydrous ethanol and sodium acetate, wherein the mass percentage of sodium acetate is 0.6%; and the pH value of the binding solution is 6.5. In the second embodiment of the present invention, the binding solution is composed of anhydrous ethanol and sodium acetate, wherein the mass percentage of sodium acetate is 0.8%; and the pH value of the binding solution is 7.5. In the third embodiment of the present invention, the binding solution is composed of anhydrous ethanol and sodium acetate, wherein the mass percentage of sodium acetate is 1.0%; and the pH value of the binding solution is 8.5.

[0011] Furthermore, the lysis solution may contain Tris-HCl, guanidine salt, EDTA, nonionic surfactant, and has a pH value of 5.0-8.0.

[0012] Furthermore, the rinsing solution is composed of rinsing solution A1 and rinsing solution A2; rinsing solution A1 may contain guanidine hydrochloride and anhydrous ethanol, with a pH value of 6.0-8.0; rinsing solution A2 may contain KAC, Tris-HCl and anhydrous ethanol, with a pH value of 7.0-9.0.

[0013] Furthermore, the eluent may contain Tris-HCl and have a pH of 7.0-9.0.

[0014] In a specific embodiment of the present invention, the solvent of the lysis buffer is water, and the solutes and their concentrations are as follows: 1.0-4.0 mol / L guanidine salt, 0.1-0.4 mol / L EDTA, 5-10% (v / v) nonionic surfactant, and 0.1-0.4 mol / L Tris-HCl. Each concentration represents the final concentration in the lysis buffer.

[0015] Specifically, in the first embodiment of the present invention, the solvent of the lysis buffer is water, and the solutes and concentrations are as follows: 1.0 mol / L guanidine salt (guanidine hydrochloride), 0.1 mol / L EDTA, 5.0% by volume of nonionic surfactants (0.25% Triton 100, 4.5% Tween 20, 0.25% NP40), and 0.1 mol / L Tris-HCl; the pH of the lysis buffer is 5.0. In the second embodiment of the present invention, the solvent of the lysis buffer is water, and the solutes and concentrations are as follows: 2.5 mol / L guanidine salt (guanidine hydrochloride), 0.25 mol / L EDTA, 7% by volume of nonionic surfactants (0.5% Triton 100, 6% Tween 20, 0.5% NP40), and 0.25 mol / L Tris-HCl; the pH of the lysis buffer is 6.5. In a third embodiment of the present invention, the solvent of the lysis buffer is water, and the solutes and their concentrations are as follows: 4 mol / L guanidine salt (guanidine hydrochloride), 0.4 mol / L EDTA, 10% by volume of nonionic surfactants (1% Triton 100, 8% Tween 20, 1% NP40), and 0.4 mol / L Tris-HCl; the pH value of the lysis buffer is 8.0. The above concentrations are the final concentrations in the lysis buffer.

[0016] In a specific embodiment of the present invention, the solvent of the rinsing solution A1 is water, and the solutes and concentrations are as follows: 2-8 mol / L guanidine hydrochloride, and 60%-80% anhydrous ethanol by volume. Each concentration represents the final concentration in the rinsing solution A1.

[0017] Specifically, in the first embodiment of the present invention, the solvent of the rinsing solution A1 is water, and the solute and concentration are as follows: 2 mol / L guanidine hydrochloride, 60% anhydrous ethanol (volume percentage); the pH of the rinsing solution A1 is 6.0. In the second embodiment of the present invention, the solvent of the rinsing solution A1 is water, and the solute and concentration are as follows: 5 mol / L guanidine hydrochloride, 70% anhydrous ethanol (volume percentage); the pH of the rinsing solution A1 is 7.0. In the third embodiment of the present invention, the solvent of the rinsing solution A1 is water, and the solute and concentration are as follows: 8 mol / L guanidine hydrochloride, 80% anhydrous ethanol (volume percentage); the pH of the rinsing solution A1 is 8.0. The above concentrations are the final concentrations in the rinsing solution A1.

[0018] In a specific embodiment of the present invention, the solvent of the rinsing solution A2 is water, and the solutes and concentrations are as follows: 0.05-1.5 mol / L KAC, 70-80% anhydrous ethanol (volume percentage), and 2.5-8.5 mol / L Tris-HCl. The above concentrations are the final concentrations in the rinsing solution A2.

[0019] Specifically, in the first embodiment of the present invention, the solvent of the rinsing solution A2 is water, and the solutes and concentrations are as follows: 0.05 mol / L KAC, 70% anhydrous ethanol (volume percentage), and 2.5 mol / L Tris-HCl; the pH of the rinsing solution A2 is 7.0. In the second embodiment of the present invention, the solvent of the rinsing solution A2 is water, and the solutes and concentrations are as follows: 0.10 mol / L KAC, 75% anhydrous ethanol (volume percentage), and 5.5 mol / L Tris-HCl; the pH of the rinsing solution A2 is 8.0. In the third embodiment of the present invention, the solvent of the rinsing solution A2 is water, and the solutes and concentrations are as follows: 1.5 mol / L KAC, 80% anhydrous ethanol (volume percentage), and 8.5 mol / L Tris-HCl; the pH of the rinsing solution A2 is 9.0. The above concentrations are the final concentrations in the rinsing solution A2.

[0020] In a specific embodiment of the present invention, the solvent of the eluent is water, and the solute and concentration are as follows: 3-9 mol / L Tris-HCl. The concentration is the final concentration in the eluent.

[0021] Specifically, in the first embodiment of the present invention, the solvent of the eluent is water, and the solute and concentration are as follows: 3 mol / L Tris-HCl; the pH value of the eluent is 7.0. In the second embodiment of the present invention, the solvent of the eluent is water, and the solute and concentration are as follows: 6 mol / L Tris-HCl; the pH value of the eluent is 8.0. In the third embodiment of the present invention, the solvent of the eluent is water, and the solute and concentration are as follows: 9 mol / L Tris-HCl; the pH value of the eluent is 9.0. The above concentrations are the final concentrations in the eluent.

[0022] Furthermore, in the lysis solution, the guanidine salt may be at least one of guanidine isothiocyanate, guanidine hydrochloride, and guanidine thiocyanate.

[0023] In a specific embodiment of the present invention, the guanidine salt is guanidine hydrochloride.

[0024] Furthermore, in the lysis solution, the nonionic surfactant may be at least one of Triton 100, Tween 20, SDS, and NP40;

[0025] In a specific embodiment of the present invention, the nonionic surfactant is specifically composed of Triton 100, Tween 20, and NP40. In the lysis solution, the concentration of Triton 100 can be 0.1-1%, the concentration of Tween 20 can be 3-8%, and the concentration of NP40 can be 0.1-1%. All percentages above represent volume percentages.

[0026] Specifically, in the first embodiment of the present invention, the lysis buffer contains 0.1% Triton 100, 3% Tween 20, and 0.1% NP40. In the second embodiment of the present invention, the lysis buffer contains 0.5% Triton 100, 6% Tween 20, and 0.5% NP40. In the third embodiment of the present invention, the lysis buffer contains 1% Triton 100, 8% Tween 20, and 1% NP40. All percentages above represent volume percentages.

[0027] Furthermore, the digestive enzyme may be proteinase K (manufacturer: Tiangen Biotech (Beijing) Co., Ltd., product number: RT403-02).

[0028] Furthermore, the concentration of the proteinase K can be 10-20 mg / mL.

[0029] Specifically, in the first embodiment of the present invention, the concentration of proteinase K is 10 mg / mL. In the second embodiment of the present invention, the concentration of proteinase K is 15 mg / mL. In the third embodiment of the present invention, the concentration of proteinase K is 20 mg / mL.

[0030] Secondly, the present invention claims protection for a kit for nucleic acid extraction and purification.

[0031] The present invention claims a kit for nucleic acid extraction and purification, including the nucleic acid extraction and purification reagents described in the first aspect above.

[0032] Furthermore, the kit may also contain an adsorbent.

[0033] In this invention, the adsorbent refers to a substance that can specifically adsorb nucleic acids onto its surface and can specifically separate from nucleic acids under specific conditions.

[0034] Furthermore, the adsorbent can be an adsorption column.

[0035] In a specific embodiment of the present invention, the adsorption column is specifically an adsorption column TC1.

[0036] Thirdly, the present invention claims protection for the use of the nucleic acid extraction and purification reagents described in the first aspect above or the kits described in the second aspect above in the process of nucleic acid extraction and purification.

[0037] Fourthly, this invention claims protection for a method for nucleic acid extraction and purification.

[0038] The nucleic acid extraction and purification method claimed in this invention may include the following steps:

[0039] S1. Add the sample to be extracted to a centrifuge tube without RNase, add the lysis buffer and digestive enzyme described in the first part above, cap the tube, and centrifuge briefly.

[0040] S2. After incubating the sample treated in S1 in a metal bath or water bath for 10-20 minutes (e.g., 20 minutes), add the binding solution, cap the tube, and centrifuge briefly.

[0041] The incubation temperature of the metal bath or water bath can be set to 50°C.

[0042] S3. Transfer the sample treated in S2 to the adsorbent described in the second aspect above, centrifuge at 13400g for 30s to 3min (e.g., 2min), discard the filtrate, and return the adsorbent to the collection tube;

[0043] S4. Add the washing solution A1 described in the first aspect above to the adsorbate, centrifuge at 13400g for 30s to 3min (e.g., 2min), discard the filtrate, and put the adsorbate back into the collection tube;

[0044] S5. Add the washing solution A2 described in the first aspect above to the adsorbate, centrifuge at 13400g for 30s to 3min (e.g., 2min), discard the filtrate, and put the adsorbate back into the collection tube;

[0045] S6. Add the washing solution A2 described in the first aspect above to the adsorbate, centrifuge at 13400g for 2-4 min (e.g., 1.5 min), discard the filtrate, put the adsorbate into a 1-2 ml (e.g., 2 ml) EP tube, open the cap and let it stand at room temperature for 2-5 min.

[0046] S7. Add the elution solution described in the first aspect above to the center of the adsorbate, let it stand at room temperature for 2-5 minutes, centrifuge at 13400g for 1 minute, discard the adsorbate, and obtain the nucleic acid product.

[0047] More specifically, the S1 operation is as follows: Aspirate 150-300 μL (e.g., 200 μL) of the sample to be extracted into an RNase-Free centrifuge tube, add 150-300 μL (e.g., 200 μL) of lysis buffer and 10-50 μL (e.g., 20 μL) of digestive enzyme into the corresponding centrifuge tubes, cap the tubes, vortex for 10-35 seconds (e.g., 20 seconds), and then centrifuge briefly.

[0048] More specifically, the S2 operation is as follows: after incubating in a metal bath or water bath (temperature 50℃) for 10-20 minutes (e.g., 20 minutes), add 150-300μL (e.g., 200μL) of binding solution to the corresponding centrifuge tube, cover the tube, vortex for 10-15 seconds (e.g., 15 seconds), and centrifuge briefly.

[0049] More specifically, the S3 operation is as follows: transfer the above mixture to the adsorbent, centrifuge at 10000 rpm (equivalent to 13400g) for 30 seconds to 3 minutes (e.g., 2 minutes), discard the filtrate, and put the adsorbent back into the collection tube;

[0050] More specifically, the S4 operation is as follows: add 300-500 μl (e.g., 300 μL) of the washing solution A1 to the adsorbate, centrifuge at 10000 rpm (equivalent to 13400 g) for 30 seconds to 3 minutes (e.g., 2 minutes), discard the filtrate, and put the adsorbate back into the collection tube;

[0051] More specifically, the S5 operation is as follows: add 400-800 μl (e.g., 600 μl) of the washing solution A2 to the adsorbate, centrifuge at 10000 rpm (equivalent to 13400 g) for 30 seconds to 3 minutes (e.g., 2 minutes), discard the filtrate, and put the adsorbate back into the collection tube;

[0052] More specifically, the S6 operation is as follows: Add 150-400 μl (e.g., 200 μl) of the washing solution A2 to the adsorbate, centrifuge at 10000 rpm (equivalent to 13400 g) for 2-4 min (e.g., 1.5 min), discard the filtrate, put the adsorbate into a 1-2 ml (e.g., 2 ml) EP tube, open the cap and let it stand at room temperature for 2-5 min;

[0053] More specifically, the S7 operation is as follows: Add 50-100 μl (e.g., 70 μl) of elution buffer to the center of the adsorbate, let it stand at room temperature for 2-5 min, centrifuge at 10000 rpm (equivalent to 13400 g) for 1 min, discard the adsorbate, and obtain the nucleic acid product.

[0054] The sample to be extracted may be derived from an animal sample. The sample may be cells, bacteria, blood, saliva, urine, or pleural / peritoneal fluid. Preferably, the sample to be tested is blood.

[0055] In a specific embodiment of the present invention, the sample to be extracted is specifically a human whole blood sample.

[0056] The extraction and purification reagents of this invention are applicable to fresh or frozen anticoagulated whole blood samples.

[0057] In some embodiments of the present invention, the isolated nucleic acid is preferably deoxyribonucleic acid (DNA).

[0058] The beneficial effects of this invention are:

[0059] (1) Compared with existing similar kits on the market, the binding solution of the present invention contains sodium acetate reagent in addition to anhydrous ethanol. The addition of this reagent can enable DNA extract to be selectively adsorbed onto the silicon matrix membrane of the centrifuge column with high efficiency, and the yield and purity of the obtained nucleic acid are significantly improved.

[0060] (2) This invention uses a unique silica gel mold purification technology, which can effectively reduce production costs;

[0061] (3) This invention uses alkaline lysis to rapidly extract DNA. The extraction process does not involve toxic reagents such as phenol and chloroform, and does not use toxic reagents such as benzene and chloroform used in traditional methods. This minimizes the harm to experimental operators, fully complies with modern environmental protection concepts, can better meet clinical use conditions, and does not require a lot of time for alcohol precipitation. It is safe, efficient, time-saving and labor-saving.

[0062] (4) This invention can remove RNA, miscellaneous proteins, lipids and other inhibitory impurities to the maximum extent. The extracted genomic DNA has high purity and stable quality. It can be used for downstream experiments in various scenarios such as methylation, PCR, enzyme digestion, and Southern hybridization. It is suitable for efficient processing of large batches of samples and has broad application prospects. Attached Figure Description

[0063] Figure 1 The results are obtained by agarose gel electrophoresis of the nucleic acid samples extracted in Example 1 and Comparative Example 1 after loading equal amounts of samples.

[0064] Figure 2 The results are obtained by agarose gel electrophoresis of the nucleic acid samples extracted in Example 2 and Comparative Example 2 after loading equal amounts of samples.

[0065] Figure 3 The results are obtained by agarose gel electrophoresis after loading equal amounts of nucleic acid samples extracted in Example 3 and Comparative Example 3.

[0066] Figure 4 The results are obtained by agarose gel electrophoresis of nucleic acid samples extracted from different kits in Example 4 after loading equal amounts of samples. Detailed Implementation

[0067] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0069] Example 1: Extraction of DNA from blood using the nucleic acid extraction and purification kit and method of the present invention.

[0070] The nucleic acid extraction and purification reagent in this embodiment consists of lysis buffer, digestive enzyme, binding buffer, rinsing buffer A1, rinsing buffer A2, and elution buffer.

[0071] The solvent of the lysis buffer is water, and the solutes and concentrations are as follows: 1 mol / L guanidine salt (guanidine hydrochloride), 0.1 mol / L EDTA, 5% by volume of nonionic surfactants (0.25% Triton 100, 4.5% Tween 20, 0.25% NP40), and 0.1 mol / L Tris-HCl (manufacturer: Beijing Solarbio Technology Co., Ltd., catalog number: T1140, the same below); the pH of the lysis buffer is 5.0.

[0072] The digestive enzyme is proteinase K with a concentration of 10 mg / mL (manufacturer: Tiangen Biotech (Beijing) Co., Ltd., product number: RT403-02; 5 mL / pack, 20 mg / mL, the same below. Dilute with water).

[0073] The binding solution is composed of anhydrous ethanol and sodium acetate, wherein the mass percentage of sodium acetate is 0.6%; the pH of the binding solution is 6.5.

[0074] The solvent of the rinsing solution A1 is water, and the solutes and concentrations are as follows: 2 mol / L guanidine hydrochloride and 60% anhydrous ethanol by volume; the pH of the rinsing solution A1 is 6.0.

[0075] The solvent of the rinsing solution A2 is water, and the solutes and concentrations are as follows: 0.05 mol / L KAC, 70% anhydrous ethanol (volume percentage), and 2.5 mol / L Tris-HCl; the pH of the rinsing solution A2 is 7.0.

[0076] The solvent of the eluent is water, and the solute and concentration are as follows: 3 mol / L Tris-HCl; the pH of the eluent is 7.0.

[0077] In addition to the reagents mentioned above, the nucleic acid extraction and purification kit in this embodiment also contains an adsorption column TC1 (manufacturer: Beijing Aibigen Biotechnology Co., Ltd., catalog number D5-50, hereinafter the same).

[0078] The above-mentioned nucleic acid extraction and purification kit was used to extract and purify nucleic acids from human whole blood samples (sample source: Henan Academy of Medical Sciences). The extraction steps are as follows:

[0079] S1. Add 200 μL of sample to an RNase-free centrifuge tube, then add 200 μL of the above lysis buffer and 20 μL of the above digestive enzyme to the corresponding centrifuge tube, cap the tube, vortex for 20 seconds, and centrifuge briefly.

[0080] S2, incubate in a 50℃ metal bath or water bath for 20 minutes, add 200μL of the above binding solution to the corresponding centrifuge tube, cover the tube, vortex for 15 seconds, and centrifuge briefly.

[0081] S3. Transfer the mixture from S2 to the above adsorption column TC1, centrifuge at 10000 rpm (equivalent to 13400 g) for 2 min, discard the filtrate, and put the adsorption column TC1 back into the collection tube.

[0082] S4. Add 300 μl of the above washing solution A1 to the adsorption column TC1, centrifuge at 10000 rpm (equivalent to 13400 g) for 2 min, discard the filtrate, and put the adsorption column TC1 back into the collection tube.

[0083] S5. Add 600 μl of the above washing solution A2 to the adsorption column TC1, centrifuge at 10000 rpm (equivalent to 13400 g) for 2 min, discard the filtrate, and put the adsorption column TC1 back into the collection tube.

[0084] S6. Add 200 μl of the above washing solution A2 to the adsorption column TC1, centrifuge at 10000 rpm (equivalent to 13400 g) for 1.5 min, discard the filtrate, put the adsorption column TC1 into a 2 ml EP tube, open the cap and let it stand at room temperature for 2-5 min to ensure that the residual ethanol is completely removed.

[0085] S7. Add 70 μl of the above elution buffer to the center of the adsorption column TC1, let it stand at room temperature for 2-5 min, centrifuge at 10000 rpm (equivalent to 13400 g) for 1 min, discard the adsorbate, and store the DNA product at -20℃.

[0086] Example 2: Extraction of DNA from blood using the nucleic acid extraction and purification kit and method of the present invention.

[0087] The nucleic acid extraction and purification reagent in this embodiment consists of lysis buffer, digestive enzyme, binding buffer, rinsing buffer A1, rinsing buffer A2, and elution buffer.

[0088] The solvent of the lysis buffer is water, and the solutes and concentrations are as follows: 2.5 mol / L guanidine salt (guanidine hydrochloride), 0.25 mol / L EDTA, 7% by volume of nonionic surfactants (0.5% Triton 100, 6% Tween 20, 0.5% NP40), and 0.25 mol / L Tris-HCl; the pH of the lysis buffer is 6.5.

[0089] The digestive enzyme is proteinase K with a concentration of 15 mg / mL (manufacturer: Tiangen Biotech (Beijing) Co., Ltd., product number: RT403-02, 5 mL / pack, 20 mg / mL).

[0090] The binding solution is composed of anhydrous ethanol and sodium acetate, wherein the mass percentage of sodium acetate is 0.8%; the pH of the binding solution is 7.5.

[0091] The solvent of the rinsing solution A1 is water, and the solutes and concentrations are as follows: 5 mol / L guanidine hydrochloride and 70% anhydrous ethanol by volume; the pH of the rinsing solution A1 is 7.0.

[0092] The solvent of the rinsing solution A2 is water, and the solutes and concentrations are as follows: 0.1 mol / L KAC, 75% anhydrous ethanol (volume percentage), and 5.5 mol / L Tris-HCl; the pH of the rinsing solution A2 is 8.0.

[0093] The solvent of the eluent is water, and the solute and concentration are as follows: 5.5 mol / L Tris-HCl; the pH of the eluent is 8.0.

[0094] In addition to the reagents mentioned above, the nucleic acid extraction and purification kit in this embodiment also contains an adsorption column TC1 (manufacturer: Beijing Aibigen Biotechnology Co., Ltd., catalog number D5-50).

[0095] The sample and DNA extraction procedures used in this embodiment are the same as those in Embodiment 1.

[0096] Example 3: Extraction of DNA from blood using the nucleic acid extraction and purification kit and method of the present invention.

[0097] The extraction and purification reagents in this embodiment consist of cell lysis buffer, digestive enzyme, binding buffer, rinsing buffer A1, rinsing buffer A2, and elution buffer.

[0098] The solvent of the lysis buffer is water, and the solutes and their concentrations are as follows: 4 mol / L guanidine salt (guanidine hydrochloride), 0.4 mol / L EDTA, 10% by volume of nonionic surfactants (1% Triton 100, 8% Tween 20, 1% NP40), and 0.4 mol / L Tris-HCl; the pH of the lysis buffer is 8.0. The above concentrations are the final concentrations in the lysis buffer.

[0099] The digestive enzyme is proteinase K with a concentration of 20 mg / mL (manufacturer: Tiangen Biotech (Beijing) Co., Ltd., product number: RT403-02 5mL / pack, 20 mg / mL).

[0100] The binding solution is composed of anhydrous ethanol and sodium acetate, wherein the mass percentage of sodium acetate is 1%; the pH of the binding solution is 8.5.

[0101] The solvent of the rinsing solution A1 is water, and the solutes and concentrations are as follows: 8 mol / L guanidine hydrochloride and 80% anhydrous ethanol by volume; the pH of the rinsing solution A1 is 8.0.

[0102] The solvent of the rinsing solution A2 is water, and the solutes and concentrations are as follows: 1.5 mol / L KAC, 80% anhydrous ethanol (volume percentage), and 8.5 mol / L Tris-HCl; the pH of the rinsing solution A2 is 9.0.

[0103] The solvent of the eluent is water, and the solute and concentration are as follows: 9 mol / L Tris-HCl; the pH of the eluent is 9.0.

[0104] In addition to the reagents mentioned above, the nucleic acid extraction and purification kit in this embodiment also contains an adsorption column TC1 (manufacturer: Beijing Aibigen Biotechnology Co., Ltd., catalog number D5-50).

[0105] The sample and DNA extraction procedures used in this embodiment are the same as those in Embodiment 1.

[0106] Comparative Example 1

[0107] The extraction reagent formulation and DNA extraction process are the same as in Example 1, except that sodium acetate reagent is not added to the binding solution of the extraction reagent formulation (i.e., the binding solution is anhydrous ethanol); the lysis buffer formulation also contains a 1 mol / L NaCl solution.

[0108] Comparative Example 2

[0109] The extraction reagent formulation and DNA extraction process are the same as in Example 2, except that sodium acetate reagent is not added to the binding solution of the extraction reagent formulation (i.e., the binding solution is anhydrous ethanol); the lysis buffer formulation also contains a 2 mol / L NaCl solution.

[0110] Comparative Example 3

[0111] The extraction reagent formulation and DNA extraction procedure were the same as in Example 3, except that sodium acetate reagent was not added to the binding buffer of the extraction reagent formulation solution (i.e., the binding buffer was anhydrous ethanol); the lysis buffer also contained a 3 mol / L NaCl solution. The ODA260 / 280 and A260 / 230 of the nucleic acids extracted in Examples 1-3 and Comparative Examples 1-3 were detected using an ultra-micro UV spectrophotometer; the concentration of the nucleic acids extracted in Examples 1-3 and Comparative Examples 1-3 was detected using a Qubit analyzer. The results are shown in Table 1.

[0112] Table 1. Comparison of nucleic acid extraction results in Examples 1-3 and Comparative Examples 1-3

[0113]

[0114]

[0115] 1 μl of nucleic acid samples extracted from Examples 1-3 and Comparative Examples 1-3 were loaded onto the samples and detected by agarose gel electrophoresis. The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown.

[0116] As can be seen from Table 1, the DNA content extracted in Examples 1-3 is higher than that in the corresponding proportions 1-3, while the CV value is lower than that in the corresponding proportions 1-3. The CV values ​​of all Examples 1-3 are less than 3%, indicating high extraction stability.

[0117] from Figures 1 to 3 It is evident that, compared to Comparative Example 1, the nucleic acid sample obtained in Example 1 has a higher band brightness; compared to Comparative Example 2, the nucleic acid sample obtained in Example 2 has a more distinct band; and compared to Comparative Example 3, the nucleic acid sample obtained in Example 3 has a more distinct band brightness.

[0118] In summary, compared with the comparative example, the DNA concentration and purity extracted by the kit of the present invention were significantly improved, and the band brightness of the obtained nucleic acid samples was higher, indicating that adding sodium acetate to the binding solution can effectively improve the DNA extraction effect.

[0119] Example 4: Comparison of the DNA extraction effect of the nucleic acid extraction and purification kit and extraction method of the present invention with commercially available kits.

[0120] Commonly available kits Q, T, V, and Z were used (Kit Q is the QIAGENQIAamp DNA Blood Mini Kit, Kit T is the Blood Genomic DNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd., Kit V is the FastPure Blood DNA Isolation Mini Kit from Nanjing Novizan Biotechnology Co., Ltd., and Kit Z is the Universal DNA Extraction Kit from Beijing Tianmo Technology Development Co., Ltd.). Experimental procedures were performed according to the corresponding kit instructions and compared with the DNA extraction kit of this invention. The extraction reagent formulation and experimental procedures were the same as in Example 2. The same volume of the same blood sample was extracted, with four samples extracted from each kit. The results are shown in Table 2.

[0121] Table 2. Comparison of DNA extraction results between the nucleic acid extraction and purification kit and extraction method of the present invention and commercially available kits.

[0122]

[0123]

[0124] As shown in Table 2 above, the DNA extraction concentrations of kits Q, T, V, and Z are all below 65 ng / μl, while the average DNA extraction concentration of the kit of this invention is above 75 ng / μl. Compared to kits Q, T, V, and Z, the OD detection values ​​obtained after DNA extraction using the kit of this invention are more stable.

[0125] 1 μl of nucleic acid samples extracted using different kits were taken and analyzed by agarose gel electrophoresis. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the bands of the kit group of the present invention are brighter when the same volume of nucleic acid sample is taken.

[0126] In summary, the DNA extracted using the kit and method of this invention has higher purity and better extraction efficiency. Furthermore, the kit is simple in composition, low in cost, and convenient for large-scale nucleic acid extraction. In addition, the kit does not contain toxic or harmful reagents such as toluene, xylene, phenol, and chloroform, minimizing harm to the environment and operators, aligning with modern environmental protection principles, and better meeting market demands.

[0127] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A reagent for nucleic acid extraction and purification, comprising cell lysis buffer, digestive enzyme, binding buffer, washing buffer, and elution buffer, characterized in that: The binding solution contains an alcohol solution and a sodium salt.

2. The reagent according to claim 1, characterized in that: The alcohol solution is anhydrous ethanol or isopropanol; And / or, the sodium salt may be at least one of sodium acetate, sodium sulfate, sodium phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

3. The reagent according to claim 1 or 2, characterized in that: The binding solution is composed of anhydrous ethanol and sodium acetate, wherein the mass percentage of sodium acetate is 0.6-1.0%; the pH value of the binding solution is 6.5-8.

5.

4. The reagent according to any one of claims 1-3, characterized in that: The cell lysate contains Tris-HCl, guanidine salt, EDTA, and nonionic surfactant, with a pH of 5.0-8.0; And / or, the rinsing solution is composed of rinsing solution A1 and rinsing solution A2; rinsing solution A1 contains guanidine hydrochloride and anhydrous ethanol, and has a pH value of 6.0-8.0; rinsing solution A2 contains KAC, Tris-HCl and anhydrous ethanol, and has a pH value of 7.0-9.

0. And / or, the eluent contains Tris-HCl with a pH of 7.0-9.

0.

5. The reagent according to claim 4, characterized in that: The solvent of the lysis buffer is water, and the solutes and concentrations are as follows: 1-4.0 mol / L guanidine salt, 0.1-0.4 mol / L EDTA, 5-10% by volume of nonionic surfactant, and 0.1-0.4 mol / L Tris-HCl; And / or, the solvent of the rinsing solution A1 is water, and the solute and concentration are as follows: 2-8 mol / L guanidine hydrochloride, and 60%-80% anhydrous ethanol by volume. And / or, the solvent of the rinsing solution A2 is water, and the solute and concentration are as follows: 0.05-1.5 mol / L KAC, 70-80% anhydrous ethanol (volume percentage), and 2.5-8.5 mol / L Tris-HCl; And / or, the solvent of the eluent is water, and the solute and concentration are as follows: 3-9 mol / L Tris-HCl.

6. The reagent according to claim 5, characterized in that: In the lysis buffer, the guanidine salt is at least one of guanidine isothiocyanate, guanidine hydrochloride, and guanidine thiocyanate; And / or, in the lysis solution, the nonionic surfactant is at least one of Triton 100, Tween 20, SDS and NP40; And / or, the digestive enzyme is proteinase K.

7. A kit for nucleic acid extraction and purification, characterized in that: The kit includes the reagents described in any one of claims 1-6.

8. The reagent kit according to claim 7, characterized in that: The kit also contains an adsorbent; Furthermore, the adsorbent is an adsorption column.

9. The use of the reagent according to any one of claims 1-6 or the kit according to claim 7 or 8 in the extraction and purification of nucleic acids.

10. A method for nucleic acid extraction and purification, comprising the following steps: S1. Add the sample to be extracted to a centrifuge tube without RNase, add the cell lysis buffer and the digestive enzyme as described in any one of claims 1-6 in sequence, cap the tube, and centrifuge briefly. S2. After incubating the sample treated in S1 in a metal bath or water bath for 10-20 minutes, add the binding solution, cap the tube, and centrifuge briefly. S3. Transfer the sample treated in S2 to the adsorbent described in claim 8, centrifuge at 13400g for 30s to 3min, discard the filtrate, and return the adsorbent to the collection tube; S4. Add the rinsing solution A1 as described in any one of claims 1-6 to the adsorbate, centrifuge at 13400g for 30s to 3min, discard the filtrate, and return the adsorbate to the collection tube; S5. Add the rinsing solution A2 as described in any one of claims 1-6 to the adsorbate, centrifuge at 13400g for 30s to 3min, discard the filtrate, and return the adsorbate to the collection tube; S6. Add the rinsing solution A2 as described in any one of claims 1-6 to the adsorbate, centrifuge at 13400g for 2-4 min, discard the filtrate, put the adsorbate into a 1-2 ml EP tube, and leave it at room temperature for 2-5 min with the cap off. S7. Add the elution solution according to any one of claims 1-6 to the center of the adsorbate, let it stand at room temperature for 2-5 minutes, centrifuge at 13400g for 1 minute, discard the adsorbate, and obtain the nucleic acid product.

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