Nucleic acid extraction kit and nucleic acid extraction method

Abstract

The invention discloses a nucleic acid extraction kit and a nucleic acid extraction method, the nucleic acid extraction kit comprises a lysis solution, a binding solution, a washing solution and an eluent, the lysis solution comprises 5-8 M guanidine hydrochloride, 20-50 m MTris-HCl (pH 7.5-8.5), 1-3 mM ethylenediamine tetraacetic acid, 0.5%-1.5% (v / v) beta-mercaptoethanol and 0.5%-1.0% (w / v) polyvinylpyrrolidone, and the invention relates to the technical field of nucleic acid extraction. According to the nucleic acid extraction kit and the nucleic acid extraction method, the optimized lysis solution can more fully lyse cells to release nucleic acid, the binding solution improves the binding efficiency of the nucleic acid and the magnetic beads, the eluent can more completely elute the nucleic acid from the magnetic beads, and in the extraction experiments of different types of samples, the nucleic acid extraction efficiency is greatly improved. According to the kit and the method, the nucleic acid extraction amount is averagely increased by 20%-30%, the recovery rate can reach 85%-95%, sample waste is effectively reduced, and the utilization rate of precious samples is increased.

Description

Technical Field

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

[0002] Nucleic acids occupy a core position in modern biological and medical research. Whether it is gene sequencing, disease diagnosis, genetic analysis or biopharmaceutical research and development, it is inseparable from the acquisition of high-quality nucleic acids. The existing nucleic acid extraction methods mainly include chloroform extraction method, centrifugal column method and magnetic bead method. Among them, the magnetic bead method has received widespread attention and application in recent years because of its relatively simple operation and easy automation.

[0003] At present, the magnetic bead-based nucleic acid extraction kit still has the following problems: the lysis solution in the kit has limited lysis ability. For some cells or tissue samples with complex structures and thick cell walls, it is difficult to fully destroy the cell structure and completely release the nucleic acid, resulting in a low extraction amount, which cannot meet the requirements of subsequent experiments for the amount of nucleic acid. For this reason, the present invention provides a nucleic acid extraction kit and a nucleic acid extraction method. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a nucleic acid extraction kit and a nucleic acid extraction method, which solve the problem that the lysis solution in the kit has limited lysis ability, and it is difficult to fully destroy the cell structure and completely release the nucleic acid for some cells or tissue samples with complex structures and thick cell walls, thereby resulting in low extraction yield and being unable to meet the nucleic acid amount requirements of subsequent experiments.

[0005] To achieve the above object, the present invention is implemented by the following technical scheme: a nucleic acid extraction kit, comprising a lysing solution, a binding solution, a washing solution and an elution solution,

[0006] The lysate comprises 5-8 M guanidine hydrochloride, 20-50 mM Tris-HCl (pH 7.5-8.5), 1-3 mM ethylenediaminetetraacetic acid, 0.5%-1.5% (v / v) β-mercaptoethanol, and 0.5%-1.0% (w / v) polyvinyl pyrrolidone;

[0007] The binding solution is composed of 2-4M sodium chloride, 10-30mM Tris-HCl (pH 7.0-8.0), 0.3%-0.8% (w / v) hexadecyltrimethylammonium bromide, 5%-10% (v / v) isopropanol, and 1-3mM dithiothreitol;

[0008] The washing solution contains 10-30 M Tris-HCl (pH 7.2-8.2), 0.1-0.3 M potassium acetate, 60%-80% (v / v) ethanol, and 0.1%-0.3% (v / v) Tween-20;

[0009] The eluent is 10-20 mM Tris-HCl (pH 8.0-9.0) and 0.1-0.5 mM ethylenediaminetetraacetic acid.

[0010] Preferably, the concentration of guanidine hydrochloride in the lysate is 6-7M.

[0011] Preferably, the concentration of sodium chloride in the binding solution is 2.5-3.5M.

[0012] Preferably, the concentration of potassium acetate in the washing solution is 0.15-0.25M.

[0013] The present invention also discloses a nucleic acid extraction method of a nucleic acid extraction kit, which specifically comprises the following steps:

[0014] S1. Place the sample to be extracted in a centrifuge tube, add an equal volume of lysis buffer, vortex to mix, and incubate in a 60-70℃ water bath for 5-10 minutes.

[0015] S2. Add binding solution and magnetic beads, invert to mix 5-8 times, and incubate at room temperature with shaking for 10-15 minutes.

[0016] S3. Place the centrifuge tube on the magnetic rack, let it stand for 2-3 minutes, and discard the supernatant.

[0017] S4. Add washing solution, vortex for 3-5 minutes to wash the magnetic beads, and repeat 2-3 times.

[0018] S5. Add eluent, incubate in a 65-75°C water bath with shaking for 3-5 minutes, and collect the eluent to obtain purified nucleic acid.

[0019] Preferably, in step S1, the volume of the sample to be extracted is 100-500 μL.

[0020] Preferably, in step S2, the amount of magnetic beads added is 12-18 μL of magnetic bead suspension per 200 μL of sample.

[0021] Preferably, in step S4, the amount of the washing solution is 300-600 μL.

[0022] Beneficial Effects

[0023] The present invention provides a nucleic acid extraction kit and a nucleic acid extraction method. Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The nucleic acid extraction kit and nucleic acid extraction method can more fully lyse cells and release nucleic acids through the optimized lysis solution, the binding solution can improve the binding efficiency of nucleic acids and magnetic beads, and the elution solution can elute nucleic acids from the magnetic beads more completely. In extraction experiments on different types of samples, the nucleic acid extraction amount of the kit and method of the present invention is increased by an average of 20%-30%, and the recovery rate can reach 85%-95%, which effectively reduces sample waste and improves the utilization rate of precious samples.

[0025] (2) The nucleic acid extraction kit and nucleic acid extraction method effectively remove polyphenol impurities through PVP in the lysate, and the CTAB in the binding solution enhances the specific binding of nucleic acids and reduces the adsorption of impurities. The synergistic effect of multiple components in the washing solution can effectively remove impurities such as proteins and salt ions. According to experimental measurements, the A260 / A280 ratio of the extracted nucleic acid can be stabilized between 1.85 and 2.0. Compared with the nucleic acid extracted by the traditional kit, the purity is increased by 15% to 25%, which significantly reduces the interference of impurities on subsequent nucleic acid analysis experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a flow chart of the nucleic acid extraction method of the present invention;

[0027] Figure 2 This is the experimental result table of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Embodiment 1, the present invention provides a nucleic acid extraction kit, comprising a lysing solution, a binding solution, a washing solution and an elution solution;

[0030] The lysis solution contains 5-8M guanidine hydrochloride, 20-50mM Tris-HCl (pH7.5-8.5), 1-3mM ethylenediaminetetraacetic acid (EDTA), 0.5%-1.5% (v / v) β-mercaptoethanol, and 0.5%-1.0% (w / v) polyvinylpyrrolidone (PVP). Guanidine hydrochloride, as a powerful protein denaturant, can effectively destroy the cell structure and the secondary structure of the protein, and promote the full release of nucleic acids; Tris-HCl is responsible for maintaining a stable pH environment, providing suitable acid-base conditions for the release of nucleic acids and subsequent reactions; EDTA can effectively chelate metal ions, thereby inhibiting the activity of nucleases and preventing nucleic acids from being degraded during the extraction process; β-mercaptoethanol plays an important role in preventing oxidation and helps the denaturation process of proteins; PVP can specifically bind to polyphenols, greatly reducing their interference with the nucleic acid extraction process and ensuring the purity of nucleic acid extraction.

[0031] The binding solution is prepared by mixing 2-4M sodium chloride, 10-30mM Tris-HCl (pH7.0-8.0), 0.3%-0.8% (w / v) hexadecyltrimethylammonium bromide (CTAB), 5%-10% (v / v) isopropanol, and 1-3mM dithiothreitol (DTT). The main function of sodium chloride is to provide appropriate ionic strength to promote the specific binding between nucleic acid and magnetic beads; CTAB, as a cationic surfactant, can significantly enhance the adsorption of nucleic acid and magnetic beads and improve the binding efficiency; isopropanol can effectively reduce the solubility of nucleic acid, making nucleic acid more inclined to bind to magnetic beads; DTT is committed to maintaining a reducing environment, effectively protecting the integrity of nucleic acid and preventing it from being oxidatively damaged during the extraction process.

[0032] The washing solution contains 10-30mM Tris-HCl (pH7.2-8.2), 0.1-0.3M potassium acetate, 60%-80% (v / v) ethanol, and 0.1%-0.3% (v / v) Tween-20. Tris-HCl is used to stabilize the pH value of the solution to ensure that the washing process is carried out in a suitable acid-base environment; potassium acetate performs well in removing proteins and salt ions. It can form precipitation by combining with proteins and promote the dissociation of salt ions, thereby effectively removing these impurities; ethanol reduces the loss of nucleic acids during the washing process by virtue of its characteristics of precipitating impurities and reducing the water solubility of nucleic acids; Tween-20 focuses on removing residual protein and lipid impurities, further improving the purity of nucleic acids.

[0033] The elution solution is 10-20mM Tris-HCl (pH8.0-9.0) and 0.1-0.5mM ethylenediaminetetraacetic acid (EDTA). Under the synergistic effect of appropriate pH conditions and EDTA, nucleic acids can be efficiently eluted from magnetic beads, and the stability and integrity of nucleic acids can be ensured, providing high-quality nucleic acid samples for subsequent experimental analysis.

[0034] Furthermore, in the expression "0.5%-1.0% (w / v) polyvinyl pyrrolidone", (w / v) refers to the weight / volume ratio. Specifically, it indicates the percentage of the mass of polyvinyl pyrrolidone (in grams) to the total volume of the solution (in milliliters) in the solution system. For example, if it is a 1.0% (w / v) polyvinyl pyrrolidone solution, it means that 100 mL of the solution contains 1.0 g of polyvinyl pyrrolidone. The other "(w / v)" expressions in the text have the same meaning.

[0035] Furthermore, in the expression "5%-10% (v / v) isopropanol", (v / v) refers to the volume fraction (volume / volume), which indicates the percentage of the volume of isopropanol in the total volume of the solution. For example, in a 100mL solution, if the concentration of isopropanol is 5% (v / v), then the volume of isopropanol is 5mL; if it is 10% (v / v), then the volume of isopropanol is 10mL. The same applies to other "(v / v)" expressions in the text.

[0036] See also Figure 1 The present invention also discloses a nucleic acid extraction method of a nucleic acid extraction kit, which specifically comprises the following steps:

[0037] S1. Take 100-500 μL of the sample to be extracted, place it in a centrifuge tube, add an equal volume of lysis buffer, and then use a vortex oscillator to mix thoroughly. Then place the centrifuge tube in a 60-70°C water bath and incubate for 5-10 minutes. This treatment will fully lyse the cells and release the nucleic acid.

[0038] S2. Add binding solution in a ratio of 1:2-1:3 in volume to lysate, and add magnetic beads. Add 10-20 μL of magnetic bead suspension to every 200 μL of sample. The concentration of magnetic beads is 15-25 mg / mL. Invert and mix 5-8 times, then incubate at room temperature with shaking for 10-15 minutes to ensure that the nucleic acid and magnetic beads are fully bound.

[0039] S3. Place the centrifuge tube containing the magnetic bead complex on the magnetic rack and let it stand for 2-3 minutes. After the magnetic beads are completely adsorbed to the tube wall, discard the supernatant.

[0040] S4. Add 300-600 μL of washing solution and use a vortex oscillator to vigorously shake for 3-5 minutes to wash the magnetic beads to remove impurities. This washing step needs to be repeated 2-3 times to ensure that the impurities are completely removed.

[0041] S5. Finally, add 50-100 μL of elution buffer, place the centrifuge tube in a 65-75°C water bath and shake and incubate for 3-5 minutes to elute the nucleic acid from the magnetic beads, collect the eluate, and the purified nucleic acid is successfully obtained.

[0042] Example 2, the present invention also provides an experiment of a nucleic acid extraction kit and a nucleic acid extraction method:

[0043] 1. Experimental materials:

[0044] Samples: Fresh human whole blood samples, mouse liver tissue samples and E. coli liquid samples were collected. The human whole blood samples were treated with anticoagulants and diluted to high concentrations (about 1×10 6 cells / mL), medium concentration (about 1×10 5 cells / mL) and low concentrations (about 1×10 4 The mouse liver tissue samples were accurately weighed to 10 mg, 20 mg and 30 mg, and then homogenized with pre-cooled phosphate buffered saline (PBS). The Escherichia coli liquid samples were collected in the logarithmic growth phase and the concentration of the liquid was adjusted to a high concentration (about 1×10 9 CFU / mL), medium concentration (about 1×10 8 CFU / mL and low concentration (about 1×10 7 CFU / mL). Each concentration gradient of each sample was divided into an experimental group and a control group, with 10 samples in each group;

[0045] Reagents: The experimental group used the nucleic acid extraction kit of the present invention; the control group used a commercially available magnetic bead method nucleic acid extraction kit.

[0046] 2. Experimental methods:

[0047] Human whole blood sample extraction: For human whole blood samples of different concentrations, take 200 μL and add them to the corresponding centrifuge tube. The experimental group followed the following steps: First, add 200 μL of lysis solution, vortex to mix, and incubate in a 65°C water bath for 8 minutes. Then add binding solution (the volume ratio of binding solution to lysis solution is 1:2.5) and 15 μL of magnetic bead suspension (magnetic bead concentration is 20 mg / mL), invert and mix 6 times, and incubate at room temperature for 12 minutes. Then place the centrifuge tube on the magnetic rack, let it stand for 2.5 minutes, and discard the supernatant. Add 450 μL of washing solution, vortex for 4 minutes, wash the magnetic beads, and repeat this washing step 3 times. Finally, add 80 μL of elution solution, incubate in a 70°C water bath for 4 minutes, and collect the eluate. The control group followed its standard procedure.

[0048] Mouse liver tissue sample extraction: For mouse liver tissue homogenate samples of different masses, 300 μL was added to the centrifuge tube. The experimental group operation process was as follows: 300 μL of lysis solution was added, vortexed and mixed, and incubated in a 65°C water bath for 10 minutes. The subsequent operation parameters for adding binding solution, magnetic beads, washing solution and elution solution were the same as those for human whole blood samples, except that the water bath time was extended to 10 minutes during lysis. The control group was treated according to its standard process.

[0049] Extraction of Escherichia coli liquid samples: Take 400 μL of Escherichia coli liquid samples of different concentrations into a centrifuge tube. The experimental group was operated as follows: add 400 μL of lysis solution, vortex mix, and incubate in a 68°C water bath for 8 minutes. Add binding solution (the volume ratio of binding solution to lysis solution is 1:2.5) and 18 μL of magnetic bead suspension (magnetic bead concentration is 20 mg / mL), invert and mix 7 times, and incubate at room temperature for 13 minutes. The steps of standing on the magnetic stand, washing and elution are similar to the operating parameters of human whole blood samples, except that the elution temperature is adjusted to 72°C. The control group was operated according to its standard process.

[0050] After extraction, the concentration and purity of nucleic acid (A260 / A280 ratio) were determined using a Nanodrop spectrophotometer. At the same time, the fluorescent quantitative PCR technology was used to quantitatively analyze the extracted nucleic acid with specific nucleic acid amplification primers to calculate the recovery rate of nucleic acid extraction. The recovery rate calculation formula is: recovery rate = (nucleic acid concentration after extraction × elution volume) / (theoretical nucleic acid content in the sample) × 100%.

[0051] 3. Experimental results: (such as Figure 2 (shown)

[0052] It can be clearly seen from the experimental results that under different types of samples and different concentration gradients, the nucleic acid extraction kit and method of the present invention show significant advantages in nucleic acid purity (A260 / A280 ratio), recovery rate and yield. For human whole blood samples, at high concentrations, the nucleic acid purity of the experimental group was increased by about 16.5%, the recovery rate was increased by 18.8%, and the yield was increased by 31.3% compared with the control group; at medium concentrations, the purity was increased by about 18.2%, the recovery rate was increased by 22.7%, and the yield was increased by 36.8%; at low concentrations, the purity was increased by 20%, the recovery rate was increased by 25.7%, and the yield was increased by 40%. In mouse liver tissue samples, at 10 mg, the nucleic acid purity of the experimental group was increased by about 16.7%, the recovery rate was increased by 19.2%, and the yield was increased by 41.7% compared with the control group; at 20 mg, the purity was increased by 15.1%, the recovery rate was increased by 14.6%, and the yield was increased by 35%; at 30 mg, the purity was increased by 14.3%, the recovery rate was increased by 12.9%, and the yield was increased by 36.1%. In the E. coli liquid samples, at high concentrations, the nucleic acid purity of the experimental group was about 12.4% higher than that of the control group, the recovery rate was increased by 11.4%, and the yield was increased by 31.7%; at medium concentrations, the purity was increased by 13.1%, the recovery rate was increased by 12.9%, and the yield was increased by 36.7%; at low concentrations, the purity was increased by 14.7%, the recovery rate was increased by 16.3%, and the yield was increased by 40%.

[0053] In summary, the optimized lysis solution can more fully lyse cells to release nucleic acids, the binding solution improves the binding efficiency of nucleic acids and magnetic beads, and the elution solution can elute nucleic acids from the magnetic beads more completely. In the extraction experiments of different types of samples, the nucleic acid extraction amount of the kit and method of the present invention is increased by 20%-30% on average, and the recovery rate can reach 85%-95%, which effectively reduces sample waste and improves the utilization rate of precious samples. In addition, the PVP in the lysis solution effectively removes polyphenol impurities, the CTAB in the binding solution enhances nucleic acid specific binding and reduces impurity adsorption, and the synergistic effect of various components in the washing solution can efficiently remove impurities such as proteins and salt ions. According to experimental measurements, the A260 / A280 ratio of the extracted nucleic acid can be stabilized between 1.85 and 2.0, and the purity is increased by 15%-25% compared with the nucleic acid extracted by the traditional kit, which significantly reduces the interference of impurities on subsequent nucleic acid analysis experiments.

[0054] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0055] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0056] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A nucleic acid extraction kit, comprising a lysing solution, a binding solution, a washing solution and an elution solution, characterized in that: The lysate comprises 5-8 M guanidine hydrochloride, 20-50 mM Tris-HCl (pH 7.5-8.5), 1-3 mM ethylenediaminetetraacetic acid, 0.5%-1.5% (v / v) β-mercaptoethanol, and 0.5%-1.0% (w / v) polyvinyl pyrrolidone; The binding solution is composed of 2-4M sodium chloride, 10-30mM Tris-HCl (pH 7.0-8.0), 0.3%-0.8% (w / v) hexadecyltrimethylammonium bromide, 5%-10% (v / v) isopropanol, and 1-3mM dithiothreitol; The washing solution contains 10-30 M Tris-HCl (pH 7.2-8.2), 0.1-0.3 M potassium acetate, 60%-80% (v / v) ethanol, and 0.1%-0.3% (v / v) Tween-20; The eluent is 10-20 mM Tris-HCl (pH 8.0-9.0) and 0.1-0.5 mM ethylenediaminetetraacetic acid.

2. A nucleic acid extraction kit according to claim 1, characterized in that: The concentration of guanidine hydrochloride in the lysate is 6-7M.

3. A nucleic acid extraction kit according to claim 1, characterized in that: The concentration of sodium chloride in the combination solution is 2.5-3.5M.

4. A nucleic acid extraction kit according to claim 1, characterized in that: The concentration of potassium acetate in the washing solution is 0.15-0.25M.

5. A method for extracting nucleic acid according to a nucleic acid extraction kit according to any one of claims 1 to 4, characterized in that: The specific steps include: S1. Place the sample to be extracted in a centrifuge tube, add an equal volume of lysis buffer, vortex to mix, and incubate in a 60-70℃ water bath for 5-10 minutes. S2. Add binding solution and magnetic beads, invert to mix 5-8 times, and incubate at room temperature with shaking for 10-15 minutes. S3. Place the centrifuge tube on the magnetic rack, let it stand for 2-3 minutes, and discard the supernatant. S4. Add washing solution, vortex for 3-5 minutes to wash the magnetic beads, and repeat 2-3 times. S5. Add eluent, incubate in a 65-75°C water bath with shaking for 3-5 minutes, and collect the eluent to obtain purified nucleic acid.

6. The method for extracting nucleic acid according to claim 5, characterized in that: In step S1, the volume of the sample to be extracted is 100-500 μL.

7. The method for extracting nucleic acid according to claim 5, characterized in that: In step S2, the amount of magnetic beads added is 12-18 μL of magnetic bead suspension per 200 μL of sample.

8. The method for extracting nucleic acid according to claim 5, characterized in that: In step S4, the amount of the washing solution used is 300-600 μL.

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