Nucleic acid extraction and purification kit and application thereof

Abstract

The invention discloses a nucleic acid extraction and purification kit and application thereof. The nucleic acid extraction and purification kit is composed of a lysis solution, magnetic beads (hydroxyl magnetic beads), a deproteinized washing solution, a washing solution and an eluent. The nucleic acid extraction and purification kit is matched with a nucleic acid extraction and purification instrument for nucleic acid extraction and purification, compared with a centrifugal column method, long-time centrifugation is not needed, high-throughput and automatic operation is facilitated, and the probability of errors caused by manual operation is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biological detection and relates to a nucleic acid extraction and purification kit and application thereof. Background Art

[0002] Humans’ understanding of nucleic acid substances began in 1869, when Friederich Miescher isolated a chemical substance he called “nuclide” from the nucleus of white blood cells. This was the first time in human history that nuclear substances in cells were purposefully extracted. However, the method used at the time was very simple. Nucleic acids were precipitated from acidic solutions simply by changing the pH of the solution. This was also the earliest description of the properties of nucleic acids, that is, a substance that is soluble in alkaline solutions but insoluble in acidic solutions.

[0003] In 1953, Watson and Crick elucidated the structure of DNA, thus opening the first year of molecular biology. Soon thereafter, standard laboratory procedures for isolating nucleic acids emerged. In 1958, Meselson and Stahl, who discovered the principle of semiconservative replication of DNA, pioneered the use of density gradient centrifugation to obtain DNA. This method uses the principle of different buoyancy density differences of each component in the centrifugal field to purify DNA. Since cesium ions (Cs) are more dense than water, the ultra-high centrifugal force applied to the CsCl solution leads to the formation of a density gradient. Nucleic acids migrate on this gradient until they reach neutral buoyancy, i.e., the isopycnic point. This method has the advantage of providing very pure, high-yield DNA at a low cost. Other liquid phase purification methods include the classic phenol-chloroform method, hexadecyltrimethylammonium bromide method, etc. However, until the 1990s, DNA extraction was still a time-consuming, cumbersome, and inefficient operation. In 1989, McCormick established a method for extracting genomic DNA by adsorbing phenolized silica to remove proteins. It is similar to the classic phenol / chloroform extraction method, that is, phenol is replaced by phenolized silica phenol. This is the earliest solid-phase adsorption extraction method. Compared with liquid-phase extraction, the advantages of solid-phase adsorption are obvious. Therefore, the traditional liquid-phase separation and extraction of nucleic acid methods are gradually replaced by new methods based on solid-phase adsorption supports. Currently common solid-phase materials include silica gel, glass particles, diatomaceous earth, anion exchange carriers, etc. However, the solid-phase method usually requires several steps of rapid centrifugation, vacuum filtration, etc. to achieve separation, and it has a large demand for samples, consumes a lot of samples, is not convenient for high-throughput and automated operations, and severely limits its application in the field of clinical genetic diagnosis.

[0004] In order to meet the needs of high throughput, high sensitivity and automated operation in modern molecular biology detection experiments, the magnetic bead method came into being. The first commercial reagent that used the magnetic bead method to extract DNA and successfully applied for a patent in the United States appeared in 1998. The magnetic bead method first lyses cells and specifically adsorbs the free nucleic acid molecules to the surface of magnetic particles, while impurities such as proteins and polysaccharides remain in the solution; under the action of an external magnetic field, the magnetic particles are separated from the liquid, and after discarding the liquid, the purified nucleic acid molecules are eluted.

[0005] The magnetic bead method utilizes the principle that the active groups of magnetic particles can bind to and dissociate with nucleic acids under certain conditions, thus avoiding the loss of nucleic acids during the extraction process as much as possible. It can also effectively remove the influence of interfering substances (such as hemoglobin, bilirubin, and lipemia) in the specimen to obtain high-quality nucleic acid templates. With the development of magnetic bead extraction technology, DNA extraction has truly begun to be standardized, rapid, and automated. Commercial kits based on magnetic bead extraction have also been widely used. This kit does not require any organic solvents or repeated centrifugation. It can currently extract high-quality DNA and RNA from whole blood, serum, plasma, saliva, urine, feces, cerebrospinal fluid, tissues, and cells, and it takes less time and has a higher recovery rate. The most significant advantage is that the extraction process can be automated and unmanned by mechanical equipment.

[0006] In recent years, molecular diagnostic technology has developed rapidly. With the maturity of technologies such as gene sequencing, the cost has been further reduced, and its application in clinical practice has become more and more common. There are dozens of sample types for molecular diagnostic testing, and the test items applicable to the processed products also cover various molecular biology technology platforms such as fluorescent quantitative PCR (polymerase chain reaction), gene sequencing, gene chips, and biological mass spectrometry. However, no matter which type of test project, accurate test results undoubtedly rely on high-quality specimens and specimen pretreatment processes. Therefore, the trend of automation in clinical molecular diagnosis will gradually become dominant in domestic clinical laboratories, and automation will first be realized in nucleic acid extraction, which is currently more popular with manual operations and has the greatest impact on the results. Magnetic bead extraction reagents plus automatic nucleic acid extractors have become the perfect combination to solve the pretreatment of clinical molecular diagnostic samples. Summary of the invention

[0007] The purpose of the invention is to provide a nucleic acid extraction and purification kit and application thereof.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A nucleic acid extraction and purification kit, consisting of the following parts:

[0010] Lysis buffer: 2-5M chaotropic salt, 10-300mM buffer, 1-100mM EDTA, 20-60% (v / v) lower alcohols, 3-10% (v / v) surfactant, pH 4-8;

[0011] Magnetic beads;

[0012] Deproteinization washing solution: 0.5-3M chaotropic salt, 10-300mM buffer, 1-100mM EDTA, 50-500mM sodium chloride, 20-60% (v / v) lower alcohols, 1-10% (v / v) surfactant, pH 4-8; washing solution: 50-100% (v / v) anhydrous ethanol;

[0013] Eluent: 10-100 mM Tris(OHM), 1-50 mM EDTA, pH 8-10.

[0014] As a preferred embodiment of the present invention, the chaotropic salt is selected from any one or two of guanidine isothiocyanate and guanidine hydrochloride.

[0015] As a preferred embodiment of the present invention, the buffer is selected from sodium citrate dihydrate buffer or tris(hydroxymethylaminomethane) buffer.

[0016] As a preferred embodiment of the present invention, the lower alcohols are ethanol and / or isopropanol.

[0017] As a preferred embodiment of the present invention, the surfactant is selected from any one or more of ethylphenyl polyethylene glycol, polyethylene glycol octylphenyl ether, and Tween 20.

[0018] As a preferred embodiment of the present invention, the magnetic beads are hydroxy magnetic beads.

[0019] As a further preferred embodiment of the present invention, the nucleic acid extraction and purification kit is composed of the following parts: lysis solution: 2-5M guanidine thiocyanate, 10-300mM sodium citrate dihydrate, 1-100mM ethylenediaminetetraacetic acid, 20-60% (v / v) anhydrous ethanol, 3-10% (v / v) Tween 20, pH 4-8;

[0020] Hydroxyl magnetic beads;

[0021] Protein removal solution: 0.5-3M guanidine thiocyanate, 10-300mM sodium citrate dihydrate, 1-100mM ethylenediaminetetraacetic acid, 50-500mM sodium chloride, 20-60% (v / v) anhydrous ethanol, 1-10% (v / v) Tween 20, pH 4-8;

[0022] Washing solution: 50-100% (v / v) anhydrous ethanol;

[0023] Eluent: 10-100 mM Tris(OHM), 1-50 mM EDTA, pH 8-10.

[0024] As a further preferred embodiment of the present invention, the nucleic acid extraction and purification kit is composed of the following parts:

[0025] Lysis buffer: 3 M guanidine thiocyanate, 50 mM sodium citrate dihydrate, 1 mM ethylenediaminetetraacetic acid, 40% (v / v) anhydrous ethanol, 5% (v / v) Tween 20, pH 5.0;

[0026] Hydroxyl magnetic beads;

[0027] Deproteinization washing solution: 1 M guanidine thiocyanate, 20 mM sodium citrate dihydrate, 1 mM ethylenediaminetetraacetic acid, 0.1 M sodium chloride, 30% (v / v) anhydrous ethanol, 3% (v / v) Tween 20, pH 5.0;

[0028] Washing solution: 80% (v / v) anhydrous ethanol;

[0029] Eluent: 10 mM Tris(2-hydroxymethylaminomethane), 1 mM EDTA, pH 8.0.

[0030] The invention discloses an application of the nucleic acid extraction and purification kit in extracting nucleic acid.

[0031] A nucleic acid extraction method uses the nucleic acid extraction and purification kit in combination with a nucleic acid extraction and purification instrument to extract and purify nucleic acid.

[0032] Beneficial effects:

[0033] (1) Low sample volume requirement: The nucleic acid extraction system can be easily scaled up or down, and a small amount of material can be used to produce high-concentration nucleic acids.

[0034] (2) Simple and fast operation: The entire operation process is basically divided into three steps (lysis and binding, washing, and elution), which can be completed manually or automatically within 30 minutes without centrifugation.

[0035] (3) Fully automated operation: The nucleic acid extractor can achieve automated, high-throughput operation with one-click start.

[0036] (4) Safe, non-toxic and harmless: The reagents do not contain toxic chemical reagents such as phenol and chloroform, which fully complies with modern environmental protection concepts. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1The nucleic acid extraction and purification kit of the present invention was used to extract HBV (hepatitis B virus) reference products of different concentrations, and the extracted nucleic acids were amplified by PCR. The nucleic acid extraction and purification kit of the present invention was used to extract four reference products of different concentrations (A: 5*10 6 IU / mL, B:5*10 5 IU / mL, C:5*10 4 IU / mL, D:5*10 3 IU / mL), positive quality control and negative quality control, and then use the amplification reagent in Shengxiang Bio's "Hepatitis B Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method)" to perform PCR amplification on the extracted nucleic acid. Figure 2 Linear regression analysis of the Ct value of PCR amplification after extraction and the logarithmic concentration. With the Ct value of PCR amplification as the horizontal axis and the logarithmic value of the theoretical concentration of the reference as the vertical axis, a linear regression analysis was performed on the Ct value of PCR amplification of the reference after extraction and the logarithmic value of the theoretical concentration. 2 =0.9999≥0.98.

[0038] Figure 3 Amplification diagram of 1000 IU / mL HBV (hepatitis B virus) extracted using the nucleic acid extraction and purification kit of the present invention. The national HBV standard diluted to 1000 IU / mL was extracted 10 times using the present invention, and then the extracted nucleic acid was PCR amplified using the "Hepatitis B Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method)" of Shengxiang Bio.

[0039] Figure 4 Amplification diagram of 1000 IU / mL HCV (hepatitis C virus) extracted using the nucleic acid extraction and purification kit of the present invention. The HCV national standard product diluted to 1000 IU / mL was extracted 10 times using the present invention, and then the extracted nucleic acid was PCR amplified using the "Hepatitis C Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method)" of Shengxiang Biotechnology.

[0040] Figure 5 Extraction of nucleic acid using nucleic acid extraction and purification instrument 96 deep well plate loading diagram

[0041] Figure 6Amplification diagram after extracting 1000 IU / mL HBV (hepatitis B virus) national standard using an automated nucleic acid extractor in combination with the nucleic acid extraction and purification kit of the present invention. GenePure Pro nucleic acid extraction and purification instrument (model: NPA-32P, manufacturer: Hangzhou Biori Technology Co., Ltd.) was used in combination with the present invention to extract 1000 IU / mL HBV (hepatitis B virus) national standard, and then Shengxiang Bio's "Hepatitis B Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method)" was used to perform PCR amplification on the extracted nucleic acid.

[0042] Figure 7 Amplification diagram after extracting 1000 IU / mL HCV (hepatitis C virus) national standard using an automated nucleic acid extractor in combination with the nucleic acid extraction and purification kit of the present invention. GenePure Pro nucleic acid extraction and purification instrument (model: NPA-32P, manufacturer: Hangzhou Biori Technology Co., Ltd.) was used in combination with the present invention to extract 1000 IU / mL HCV (hepatitis C virus) national standard, and then Shengxiang Bio's "Hepatitis C Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method)" was used to perform PCR amplification on the extracted nucleic acid. DETAILED DESCRIPTION

[0043] Example 1

[0044] 1The nucleic acid extraction and purification kit consists of the following parts:

[0045] Lysis buffer: 3 M guanidine thiocyanate, 50 mM sodium citrate dihydrate, 1 mM ethylenediaminetetraacetic acid, 40% (v / v) anhydrous ethanol, 5% (v / v) Tween 20, pH 5.0;

[0046] Magnetic beads (hydroxy magnetic beads);

[0047] Deproteinization washing solution: 1 M guanidine thiocyanate, 20 mM sodium citrate dihydrate, 1 mM ethylenediaminetetraacetic acid, 0.1 M sodium chloride, 30% (v / v) anhydrous ethanol, 3% (v / v) Tween 20, pH 5.0;

[0048] Washing solution: 80% (v / v) anhydrous ethanol;

[0049] Eluent: 10 mM Tris(2-hydroxymethylaminomethane), 1 mM EDTA, pH 8.0.

[0050] 2 The experimental steps are as follows:

[0051] 2.1 Take an appropriate amount of 1.5mL centrifuge tubes and add 500μL lysis buffer and 10μL magnetic beads to each tube according to the number of samples to be tested;

[0052] 2.2 Add 200 μL of the sample to be tested to each tube, shake and mix for 10 seconds, and then let it stand at room temperature for 10 minutes.

[0053] 2.3 After instant centrifugation, place the centrifuge tube on the magnetic rack and slowly aspirate the solution after 2 minutes (be careful not to touch the brown substance adsorbed on the tube wall);

[0054] 2.4 Add 500 μL of protein removal solution to each tube and shake for 5 seconds. Centrifuge for a moment and place the centrifuge tube on a magnetic rack. After 2 minutes, slowly aspirate the solution (be careful not to touch the brown material adsorbed on the tube wall).

[0055] 2.5 Add 600 μL of washing solution to each tube and shake for 5 seconds. Centrifuge for a short time and place the centrifuge tube on a magnetic rack. After 2 minutes, slowly aspirate the solution (be careful not to touch the brown material adsorbed on the tube wall).

[0056] 2.6 Repeat step 4.2.5, place the centrifuge tube at room temperature, open the lid and let it stand for 1 minute.

[0057] 2.7 Add 50 μL of elution buffer to each tube, elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, pipette and mix 3-4 times, and then place in a 56℃ metal bath for 5 minutes.

[0058] 2.8 Place the EP tube on the magnetic rack. After 2 minutes, transfer the liquid to a new EP tube and store it at -20±5℃ for later use.

[0059] 3 Extraction effect

[0060] 3.1 Linearity

[0061] The nucleic acid extraction and purification kit of the present invention was used to extract four reference samples of different concentrations (A: 5*10 6 IU / mL, B:5*10 5 IU / mL, C:5*10 4 IU / mL, D:5*10 3 IU / mL), positive quality control and negative quality control, and then use the amplification reagent in Shengxiang Bio's "Hepatitis B Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method)" to perform PCR amplification on the extracted nucleic acid. Results ( Figure 1 and Figure 2 ) analysis showed that the nucleic acid concentration of the nucleic acid extraction kit of the present invention was still linear after extracting the linear concentration nucleic acid sample (linear regression correlation coefficient R 2 =0.9999). Therefore, the nucleic acid extracted by the nucleic acid extraction and purification kit of the present invention can truly reflect the actual concentration of nucleic acid in different samples.

[0062] 3.2 Precision

[0063] Extraction precision of DNA samples: The present invention was used to extract and dilute the HBV (hepatitis B virus) national standard product to 1000 IU / mL 10 times, and then the extracted nucleic acid was amplified by PCR using the Hepatitis B Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method) of Shengxiang Biotechnology ( Figure 3 ). The coefficient of variation (CV%) of the amplified data was 0.95% (Table 1).

[0064] Extraction precision of RNA samples: The present invention was used to extract and dilute the HCV (hepatitis C virus) national standard to 1000 IU / mL 10 times, and then the extracted nucleic acid was amplified by PCR using the Hepatitis C Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method) of Shengxiang Biotechnology ( Figure 4 ). The coefficient of variation (CV%) of the amplified data was 1.62% (Table 2).

[0065] Therefore, the precision of the repeatedly extracted DNA samples and RNA samples was ≤5%, which proved that the nucleic acid extracted by the nucleic acid extraction and purification kit of the present invention had good repeatability, small extraction deviation, and could stably reflect the nucleic acid concentration in the sample.

[0066] Table 1. Data analysis of amplification results of HBV standard products extracted by the present invention

[0067]

[0068] Table 2. Data analysis of amplification results of HCV standard products extracted by the present invention

[0069]

[0070] 3.3 Recovery rate

[0071] The extraction recovery rate was calculated by comparing the extraction effects of different extraction kits on double-stranded DNA (dsDNA). dsDNA was quantified using a nucleic acid quantifier (Thermo Fisher's Qubit4.0 Fluorometer) in combination with Thermo Fisher's Qubit TM The dsDNA quantification kit was used for accurate quantification, and then 200 μL of dsDNA samples were extracted 3 times using Tiangen's "Serum and Plasma Free DNA Extraction Kit", Shengxiang Bio's "DNA Extraction Kit", Biori's "Whole Blood Genomic DNA Universal Kit" and the nucleic acid extraction and purification kit of the present invention, and eluted with the same volume of eluent. Then a nucleic acid quantification instrument (Thermo Fisher's Qubit4.0 Fluorometer) was used in conjunction with Thermo Fisher's Qubit TMThe dsDNA quantitative kit accurately quantifies the dsDNA extracted by each extraction kit, and finally calculates the extraction recovery rates of different kits (Table 3). According to the analysis results, it can be seen that the present invention has a higher recovery rate under the same conditions.

[0072] Table 3. Analysis of recovery rates of dsDNA extracted by different kits

[0073]

[0074] Example 2 Analysis of the automated extraction precision of the nucleic acid extraction and purification kit of the present invention

[0075] The nucleic acid extraction and purification kit of the present invention was used in conjunction with GenePure Pro nucleic acid extraction and purification instrument (model: NPA-32P; manufacturer: Hangzhou Bioray Technology Co., Ltd.) to extract 1000 IU / mL HBV (hepatitis B virus) national standard and 1000 IU / mL HCV (hepatitis C virus) national standard 10 times respectively, and then amplified using Shengxiang Bio's "Hepatitis B Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method)" and "Hepatitis C Virus Nucleic Acid Quantitative Detection Kit (PCR-Fluorescent Probe Method)" respectively ( Figure 6 and Figure 7 ), and the coefficient of variation (CV%) of the amplification results was analyzed.

[0076] The steps of using the nucleic acid extraction and purification kit of the present invention in conjunction with the GenePure Pro nucleic acid extraction and purification instrument to extract nucleic acid are as follows:

[0077] (1) According to Figure 5 As shown, confirm the direction of the 96-deep-well plate, and then add the corresponding reagents to the corresponding wells;

[0078] (2) Add the samples to be extracted into the wells of the lysis buffer (columns 1 and 7) in the form of one well per sample, 200 μL per sample;

[0079] (3) Place the 96-deep-well plate into the nucleic acid extraction instrument and install the magnetic rod sheath;

[0080] (4) Carry out the automated extraction experiment according to the following procedure in Table 4:

[0081] Table 4. Automated extraction experimental parameters

[0082]

[0083] Heating setting: elution temperature 70°C; elution start heating step: 5;

[0084] (5) After the automated program is completed, transfer the eluate from columns 5 and 11 to new nuclease-free 1.5 mL centrifuge tubes and store at -20 ± 5 °C for later use.

[0085] The data analysis of the amplification showed that the coefficient of variation (CV%) of the HBV national standard product extracted by the present invention after amplification was 0.96% (Table 5); the coefficient of variation (CV%) of the HCV national standard product after amplification was 1.11% (Table 6). The precision of the DNA sample and RNA sample extracted automatically was ≤5%, which proved that the nucleic acid extracted by the nucleic acid extraction and purification kit of the present invention in combination with the automatic extraction instrument had good repeatability, small extraction deviation, and could stably reflect the nucleic acid concentration in the sample.

[0086] Table 5. Data analysis of amplification results of HBV standard extracted by the automatic extractor of the present invention

[0087]

[0088] Table 6. Data analysis of amplification results of HCV standard extracted by the automatic extractor of the present invention

[0089]

Claims

1. A nucleic acid extraction and purification kit, characterized in that It consists of the following parts: Lysis buffer: 2-5M chaotropic salt, 10-300mM buffer, 1-100mM EDTA, 20-60% (v / v) lower alcohols, 3-10% (v / v) surfactant, pH 4-8; Magnetic beads; Deproteinization washing solution: 0.5-3M chaotropic salt, 10-300mM buffer, 1-100mM EDTA, 50-500mM sodium chloride, 20-60% (v / v) lower alcohols, 1-10% (v / v) surfactant, pH 4-8; washing solution: 50-100% (v / v) anhydrous ethanol; Eluent: 10-100 mM Tris(OHM), 1-50 mM EDTA, pH 8-10.

2. The nucleic acid extraction and purification kit according to claim 1, characterized in that The chaotropic salt is selected from any one or two of guanidine isothiocyanate and guanidine hydrochloride; the buffer is selected from sodium citrate dihydrate buffer or tris(hydroxymethyl)aminomethane buffer.

3. The nucleic acid extraction and purification kit according to claim 1, characterized in that The lower alcohols are ethanol and / or isopropanol.

4. The nucleic acid extraction and purification kit according to claim 1, characterized in that The surfactant is selected from any one or more of ethylphenyl polyethylene glycol, polyethylene glycol octylphenyl ether, and Tween 20.

5. The nucleic acid extraction and purification kit according to claim 1, characterized in that The magnetic beads are hydroxy magnetic beads.

6. The nucleic acid extraction and purification kit according to any one of claims 1 to 5, characterized in that The nucleic acid extraction and purification kit is composed of the following parts: Lysis buffer: 2-5 M guanidine thiocyanate, 10-300 mM sodium citrate dihydrate, 1-100 mM ethylenediaminetetraacetic acid, 20-60% (v / v) anhydrous ethanol, 3-10% (v / v) Tween 20, pH 4-8; Hydroxyl magnetic beads; Protein removal solution: 0.5-3M guanidine thiocyanate, 10-300mM sodium citrate dihydrate, 1-100mM ethylenediaminetetraacetic acid, 50-500mM sodium chloride, 20-60% (v / v) anhydrous ethanol, 1-10% (v / v) Tween 20, pH 4-8; Washing solution: 50-100% (v / v) anhydrous ethanol; Eluent: 10-100 mM Tris(OHM), 1-50 mM EDTA, pH 8-10.

7. The nucleic acid extraction and purification kit according to claim 6, characterized in that The nucleic acid extraction and purification kit is composed of the following parts: Lysis buffer: 3 M guanidine thiocyanate, 50 mM sodium citrate dihydrate, 1 mM ethylenediaminetetraacetic acid, 40% (v / v) anhydrous ethanol, 5% (v / v) Tween 20, pH 5.0; Hydroxyl magnetic beads; Deproteinization washing solution: 1 M guanidine thiocyanate, 20 mM sodium citrate dihydrate, 1 mM ethylenediaminetetraacetic acid, 0.1 M sodium chloride, 30% (v / v) anhydrous ethanol, 3% (v / v) Tween 20, pH 5.0; Washing solution: 80% (v / v) anhydrous ethanol; Eluent: 10 mM Tris(2-hydroxymethylaminomethane), 1 mM EDTA, pH 8.

0.

8. Use of the nucleic acid extraction and purification kit according to any one of claims 1 to 5 in extracting nucleic acids.

9. A method for extracting nucleic acid, characterized in that: The following steps are involved: (1) Take an appropriate number of 1.5 mL centrifuge tubes and add 500 μL of lysis buffer and 10 μL of magnetic beads to each tube according to the number of samples to be tested; (2) Add 200 μL of the sample to be tested to each tube, shake and mix for 10 seconds, and then let stand at room temperature for 10 minutes; (3) After instant centrifugation, place the centrifuge tube on a magnetic rack and slowly aspirate the solution after 2 minutes; (4) Add 500 μL of protein removal solution to each tube and shake for 5 seconds. Centrifuge briefly and place the tube on a magnetic rack. After 2 minutes, slowly aspirate the solution. (5) Add 600 μL of washing solution to each tube and shake for 5 seconds. Centrifuge briefly and place the tube on a magnetic rack. After 2 minutes, slowly aspirate the solution. (6) Repeat step (5) and place the centrifuge tube at room temperature with the lid open for 1 minute; (7) Add 50 μL of elution buffer to each tube, elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, pipette and mix 3-4 times, and then bathe at 56°C for 5 minutes; (8) Place the EP tube on a magnetic rack. After 2 minutes, transfer the liquid to a new EP tube and store it at -20±5℃ for later use.

10. A method for extracting nucleic acid, characterized in that: The nucleic acid extraction and purification kit according to any one of claims 1 to 5 is used in combination with a nucleic acid extraction and purification instrument to extract and purify nucleic acid.

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