Preparation and application of nucleic acid extraction material based on CTAB modified attapulgite

Abstract

The present invention discloses a preparation and application of a nucleic acid extraction material based on CTAB-modified attapulgite, and belongs to the field of biotechnology. The specific steps of the method are as follows: S1, acid activation modification: stirring and reacting attapulgite powder with HCl aqueous solution, filtering, washing the solid, drying, and then grinding and sieving to obtain acid-activated attapulgite. S2, CTAB modification: adding CTAB and acid-activated attapulgite to hydrochloric acid, stirring, adjusting the pH to neutral, drying the solution, grinding and drying the obtained solid, sieving, and obtaining CTAB-modified attapulgite. The present invention utilizes CTAB to modify attapulgite, optimizes its physical and chemical properties, greatly improves its nucleic acid adsorption performance, and ensures low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid extraction material preparation, and specifically relates to the preparation and application of a nucleic acid extraction material based on CTAB modified attapulgite. Background Art

[0002] Nucleic acid extraction is currently mainly concentrated on the solid phase carrier adsorption method, and its operation steps can be divided into three parts: cell lysis, nucleic acid adsorption and elution. It is reported that the solid phase materials for nucleic acid adsorption mainly include: silica and its derivatives, metal-based materials, polymer materials and other inorganic non-metallic materials. Among them, the more commonly used commercial methods are the column extraction method with silica gel filter membrane as the solid phase carrier and the metal-based magnetic bead method. The column extraction method requires tedious operations such as centrifugation and filtration, while the automated, high-throughput magnetic bead method has a higher cost. Therefore, there is an urgent need to develop a low-cost, efficient material and method for extracting nucleic acids. Summary of the invention

[0003] In order to solve the problems of high cost or low efficiency of current nucleic acid extraction materials, the present invention provides a preparation method and application of a hexadecyltrimethylammonium bromide (CTAB) modified attapulgite adsorption material by improving attapulgite, so as to achieve the purpose of low-cost and high-efficiency extraction of nucleic acids using modified attapulgite.

[0004] The present invention provides a method for preparing a CTAB-modified attapulgite adsorption material, and the specific operation steps are as follows:

[0005] S1. Acid activation modification: The attapulgite powder is stirred and reacted with an aqueous solution of HCl, filtered, the solid is washed, dried, and then ground and sieved to obtain acid-activated attapulgite.

[0006] S2. CTAB modification: adding CTAB and acid-activated attapulgite to hydrochloric acid, stirring, adjusting the pH to neutral, drying the solution, grinding the dried solid, and sieving to obtain CTAB-modified attapulgite.

[0007] Furthermore, the mesh size of the attapulgite powder in step S1 is greater than 325 meshes.

[0008] Furthermore, the concentration of the HCl aqueous solution in step S1 is 5-15 wt %, and can be specifically 10 wt %.

[0009] Furthermore, in step S1, the mass volume ratio of the attapulgite powder and the HCl aqueous solution is 1 g: (5-15) mL, and can be specifically 1 g: 10 mL.

[0010] Furthermore, in step S1, the stirring is carried out at 50-90° C. for 1-3 hours, and specifically at 70° C. for 2 hours.

[0011] Furthermore, the sieving in step S1 is through a 325-mesh standard sieve.

[0012] Furthermore, in step S2, the mass ratio of acid-activated attapulgite to CTAB is 10:(0.5-1.5), and can be specifically 10:1.

[0013] Furthermore, in step S2, the concentration of hydrochloric acid is 0.5-1.5 wt %, and can be specifically 1 wt %.

[0014] Furthermore, in step S2, the mass volume ratio of the acid-activated attapulgite and the hydrochloric acid is 2g:(100-500)mL, and can be specifically 2g:300mL.

[0015] Furthermore, in step S2, the stirring is carried out at 50-70° C. for 20-28 hours, and specifically at 60° C. for 24 hours.

[0016] Furthermore, the sieving in step S2 is through a 325-mesh standard sieve.

[0017] The invention provides a CTAB modified attapulgite adsorption material.

[0018] The invention provides application of the CTAB modified attapulgite adsorption material in the field of nucleic acid adsorption.

[0019] The present invention provides a method for extracting DNA from a CTAB-modified attapulgite adsorption material, and the specific operating steps are as follows:

[0020] The attapulgite suspension is prepared, and the attapulgite suspension, the solution containing DNA and the buffer solution are mixed, shaken, centrifuged and the precipitate after centrifugation is collected.

[0021] Furthermore, the concentration of the attapulgite sample suspension is 10-50 mg / mL, and can be specifically 30 mg / mL.

[0022] Furthermore, the solution containing DNA is a solution containing 1 to 900 μg / mL DNA.

[0023] Specifically, the solution containing DNA is a solution containing 100-200 μg / mL fish sperm DNA, or a Staphylococcus aureus cell lysate containing DNA.

[0024] Furthermore, the volume ratio of the attapulgite sample suspension to the solution containing DNA is 1:(1-20).

[0025] Preferably, the volume ratio of the attapulgite sample suspension to the solution containing DNA is 1:(5-15).

[0026] Furthermore, the buffer solution is a CH3COOH-CH3COONa solution.

[0027] Further, after adding the buffer solution, the pH of the system is 3-8, Na + The concentration is 10~600mM.

[0028] Specifically, the pH of the mixed liquid system is 5, Na + The concentration is 60mM.

[0029] Furthermore, the oscillation was performed at 1000 r / min for 10 min.

[0030] The present invention provides a method for desorbing DNA from a CTAB-modified attapulgite adsorption material, and the specific steps are as follows:

[0031] The precipitate obtained after centrifugation in the DNA extraction method is washed with ethanol, dried, and the elution solution and the dried precipitate are mixed and centrifuged to obtain the supernatant.

[0032] Furthermore, the concentration of ethanol is 50-80%, and specifically can be 70%.

[0033] Furthermore, the eluent is one of water, Tris-EDTA buffer or sodium tripolyphosphate aqueous solution.

[0034] Preferably, the eluent is one of ultrapure water, Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0) or a sodium tripolyphosphate aqueous solution with a concentration of 5 to 50 mM.

[0035] Specifically, the eluent is a 30 mM sodium tripolyphosphate aqueous solution.

[0036] Furthermore, the mass volume ratio of the dried precipitate and the eluent is 300 μg: (80-200) μL.

[0037] Specifically, the mass volume ratio of the dried precipitate and the eluent is 300 μg:100 μL.

[0038] A DNA extraction method comprises the following steps: firstly, DNA is adsorbed according to a DNA extraction method of a CTAB-modified attapulgite adsorption material, and then DNA is desorbed according to a DNA desorption method of a CTAB-modified attapulgite adsorption material.

[0039] Advantages and effects of the present invention:

[0040] (1) The present invention utilizes CTAB to modify attapulgite, optimizes its physical and chemical properties, and greatly improves its nucleic acid adsorption performance while ensuring low cost.

[0041] (2) The saturated adsorption capacity of CTAB-modified attapulgite for DNA is 18.6566 μg / mg. The desorption rate of CTAB-modified attapulgite for DNA can reach more than 75% by using a suitable desorbent. It is reported that the saturated adsorption capacity of silica-coated magnetic beads (Optimization of influencing factors of nucleic acid adsorption onto silica-coated magnetic particles: Application to viral nucleic acid extraction from serum) for DNA is 10.6 μg / mg, and the desorption rate is 93% ± 3.1%; the reported saturated adsorption capacity of lithium-modified montmorillonite for DNA is 8 to 10 μg / mg, which indicates that the saturated adsorption capacity of DNA of the present invention is better than that of silica-coated magnetic beads and lithium-modified montmorillonite.

[0042] (3) When CTAB-modified attapulgite was used for nucleic acid extraction of Staphylococcus aureus, a high yield of genomic DNA (5.45±0.46) μg was obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the X-ray diffraction (XRD) characterization diagram of the attapulgite before and after modification prepared in Example 1 of the present invention.

[0044] Figure 2 This is a Fourier transform infrared spectrum (FTIR) analysis diagram of the attapulgite before and after modification prepared in Example 1 of the present invention.

[0045] Figure 3 This is a zeta potential analysis diagram of the attapulgite before and after modification prepared in Example 1 of the present invention.

[0046] Figure 4 (a) is a scanning electron microscope (SEM) image of the attapulgite used. Figure 4 (b) is a SEM image of the acid-modified attapulgite obtained in Example 1 of the present invention, Figure 4 (c) is a scanning electron microscope (SEM) image of the CTAB-modified attapulgite prepared in Example 1.

[0047] Figure 5This is the effect of initial pH on DNA adsorption in the adsorption system of Example 2 of the present invention.

[0048] Figure 6 is the Na in the adsorption system of Example 2 of the present invention + Effect of concentration on DNA adsorption.

[0049] Figure 7 This is a graph showing the adsorption kinetics of DNA adsorbed by CTAB-modified attapulgite prepared in Example 2 of the present invention.

[0050] Figure 8 This is a diagram showing the desorption conditions of DNA from CTAB-modified attapulgite obtained in Example 2 of the present invention. Figure 8 (a) The effect of different desorption agents on DNA desorption. Figure 8 (b) The effect of the concentration of the optimal desorption agent STPP on DNA desorption. Figure 8 (c) is the desorption equilibrium time of DNA by the desorbent on CTAB-modified attapulgite.

[0051] Fig. 9 This is the electrophoresis diagram of bacterial genomic DNA extraction in Example 3 of the present invention. DETAILED DESCRIPTION

[0052] Materials and Reagents:

[0053] Attapulgite was produced from Oubaite in Xuyi, Jiangsu Province; fish sperm DNA, sodium tripolyphosphate (Reagent Grade, Purity ≥ 98.0%), hexadecyltrimethylammonium bromide (CTAB), column-based bacterial genomic DNA extraction kit (NO.B518255Ezup), and magnetic bead-based bacterial genomic DNA extraction kit (NO.B518725) were purchased from Shanghai Bioengineering Co., Ltd.; DNA loading buffer (instant view TM Red fluorescent DNA loading buffer (6× containing bromophenol blue) was purchased from Shanghai Biyuntian Co., Ltd.; anhydrous ethanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); glacial acetic acid (analytical grade, Shanghai Aladdin Reagent Co., Ltd.); anhydrous sodium acetate (analytical grade, Beijing Inokai Technology Co., Ltd.); ultrapure water used in the experiment was purified using a Milli-Q A10 filtration system (18.2 MΩ·cm); Staphylococcus aureus ATCC 6538: preserved in the Food Safety and Quality Control Research Center of the School of Food Science and Technology, Jiangnan University.

[0054] Instruments and equipment:

[0055] X-ray diffractometer (D2 PHASER, Bruker AXS GmbH, Germany), Fourier transform infrared spectrometer (IS10, Nicolet, USA), field emission scanning electron microscope (Quanta 250FEG, FEI, USA), nanoparticle size and zeta potential analyzer (Zetasizer nano ZS, Malvern, UK).

[0056] Example 1

[0057] S1. Acid activation modification: 10 g of attapulgite powder (>325 mesh) was mixed with 100 mL of 10 wt% HCl, stirred and reacted at 70 ° C for 2 h, filtered and washed with deionized water to pH = 6.0, and the washed attapulgite powder was placed in an oven at 105 ° C for 12 h, and then ground through a 325 mesh standard sieve to obtain acid-activated attapulgite powder.

[0058] S2, CTAB modification: 2g of acid-activated attapulgite powder was mixed with 100mL of ultrapure water to prepare a 2% (w / w) clay slurry, stirred at 80°C for 4h, the product was centrifuged, and washed with ultrapure water for more than 10 times to remove the CTAB on the surface of the attapulgite powder. The product was placed in an oven at 60°C and dried for 12h, and then ground through a 325 mesh sieve to obtain CTAB-modified attapulgite powder.

[0059] The attapulgite, acid-activated attapulgite, and CTAB-modified attapulgite were characterized. X-ray diffraction analysis (XRD) of the attapulgite before and after modification was performed. Figure 1 As shown in Figure 1, the XRD pattern of the attapulgite sample was compared with that of attapulgite (PDF standard card number #31-0783), quartz (PDF standard card number #85-0798) and dolomite (PDF standard card number #36-0426). The results showed that the main mineral composition of the attapulgite sample contained attapulgite, quartz and dolomite. After acid modification, the peak of dolomite disappeared, indicating that hydrochloric acid reacted with dolomite, dissolving dolomite and purifying attapulgite. After further modification by CTAB, the structure of attapulgite did not change significantly.

[0060] The FTIR analysis of the attapulgite before and after modification showed that Figure 2 As shown, the attapulgite sample has a -1 and 3543.5cm -1 The two stretching vibration peaks of (Mg, Al)-OH, 3417 cm -1 The peak at 1645cm-1 is the stretching vibration peak of zeolite water in attapulgite; the peak at 1033cm-1 is the bending vibration absorption peak of adsorbed water and crystal water; -1The peak at 790 cm is the asymmetric stretching vibration peak of Si-O between the tetrahedral layers in the single chain; -1 and 516cm -1 is the bending vibration peak of Si-O. After HCl modification, the clay mineral still shows the characteristic absorption peak of the original attapulgite, and the dolomite CO3 2- The characteristic peaks at 1448cm-1 and 730cm-1 disappeared, indicating that the modified material was purified without destroying the structure and the basic skeleton of the clay mineral was still preserved. -1 and 2922cm -1 The asymmetric and symmetric stretching vibration peaks corresponding to the methylene CH2 appeared respectively. Since there are three methyl groups in the CTAB structure, it shows that after organic modification, CTAB was successfully modified on attapulgite.

[0061] The Zeta potential test of attapulgite before and after modification was carried out, such as Figure 3 As shown in the figure, within the measured range, the surface of the attapulgite is negatively charged, and the attapulgite modified by HCl and CTAB changes its original charge. 2+ Mg 2+ The dissolution leads to an increase in the overall negative charge of the attapulgite, which increases the negative charge of the HCl-attapulgite. Compared with the HCl-attapulgite, the negative charge of the CTAB-attapulgite is also significantly reduced. This is because CTAB (C 16 H 37 N(CH3)3Br) contains a permanent positive charge that does not change with pH value. It compensates for most of the permanent negative charge of attapulgite, making its charge zero point at pH 2-3.

[0062] Scanning electron microscope (SEM) analysis of attapulgite before and after modification was performed. Figure 4 As shown in Figure (a), nanorods with an average diameter of 50nm and a length of 200-300nm are entangled and aggregated. Figure (b) shows that after acid modification, dolomite is dissolved, the attapulgite is purified, its basic skeleton is still preserved, the gap is enlarged, and the attapulgite crystal bundles are disintegrated to looseness. Figure (c) shows that after the acid-modified attapulgite is modified by CTAB, the attapulgite becomes short and thick, and relatively smooth.

[0063] Example 2

[0064] Adsorption of fish sperm DNA by modified attapulgite: 30 mg of CTAB-attapulgite sample was added to 1 mL of ultrapure water to prepare an attapulgite sample suspension. Then, 10 μL of CTAB-attapulgite sample suspension (30 mg / mL) was mixed with 50 μL of adsorption solution (containing 200 ng / μL fish sperm DNA), 10 μL of CH3COOH-CH3COONa buffer solution and 30 μL of ultrapure water. The system pH was 5, Na + The concentration was 60 mM and the total volume was 100 μL. The mixture was shaken at 1000 r / min in a constant temperature shaking incubator and centrifuged at 8000×g for 10 min to obtain CTAB-modified attapulgite with DNA adsorbed. The DNA concentration in the supernatant was measured using a micro-nucleic acid quantifier.

[0065] The DNA concentration in the supernatant was measured using a micro-nucleic acid quantifier. The amount of DNA adsorbed by the adsorption material was calculated based on the change in DNA content in the solution before and after adsorption equilibrium. The saturated adsorption amount of DNA by CTAB-attapulgite was 18.6566 μg / mg.

[0066] The amount of DNA adsorbed by the adsorption material is calculated by the change in DNA content in the solution before and after adsorption equilibrium:

[0067] Q e =V0(C0-C e ) / W s

[0068] Where: Q e (μg / mg) is the amount of DNA adsorbed when adsorption equilibrium is reached; C e (μg / mL) is the concentration of DNA in the supernatant when adsorption equilibrium is reached; C0 (μg / mL) is the initial concentration of the DNA solution; V0 (mL) is the volume of the DNA solution added; W s is the adsorbent dosage.

[0069] Comparative Example 1

[0070] This comparative example is to investigate the effect of the initial pH of the solution on adsorption. According to the process of Example 2, the amount of CH3COOH in the buffer solution was adjusted to adjust the pH of the system to 3, 4, 6, 7, and 8, and other operations remained unchanged.

[0071] Effect of initial pH in adsorption system on DNA adsorption Figure 5As shown in the figure, the surface groups of attapulgite modified by CTAB are pH-sensitive groups, which determine the charge at different pH values. Under acidic conditions, the hydrogen ions in the solution make the attapulgite carry more positive charges, and more DNA is adsorbed through electrostatic action. Under alkaline conditions, the attapulgite is negatively charged as a whole. There is electrostatic repulsion between the attapulgite and DNA in the solution, which hinders the adsorption of DNA and reduces the adsorption rate of DNA. Under experimental conditions, the amino or phosphate groups in the DNA molecules remove protons and carry negative charges. Therefore, it is difficult for DNA to adsorb on the surface of the clay that is also negatively charged. After modification by CTAB, CTAB-attapulgite is positively charged at pH < 3, which improves the microscopic charge environment on the surface of the attapulgite, enabling it to effectively bind anions through electrostatic interactions. At pH > 3, the amount of DNA adsorption gradually decreases with the continuous increase of pH.

[0072] Comparative Example 2

[0073] This comparative example is to investigate the solution Na + The effect on adsorption was as follows: according to the process of Example 2, the amount of CH3COONa in the buffer solution was adjusted to adjust the Na + The concentrations were 0, 20, 40, 80, 100 and 120 mM, and other operations remained unchanged.

[0074] Effect of initial pH in adsorption system on DNA adsorption Figure 6 As shown, Na + As the concentration increases from 0mM to 60mM, the amount of DNA adsorbed on the surface of the material gradually increases. + As the concentration continued to increase, the amount of DNA adsorption did not change much, so the Na + The optimum concentration is 60mM.

[0075] Comparative Example 3

[0076] This comparative example is to investigate the effect of oscillation time on adsorption. Referring to the process of Example 2, the oscillation time is adjusted to 20, 30, 40, 50, and 60 minutes, and other operations remain unchanged. Kinetic simulation can be used to investigate the adsorption rate and reaction equilibrium time during the adsorption process.

[0077] Adsorption kinetic model fitting: Pseudo-first order model and pseudo-second order model were used to fit the DNA adsorption kinetics. 2 value to judge the quality of the fit.

[0078] The pseudo-first-order model equation is as follows: ln(q e -q t )=ln qe -K1t

[0079] The pseudo-second-order model equation is as follows: t / q t =1 / Kq e 2 +t / q e

[0080] In the formula, q e (μg / mg) is the equilibrium adsorption capacity; q t (mg / g) is the amount of DNA adsorbed by clay when the reaction time is t; t (min) is the adsorption time; K1, K (g·(mg·min) -1 ) is the rate constant.

[0081] The adsorption kinetics of DNA on CTAB-modified attapulgite are shown in Figure 7 As shown in Figure 2, the adsorption data were fitted using pseudo-first-order adsorption kinetics and pseudo-second-order adsorption kinetics models. The R 2 , the pseudo-first-order model is the best fitting model, R 2 The experimental value of equilibrium adsorption is consistent with the theoretical value of the pseudo-first-order model, and the predicted saturated adsorption capacity of CTAB-attapulgite is 18.6566μg / mg. CTAB-attapulgite rapidly adsorbs in the first 10 minutes, and the adsorption percentage reaches more than 90%. The adsorption slows down gradually after 10 minutes, indicating that the adsorption gradually approaches equilibrium. In subsequent experiments, 10 minutes was selected as the reaction time.

[0082] Example 3

[0083] Desorption of fish sperm DNA by modified attapulgite: The CTAB-modified attapulgite adsorbing DNA obtained in Example 2 was washed with 70% ethanol, centrifuged and air-dried, and DNA was eluted with 100 μL of elution solution. The nucleic acid concentration in the elution solution was measured with a micro-nucleic acid quantifier.

[0084] The results of the desorption conditions of DNA by CTAB-modified attapulgite are as follows: Figure 8As shown in the figure, after washing with 70% ethanol and drying, the elution effect of 100μL TE buffer (10mM Tris, 1mM EDTA, pH 8.0) and sodium tripolyphosphate (STPP) on DNA was compared, and the concentration of nucleic acids in the eluent was measured by a micro-ultraviolet spectrophotometer. The results showed that the desorption effect of STPP on nucleic acids on modified attapulgite was significantly better than that on TE buffer, and the optimal concentration of STPP desorption was 30mM, and the desorption rate could reach (76.89±1.59)%. The desorption performance of 100mM STPP on DNA on CTAB-attapulgite was investigated, and the results showed that nucleic acids were desorbed within 10min, and the desorption rate was more than 75%.

[0085] Example 4 Application of CTAB-modified attapulgite adsorption material for nucleic acid extraction

[0086] S1. Bacteria collection: Inoculate the bacterial solution into 3 mL of sterilized tryptic soy broth (TSB) and culture at 37°C and 160 rpm for 18 h. Take 1 mL of overnight cultured bacteria in a 1.5 mL centrifuge tube, centrifuge at 8,000 rpm for 1 min at room temperature, discard the supernatant, and collect the bacteria.

[0087] S2, bacterial lysis: Add 100μl Buffer Digestion, 80μL lysozyme solution (20mg / ml) and 2μL lysostaphin solution (2mg / mL) to resuspend the bacterial solution, and place in a 37℃ water bath for 30min. Add 20μl Proteinase K solution and oscillate to mix. To obtain RNA-free DNA, add 20μl RNase A (10mg / ml) after the water bath and place at room temperature for 2-5min. Place in a 56℃ water bath for 30min until the cells are completely lysed. Add 200μl Buffer BD, mix thoroughly by inversion, and place in a 70℃ water bath for 10min until the solution becomes clear, and obtain bacterial lysis solution.

[0088] S3. Adsorption of bacterial genomic DNA by modified attapulgite

[0089] 30 mg of CTAB-attapulgite sample was added to 1 mL of ultrapure water to make a suspension. Then, 30 μL of CTAB-attapulgite sample suspension (30 mg / mL), bacterial lysate (400 μL) and 70 μL of buffer solution were mixed to a total system of 500 μL. The mixture was shaken at 1000 r / min in a constant temperature shaking incubator and centrifuged at 8000 × g for 10 min. The DNA concentration in the supernatant was measured using a micro-nucleic acid quantifier. The amount of adsorbed DNA can be calculated by the difference between the amount of added DNA and the amount of supernatant DNA.

[0090] S4. Desorption of bacterial genomic DNA by modified attapulgite

[0091] The precipitate was washed with 70% ethanol, dried after centrifugation, and the DNA was eluted with 100 μL of elution solution. The nucleic acid concentration in the elution solution was measured using a micro-nucleic acid quantifier.

[0092] Comparative Example 4

[0093] The centrifugal column method and magnetic bead method for extracting genomic DNA were used as controls, and Staphylococcus aureus was used as the object of nucleic acid extraction. The yield of bacterial genomic DNA extracted by modified attapulgite and the quality of nucleic acid were evaluated.

[0094] Centrifugal column method: The centrifugal column method for adsorbing nucleic acids is to place the adsorption column in a collection tube, use a pipette to add all 400 μL of the cell lysate obtained in step (1) of Example 4 to the adsorption column, let it stand for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the waste liquid in the collection tube. Then put the adsorption column back into the collection tube, add 500 μl PW Solution, centrifuge at 10,000 rpm for 30 seconds, and discard the filtrate. Then put the adsorption column back into the collection tube, add 500 μl Wash Solution, centrifuge at 10,000 rpm for 30 seconds, and discard the filtrate. Put the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 2 minutes at room temperature, and remove the residual Wash Solution. Finally, take out the adsorption column, put it into a new 1.5 ml centrifuge tube, add 50-100 μl CEBuffer, let it stand for 3 minutes, centrifuge at 12,000 rpm for 2 minutes at room temperature, and collect the DNA solution. The extracted DNA can be used for the next experiment immediately or stored at -20°C.

[0095] Magnetic bead method: The method for adsorbing nucleic acids by the magnetic bead method is to add 500 μL Buffer MB and 10 μL magnetic beads to 400 μL of the cell lysate obtained in step (1) of Example 4, mix by aspiration or shaking, and let stand at room temperature for 1 min. Place the centrifuge tube on the magnetic rack for 30 seconds, wait for the magnetic beads to be completely absorbed to the tube wall, aspirate and discard the supernatant, and remove the centrifuge tube from the magnetic rack. Add 900 μL 70% ethanol to the centrifuge tube, aspirate or shake to mix, place the centrifuge tube on the magnetic rack for 30 seconds, wait for the magnetic beads to be completely absorbed to the tube wall, aspirate and discard the supernatant, and remove the centrifuge tube from the magnetic rack. Then repeat the above steps once, open the lid and dry at room temperature for 15-20 minutes or open the lid and dry in a 55°C constant temperature box for 5-7 minutes until there is no liquid residue in the tube. Add 50-100 μL TEBuffer to the centrifuge tube, bathe in a 65°C water bath for 5-10 minutes, and mix occasionally. Finally, take out the centrifuge tube and place it on the magnetic rack for 30 seconds. After the magnetic beads are completely absorbed onto the tube wall, carefully pipette the supernatant into a new centrifuge tube to obtain genomic DNA.

[0096] Take 1 μl of DNA extraction product, use an ultra-micro nucleic acid quantifier to determine its concentration, A260 and A280, and calculate the yield (concentration × elution volume) and purity (A260 / A280). Use 0.8% agarose gel electrophoresis to detect the extracted genomic DNA, with a sample volume of 5 μL and constant voltage electrophoresis at 3-5V / cm for 2 hours. Observe and take pictures under ultraviolet light with a wavelength of 254nm. The appearance of a single band of appropriate fragment size in the electrophoresis indicates that the DNA integrity is good. The electrophoresis diagram of bacterial genomic DNA extraction is shown below. Fig. 9 As shown, 1 is the band obtained by column extraction method, 2 is the band obtained by magnetic bead extraction method, and 3 is the band obtained by modified attapulgite adsorption method. The extracted Staphylococcus aureus genomic DNA is greater than 15,000 bp in length and is a single band, indicating that the extracted genomic DNA has good integrity.

[0097] The yield and purity of Staphylococcus aureus genomic DNA extracted by different nucleic acid adsorption methods are shown in Table 1. Compared with the commercially available column extraction method and magnetic bead method, the DNA yield of the CTAB-modified attapulgite extraction method was slightly higher than that of the centrifugal column extraction method, which was 5.45±0.46μg.

[0098] Table 1 The amount and purity of genomic DNA extracted by different methods

[0099]

[0100] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for extracting nucleic acid, It is characterized in that First, perform nucleic acid adsorption. The specific steps are as follows: Take CTAB-modified attapulgite to prepare an attapulgite suspension, mix the attapulgite suspension, a solution containing DNA, and a buffer solution, shake, centrifuge, and take the precipitate after centrifugation; The buffer solution is CH 3 COOH-CH 3 COONa solution; after adding the buffer solution, the pH of the system is 5, Na + The concentration is 60 mM; The CTAB modified attapulgite is prepared by the following steps: S1. Acid activation modification: stirring the attapulgite powder and the HCl aqueous solution to react, filtering, washing the solid, drying, and then grinding and sieving to obtain acid-activated attapulgite; the concentration of the HCl aqueous solution is 5-15 wt%; the mass volume ratio of the attapulgite powder and the HCl aqueous solution is 1g: (5-15) mL; S2, CTAB modification: 2 g of acid-activated attapulgite powder was mixed with 100 mL of ultrapure water to prepare a 2%, w / w clay slurry, and stirred at 80°C for 4 h. The product was centrifuged and washed with ultrapure water for more than 10 times to remove the CTAB on the surface of the attapulgite powder; the product was placed in an oven at 60°C and dried for 12 h, and then ground and sieved with a 325-mesh sieve to obtain CTAB-modified attapulgite; Then, the nucleic acid is desorbed. The specific steps are as follows: The precipitate after centrifugation was washed with ethanol, dried, and the dried precipitate was mixed with an eluent and centrifuged to obtain the supernatant; the eluent was a 30 mM sodium tripolyphosphate aqueous solution; the mass volume ratio of the dried precipitate to the eluent was 300 μg: (80-200) μL.

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