CN110684764A - Lysis solution for nucleic acid extraction, nucleic acid extraction kit and nucleic acid extraction method - Google Patents
Lysis solution for nucleic acid extraction, nucleic acid extraction kit and nucleic acid extraction method Download PDFInfo
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Abstract
The invention discloses a lysis solution for nucleic acid extraction, a nucleic acid extraction kit and a nucleic acid extraction method. The nucleic acid extraction lysate comprises 0.5-2M sodium salt, 2-5M guanidine salt, 1-5 mM metal ion complexing agent, 0.25-1% of nonionic surfactant and 1-3% of PEG, wherein the molecular weight of the PEG is 6000-10000 Da, preferably 8000 Da. The lysis solution has reasonable proportioning, can fully and effectively lyse cells, has high lysis efficiency, enables nucleic acid in the cells to be fully released, obtains nucleic acid with higher concentration and purity, and is beneficial to improving the quality and the extraction efficiency of the nucleic acid; in addition, the lysate and the kit do not need to digest and remove protein by using proteinase K in the whole nucleic acid extraction process, and alcohol is not used for precipitating nucleic acid, so that the obtained nucleic acid has high purity and complete fragments.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to lysis solution for nucleic acid extraction, a nucleic acid extraction kit and a nucleic acid extraction method.
Background
Nucleic acid is a kind of biopolymer, is an essential constituent substance for all known lives, is widely present in animal and plant cells and microorganisms, and is divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). At present, the detection of nucleic acid is not isolated in various research and detection fields of molecular biology, and the extraction and purification of nucleic acid is the basis of molecular biology research and clinical detection.
The existing nucleic acid extraction methods mainly comprise an alkaline lysis method, a phenol chloroform extraction method, a chelate resin method, a centrifugal column membrane adsorption method, a magnetic bead method and the like. The alkaline cracking method and the like have the defects of poor nucleic acid quality, complicated operation steps and long time consumption due to the use of toxic and harmful organic solvents in the extraction process. Compared with other methods, the magnetic bead method is simpler and more convenient to operate, is suitable for various sample types, and has lower requirements on samples. The surface-modified nano magnetic beads can specifically adsorb nucleic acid in a high-salt low-PH solution environment, separate the nucleic acid from biomacromolecules such as protein, polysaccharide, fat and the like under the action of an external magnetic field and a washing solution, and separate the magnetic beads from the nucleic acid by changing the pH value of the solution, so that high-quality nucleic acid can be obtained; and the extraction and purification of nucleic acid by using the magnetic bead method can be carried out by matching with an automatic nucleic acid extraction instrument, so that the extraction of nucleic acid is automatic and high-throughput.
Although the existing method for extracting nucleic acid by using magnetic beads is improved, the method still has many defects. Firstly, a lysis solution commonly used for extracting nucleic acid by a magnetic bead method usually contains proteinase K, the protein in a biological sample needs to be digested by the proteinase K, especially for a whole blood sample, the proteinase K needs to be added to ensure that high-quality nucleic acid can be extracted, the proteinase K is easy to remain, the extraction time is long, and the extraction process needs to be heated. Secondly, in the nucleic acid extraction operation in the prior art, the lysis of the sample and the combination of the magnetic beads and the DNA are often carried out step by step, and the corresponding kit often comprises independently subpackaged lysis solution and combination solution, so that the nucleic acid extraction process is complicated, the probability of nucleic acid pollution is increased to a great extent, and the subsequent detection and the detection accuracy are not facilitated.
Disclosure of Invention
The invention aims to provide a lysis solution for nucleic acid extraction, a nucleic acid extraction kit and a nucleic acid extraction method, so as to realize rapid, efficient and safe extraction of nucleic acid in a biological sample.
As a first aspect of the invention, the invention provides a nucleic acid extraction lysate.
Preferably, the lysis solution comprises 0.5-2M sodium salt, 2-5M guanidine salt, 1-5 mM metal ion complexing agent, 0.25-1% of nonionic surfactant and 1-3% of PEG.
Wherein the sodium salt can provide sodium ions to the solution environment to promote the negatively charged nucleic acids to be specifically combined with the nucleic acid adsorbate, and the sodium salt can change the osmotic pressure of the biological sample to make the biological sample easier to crack, and the sodium salt can be sodium acetate, sodium chloride, sodium iodide and the like; the guanidine salt is a strong protein denaturant, and the guanidine salt is used for cracking cells and releasing nucleic acid from protein; the PEG can selectively precipitate nucleic acid, so that the nucleic acid is accumulated in the solution, and the adsorption of the nucleic acid adsorbate to the nucleic acid is facilitated.
In the present invention, a nucleic acid-adsorbing substance is a substance that can specifically adsorb nucleic acids on its surface and can specifically separate from nucleic acids under specific conditions. In some preferred embodiments, the nucleic acid adsorbate is a magnetic bead. In other preferred embodiments, the nucleic acid adsorbate is an adsorption membrane, such as a silica gel adsorption membrane.
Preferably, the molecular weight of the PEG is 6000-10000 Da.
Preferably, the PEG has a molecular weight of 8000 Da.
Preferably, the sodium salt is sodium acetate, the guanidine salt is guanidine hydrochloride, the metal ion complexing agent is EDTA, and the nonionic surfactant is Triton X-100.
Guanidine hydrochloride can not only rapidly destroy cell membranes, but also denature proteins, so that the proteins are denatured and precipitated, and nucleic acid can get rid of the winding of the proteins; EDTA, i.e. ethylenediaminetetraacetic acid, capable of reacting with Mg2+、Ca2+、Mn2+、Fe2+Binding metal ions to reduce the influence of the metal ions on the quality of nucleic acid; TritonX-100, polyethylene glycol octyl phenyl ether-100, is capable of solubilizing lipids to increase the permeability of cell membranes.
Preferably, the pH value of the lysis solution is 7.0-7.5.
Preferably, the pH of the lysate is 7.4.
As a second aspect of the present invention, the present invention provides a nucleic acid extraction kit.
Preferably, the kit comprises a lysate as described above.
Preferably, the lysis solution comprises 0.5-2M sodium salt, 2-5M guanidine salt, 1-5 mM metal ion complexing agent, 0.25-1% of nonionic surfactant and 1-3% of PEG.
Preferably, the molecular weight of the PEG is 6000-10000 Da.
Preferably, the PEG has a molecular weight of 8000 Da.
Preferably, the sodium salt is sodium acetate, the guanidine salt is guanidine hydrochloride, the metal ion complexing agent is EDTA, and the nonionic surfactant is Triton X-100.
Preferably, the pH value of the lysis solution is 7.0-7.5.
Preferably, the pH of the lysate is 7.4.
Preferably, the kit further comprises a first washing buffer, wherein the first washing buffer comprises 15-30 mM Tris-base, 10-20 mM EDTA, 150-200 mM sodium chloride and 50-60% absolute ethyl alcohol, and the pH value is 9.6. The Tris-base can maintain the stability of the nucleic acid released after the sample to be extracted is cracked, so that the degradation of the nucleic acid is avoided, and the concentration and the purity of the nucleic acid are improved; the absolute ethyl alcohol can enable DNA to be in an infusible state in a solution, and can enhance the adsorption of nucleic acid adsorbate to nucleic acid, so as to ensure that the nucleic acid is not dissolved in water; the sodium chloride and EDTA remove excess protein from the surface of the nucleic acid and stabilize the nucleic acid.
Preferably, the kit further comprises a second wash buffer comprising 75% absolute ethanol. Wherein, the 75% absolute ethyl alcohol can fully dissociate salt ions and impurities on the surface of the nucleic acid.
Preferably, the kit further comprises an eluent comprising a DEPC-treated ultrapure water solution. The DEPC treated ultrapure water solution does not contain alcohol substances, and can well protect nucleic acid from being degraded.
The eluent is not limited to DEPC-treated ultrapure water solution, and may be other eluents as long as nucleic acid can be eluted from the nucleic acid-adsorbed material
Preferably, the kit further comprises a nucleic acid adsorbate capable of specifically adsorbing nucleic acids released after lysis of the biological sample and capable of separating the nucleic acids from other substances of the biological sample or other substances released after lysis of the biological sample by means of an external force.
Preferably, the nucleic acid adsorbate is a magnetic bead or an adsorption film; when the adsorbate is a magnetic bead, the lysed biological sample can be mixed with the magnetic bead to adsorb the nucleic acid on the magnetic bead, and then the magnetic bead carrying the nucleic acid is separated from the lysed biological sample by magnetic force; when the adsorbate is an adsorption film, the lysed biological sample is mixed with the adsorption film to adsorb the nucleic acid to the adsorption film, and then the adsorption film carrying the nucleic acid is separated from the lysed biological sample by an external force, for example, a certain pressure is applied to the adsorption film.
Preferably, the magnetic beads are superparamagnetic nano-particles with 100-500 nm of diameter and the adsorption film is a silica gel adsorption film.
Preferably, the diameter of the magnetic beads is 200 nm.
Illustratively, when the nucleic acid adsorbate is a magnetic bead, the method of using the kit comprises the following steps:
(1) adding a certain amount of biological sample and magnetic beads into the lysis solution, crushing sample cells under the action of the lysis solution, releasing nucleic acid, separating and aggregating the nucleic acid and protein, specifically combining the nucleic acid and the magnetic beads, and separating the magnetic beads from the lysis solution containing biological sample fragments under the action of an external magnetic field;
(2) adding a first washing buffer solution into the magnetic beads adsorbed with the nucleic acids for washing so as to remove redundant proteins on the surfaces of the nucleic acids, and separating the magnetic beads from the first washing buffer solution under the action of an external magnetic field;
(3) adding a second washing buffer solution into the magnetic beads adsorbed with the nucleic acids for washing to remove salt ions on the surfaces of the nucleic acids, and separating the nano magnetic beads from the second washing buffer solution under the action of an external magnetic field;
(4) and adding an eluent into the washed magnetic beads with the adsorbed nucleic acids, separating the nucleic acids from the magnetic beads, and recovering the solution to obtain the nucleic acids.
As a third aspect of the invention, the invention provides the use of a lysate as described above for the extraction of nucleic acids in a biological sample.
As a fourth aspect of the present invention, the present invention provides the use of the nucleic acid extraction kit as described above for extracting nucleic acids in a biological sample.
In one embodiment of the invention, the biological sample comprises cells, whole blood, animal tissue homogenate, serum, plasma, tissue extract, swab, urine, bodily fluids, viruses, mycoplasma, and chlamydia. The biological sample is not limited to the above-mentioned sample, and may be other samples to be extracted containing nucleic acid.
As a fifth aspect of the present invention, the present invention provides a nucleic acid extraction method.
Preferably, the method comprises the steps of:
1) mixing and reacting a nucleic acid extraction lysate, a nucleic acid adsorbate and a biological sample to obtain the adsorbate adsorbed with nucleic acid, wherein the lysate comprises 0.5-2M sodium salt, 2-5M guanidinium salt, 1-5 mM metal ion complexing agent, 0.25-1% of nonionic surfactant and 1-3% of PEG;
2) washing the nucleic acid adsorbate adsorbed with nucleic acid by using a washing buffer solution, wherein the washing buffer solution comprises a first washing buffer solution and a second washing buffer solution, the first washing buffer solution comprises 15-30 mM Tris-base, 10-20 mM EDTA, 150-200 mM sodium chloride and 50-60% absolute ethyl alcohol, and the pH value is 9.6; the second wash buffer comprises 75% absolute ethanol;
3) and treating the nucleic acid adsorbate adsorbed with the nucleic acid by using an eluent to separate the nucleic acid from the nucleic acid adsorbate to obtain the nucleic acid, wherein the eluent comprises DEPC treated ultrapure water solution.
Preferably, the nucleic acid-adsorbing substance is a magnetic bead.
As a sixth aspect of the invention, the invention provides the use of PEG8000 in the preparation of a lysate for the extraction of nucleic acids.
Preferably, the PEG8000 is for binding to at least a portion of the nucleic acid, wherein the lysate does not contain ethanol, methanol, propanol, or isopropanol.
The PEG8000 is used for being combined with at least one part of the nucleic acid, which means that the PEG8000 can be combined with part of the nucleic acid or all of the nucleic acid released after the biological sample is cracked, so that the part of the nucleic acid or all of the nucleic acid is precipitated, the nucleic acid is conveniently captured by the nucleic acid adsorbate, and the hydrophobic effect between the nucleic acid and the nucleic acid adsorbate can be promoted, so that the combination of the nucleic acid and the nucleic acid adsorbate is firmer.
Further, the lysate does not contain alcohols, such as ethanol, methanol, propanol, and/or isopropanol, for precipitating nucleic acids, and PEG8000 is used instead of alcohols as a nucleic acid precipitation reagent. On one hand, the precipitation of nucleic acid by alcohol is not very specific, and any organic macromolecule and some salts can be synchronously precipitated after the concentration of alcohol reaches a certain level, so that the purity of the extracted nucleic acid is not enough; on the other hand, in the case of nucleic acids, alcohol precipitation requires a certain proportion of alcohol, and the alcohol content in the system affects the precipitation effect and the nucleic acid yield. In the invention, PEG8000 is solid powder which is easy to dissolve in water, and other reagents which form the lysate are all water-soluble reagents, so that the lysate can be dissolved by using aqueous solution to the maximum extent during preparation, the volume of alcohol substances in the lysate is not considered, the quality of the lysate is stable, and the influence on the effect caused by alcohol volatilization in the storage process is avoided.
The invention has the beneficial effects that:
(1) the lysis solution has reasonable proportioning, can fully and effectively lyse cells, has high lysis efficiency, can fully release nucleic acid in the cells, obtains the nucleic acid with higher concentration and purity, is favorable for improving the quality and the extraction efficiency of the nucleic acid, and ensures the accuracy of subsequent operation.
(2) The lysate and the kit do not need to digest and remove protein by using proteinase K in the whole nucleic acid extraction process, and alcohol is not used for precipitating nucleic acid, so that the obtained nucleic acid has high purity and complete fragments; and the biological sample is cracked without being heated, so that the requirement of experimental equipment is reduced.
(3) The kit realizes the synchronous implementation of sample cracking and nucleic acid combination, reduces the reagent cost compared with the existing kit, and is more convenient to use; in addition, all reagents can be stored and transported at normal temperature, so that the storage and transportation of the kit are facilitated.
(4) The nucleic acid extraction method has simple steps and short time, can realize the automation and high-throughput quantification of nucleic acid extraction by matching with an instrument, and only needs 15-20min for extracting 32 samples; and all reagents used in the extraction process are nontoxic and harmless, so that the experiment operators are effectively protected.
Drawings
FIG. 1 is a graph showing PCR detection of African swine fever virus whole blood samples after nucleic acid extraction using the kit of the present invention and Takara kit.
FIG. 2 is a graph showing the PCR detection of HCV RNA after nucleic acid extraction using the kit of the present invention and the Takara kit.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
In the embodiment of the invention, the amount of PEG in the lysate is mass percentage, and the amount of TritonX-100 is volume percentage; the amount of absolute ethanol included in the first wash buffer and the second wash buffer is a volume percentage. Those who do not specify specific conditions in the examples of the present invention follow conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.
Example 1
In this example, saliva of a normal human origin collected by the applicant was used as a sample, and nucleic acid in the sample was extracted. The nucleic acid extraction procedure of this example was as follows:
1. adding 100 mu L of sample into a centrifuge tube, adding 400 mu L of lysis solution and superparamagnetic hydroxyl nano-magnetic material with the particle size of 200nm into the centrifuge tube, uniformly mixing for 4min at room temperature, placing the centrifuge tube on a magnetic frame, magnetically attracting for 20s, and sucking supernatant; wherein the lysis solution comprises the following components: 0.5M of sodium acetate, 1mM of EDTA, 2M of guanidine hydrochloride, TritonX-1000.25%, and PEG 80001%, wherein the pH of the lysate is 7.4;
2. adding 500 mu L of first washing buffer solution into a centrifuge tube, washing and uniformly mixing for 1min, placing the centrifuge tube on a magnetic frame, magnetically attracting for 20s, and sucking supernatant; wherein the first washing buffer comprises the following components: tris-base 15mM, EDTA10mM, sodium chloride 150mM, absolute ethanol 50%, pH 9.6;
3. adding 500 mu L of second washing buffer solution into the centrifuge tube, washing and uniformly mixing for 1min, placing the centrifuge tube on a magnetic frame, magnetically attracting for 20s, and sucking supernatant; then opening the cover and drying for 1 min; wherein the second washing buffer comprises 75% absolute ethyl alcohol;
4. adding 60 mu L of eluent into a centrifugal tube, uniformly mixing for 5min, placing the centrifugal tube on a magnetic frame, magnetically attracting for 20s, and absorbing supernatant into a clean centrifugal tube to obtain nucleic acid; wherein the eluent is DEPC treated ultrapure water solution.
Example 2
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: sodium acetate 1M, EDTA 3mM, guanidine hydrochloride 4M, triton x-1000.5%, PEG 80002%, the lysate pH 7.0.
Example 3
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: sodium acetate 2M, EDTA 5mM, guanidine hydrochloride 5M, triton x-1001%, PEG 80003%, the lysate pH 7.5.
Example 4
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: 0.5M sodium acetate, 1mM EDTA, 2M guanidine hydrochloride, TritonX-1000.25%, PEG 60001%, and the pH of the lysate was 7.4.
Example 5
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: 0.5M of sodium acetate, 1mM of EDTA, 2M of guanidine hydrochloride, TritonX-1000.25% and 100001% of PEG, wherein the pH value of the lysate is 7.4.
Example 6
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: 0.5M sodium acetate, 1mM EDTA, 2M guanidine hydrochloride, TritonX-1000.25%, and PEG 120001%, wherein the pH of the lysate is 7.4.
Example 7
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: 0.5M of sodium acetate, 1mM of EDTA, 2M of guanidine hydrochloride, TritonX-1000.25% and PEG 40001%, wherein the pH value of the lysis solution is 7.4.
Example 8
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: 0.5M sodium acetate, 1mM EDTA, 2M guanidine hydrochloride, TritonX-1000.25%, PEG 8001%, and the pH of the lysate is 7.4.
Example 9
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: 0.5M sodium acetate, 1mM EDTA, 2M guanidine hydrochloride, TritonX-1000.25%, and 2001% PEG, wherein the pH of the lysate is 7.4.
Example 10
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: 0.5M sodium chloride, 1mM EDTA, 2M guanidine hydrochloride, TritonX-1000.25%, PEG 80001%, and the pH of the lysate is 7.4.
Example 11
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: 0.5M of sodium acetate, 1mM of EDTA, 2M of guanidine hydrochloride, 1% of Triton X-1000.25% of ethanol, and the pH value of the lysate is 7.4.
Example 12
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: 0.5M of sodium acetate, 1mM of EDTA, 2M of guanidine hydrochloride, 1% of Triton X-1000.25% of isopropanol, and the pH value of the lysate is 7.4.
Example 13
In this example, the compositions of the sample, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the eluent used in the present example are the same as those in example 1, except that the lysis solution comprises the following components: sodium acetate 0.5M, EDTA 1mM, guanidine hydrochloride 2M, triton x-1000.25%, the lysate pH 7.4.
Example 14
In this embodiment, the composition of the sample, the nucleic acid extraction process, the lysis solution, the second washing buffer solution, and the eluent is the same as in embodiment 1, except that the first washing buffer solution comprises the following components: tris-base 30mM, EDTA 20mM, sodium chloride 200mM, absolute ethanol 60%, pH 9.6.
Different tests of the nucleic acids extracted in examples 1 to 14 above:
the concentrations and purities of the nucleic acids of examples 1 to 14 were measured by nanodrop spectrophotometer, in which the nucleic acid purities were ratios of A260 and A280, A260 represents the absorbance of the nucleic acid at a wavelength of 260nm, A280 represents the absorbance of the nucleic acid at a wavelength of 280nm, and the experimental results were as follows:
TABLE 1 results of concentration and purity measurement of nucleic acids extracted in different examples
| Experimental group | Concentration (ng/. mu.L) | Purity of |
| Example 1 | 89.6 | 1.87 |
| Example 2 | 90.3 | 1.85 |
| Example 3 | 87.9 | 1.88 |
| Example 4 | 80.7 | 1.81 |
| Example 5 | 82.5 | 1.80 |
| Example 6 | 68.9 | 1.64 |
| Example 7 | 71.5 | 1.72 |
| Example 8 | 69.6 | 1.61 |
| Example 9 | 67.3 | 1.59 |
| Example 10 | 76.5 | 1.77 |
| Example 11 | 56.7 | 1.48 |
| Example 12 | 59.1 | 1.52 |
| Example 13 | 55.0 | 1.43 |
| Example 14 | 87.1 | 1.82 |
From the results of examples 1 to 9, it can be seen that the concentration and purity of the nucleic acid extracted from the lysate containing PEG with the molecular weight of 6000 to 10000 are significantly higher than those of PEG12000, PEG4000, PEG800 and PEG200, and the lysate containing PEG8000 has the best effect, which indicates that PEG with a specific molecular weight is beneficial to nucleic acid precipitation and is beneficial to capture of nucleic acid by magnetic beads.
From the results of example 1, example 11 and example 12, it can be seen that the lysate of the present invention extracted nucleic acid at a higher concentration and purity than the lysate using alcohol as a precipitating agent.
From the results of example 1 and example 13, it can be seen that the concentration and purity of the nucleic acid extracted after adding PEG8000 to the lysate are significantly improved, indicating that the lysate of the present invention can specifically and strongly obtain the target nucleic acid in the sample.
In conclusion, the concentration and purity of the nucleic acid extracted by the lysis solution are higher, and the purity is higher than 1.8, which indicates that the lysis solution can extract the nucleic acid with higher quality. Although PEG can precipitate nucleic acid, the experiment shows that only the lysate containing PEG with the molecular weight of 6000-10000 is beneficial to obtaining the nucleic acid with relatively high concentration and purity.
Example 15
In this embodiment, the african swine fever virus whole blood sample is used as a sample, and the Kit of the present invention and the commercially available taka MiniBEST Viral RNA/DNA Extraction Kit ver.5.0 are respectively used for nucleic acid Extraction, and the experimental process of the Kit of the present invention is as follows:
1. adding 100 mu L of African swine fever virus whole blood sample into a centrifuge tube, adding 400 mu L of lysis solution and super-cis-hydroxy nano-magnetism with the particle size of 200nm into the centrifuge tube, uniformly mixing for 4min at room temperature, placing the centrifuge tube on a magnetic frame, magnetically attracting for 20s, and sucking supernatant; wherein the lysis solution comprises the following components: 0.5M of sodium acetate, 5mM of EDTA, 2M of guanidine hydrochloride, TritonX-1000.25%, and PEG 80001%, wherein the pH of the lysate is 7.4;
2. adding 500 mu L of first washing buffer solution into a centrifuge tube, washing and uniformly mixing for 1min, placing the centrifuge tube on a magnetic frame, magnetically attracting for 20s, and sucking supernatant; wherein the first washing buffer comprises the following components: 15mM Tris-base, 10mM EDTA, 150mM NaCl, 60% absolute ethanol, pH 9.6;
3. adding 500 mu L of second washing buffer solution into the centrifuge tube, washing and uniformly mixing for 1min, placing the centrifuge tube on a magnetic frame, magnetically attracting for 20s, and sucking supernatant; then opening the cover and drying for 1 min; wherein the second washing buffer comprises 75% absolute ethyl alcohol;
4. adding 60 mu L of eluent into a centrifugal tube, uniformly mixing for 5min, placing the centrifugal tube on a magnetic frame, magnetically attracting for 20s, and absorbing supernatant into a clean centrifugal tube to obtain nucleic acid; wherein the eluent is DEPC treated ultrapure water solution.
The control group was TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0, the Extraction procedure was as described above, the sample size was 200. mu.L, and the volume of the eluate was 50. mu.L.
After extraction, 2. mu.L of each of the extracted nucleic acids was subjected to a fluorescent quantitative PCR experiment. The results of the experiment are shown in fig. 1 and table 2 as follows:
TABLE 2 average CT values measured on African swine fever virus whole blood samples
From the experimental results, the extraction efficiency of the kit of the invention is higher compared with the control group.
Example 16
In this example, taking Extraction of HCV RNA as an example, nucleic acid Extraction was carried out using the Kit of the present invention and TaKaRa MiniBESTViral RNA/DNA Extraction Kit Ver.5.0, respectively, in combination with a BFEX-32 nucleic acid extractor, and the Extraction procedure was as follows:
TABLE 3 BFEX-32 nucleic acid extractor extraction procedure
After the extraction, 2 μ L of HCV nucleic acid was taken for fluorescent quantitative PCR, and the experimental results are shown in fig. 2 and table 4:
TABLE 4 mean CT values for HCV RNA detection
From the experimental results, the extraction efficiency of the kit of the invention is higher compared with the control group.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (12)
1. The lysis solution for nucleic acid extraction is characterized by comprising 0.5-2M of sodium salt, 2-5M of guanidine salt, 1-5 mM of metal ion complexing agent, 0.25-1% of nonionic surfactant and 1-3% of PEG.
2. The lysate of claim 1, wherein the PEG has a molecular weight of 6000-10000 Da.
3. Lysis solution according to claim 2, wherein said PEG has a molecular weight of 8000 Da.
4. The lysate of claim 1, wherein the sodium salt is sodium acetate, the guanidinium salt is guanidinium hydrochloride, the metal ion complexing agent is EDTA, and the non-ionic surfactant is triton x-100.
5. A kit for nucleic acid isolation comprising the lysate according to any one of claims 1 to 4.
6. The kit according to claim 5, wherein the kit further comprises a first wash buffer comprising 15-30 mM Tris-base, 10-20 mM EDTA, 150-200 mM sodium chloride and 50-60% absolute ethanol, pH 9.6.
7. The kit of claim 5, further comprising a second wash buffer comprising 75% absolute ethanol.
8. The kit of claim 5, further comprising an eluent comprising a DEPC treated ultrapure water solution.
9. Use of a lysate according to any one of claims 1 to 4 and a nucleic acid extraction kit according to any one of claims 5 to 8 for extracting nucleic acid from a biological sample.
10. A method for extracting nucleic acid, comprising the steps of:
1) mixing and reacting a nucleic acid extraction lysate, a nucleic acid adsorbate and a biological sample to obtain the adsorbate adsorbed with nucleic acid, wherein the lysate comprises 0.5-2M sodium salt, 2-5M guanidinium salt, 1-5 mM metal ion complexing agent, 0.25-1% of nonionic surfactant and 1-3% of PEG;
2) washing the nucleic acid adsorbate adsorbed with nucleic acid by using a washing buffer solution, wherein the washing buffer solution comprises a first washing buffer solution and a second washing buffer solution, the first washing buffer solution comprises 15-30 mM Tris-base, 10-20 mM EDTA, 150-200 mM sodium chloride and 50-60% absolute ethyl alcohol, and the pH value is 9.6; the second wash buffer comprises 75% absolute ethanol;
3) and treating the nucleic acid adsorbate adsorbed with the nucleic acid by using an eluent to separate the nucleic acid from the nucleic acid adsorbate to obtain the nucleic acid, wherein the eluent comprises DEPC treated ultrapure water solution.
11. The method of claim 10, wherein the nucleic acid adsorbent is a magnetic bead.
Use of PEG8000 in the preparation of a lysate for the extraction of nucleic acids, wherein said PEG8000 is used to bind to at least a portion of said nucleic acids, wherein said lysate is free of ethanol, methanol, propanol or isopropanol.
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