CN112063616A - Nucleic acid extraction method and extraction kit - Google Patents
Nucleic acid extraction method and extraction kit Download PDFInfo
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Abstract
The invention discloses a nucleic acid extraction method, which comprises the following steps: taking a sample to be detected, adding cell lysate, uniformly mixing, standing at room temperature to separate solid from liquid, and obtaining supernatant, wherein the supernatant is extracted nucleic acid; the volume ratio of the sample to be detected to the cell lysate is 4: 4-6; the formula of the lysis solution is as follows: 60 to 70mM of Tris-HCl with pH 8 to 9, 15 to 18mM of ammonium sulfate, 5 to 8mM of MgCl2.6H2O, 5-8 mu M EDTA, 1-2 mu M SDS and 40-60 mu g/ml proteinase K. The invention also provides a nucleic acid extraction kit, which comprises the lysate. The whole extraction process of the nucleic acid extraction method is only 10 minutes, and the supernatant can be directly used for subsequent target gene detection, so that the extraction time is greatly shortened, the experimental steps are simplified, and the targets of no loss and no pollution in nucleic acid extraction are realized.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a nucleic acid extraction method and an extraction kit.
Background
At present, nucleic acid detection is widely applied to a plurality of fields of clinical medicine, inspection and quarantine and the like, in particular to nucleic acid amplification technologies such as PCR, isothermal amplification and the like due to the characteristics of high sensitivity, strong specificity, simple and convenient operation, short required time and the like. However, the nucleic acid amplification technique is sensitive to the interfering substances in the clinical or directly obtained specimen, which may cause false negative of the detection result, and therefore, the extraction and purification of the sample nucleic acid is required before the nucleic acid amplification. However, the extraction and purification of nucleic acid from a sample has been the most time-consuming and tedious process in the whole experiment, and seriously affects the speed of sample detection and field application. Heretofore, methods for extracting nucleic acids include boiling methods, centrifugal column methods, and magnetic bead methods. During extraction by a boiling method, tube replacement, centrifugation and other steps are needed for many times, the sample processing time is long, and a small amount of substances for inhibiting amplification still exist, so that the subsequent amplification can be inhibited; the centrifugal column method has good extraction effect, but has various steps, and the process has the defects that nucleic acid is easy to lose or pollute; the magnetic bead method has simple extraction steps, high recovery rate and easy automatic extraction, but the product price is very expensive.
In summary, the existing nucleic acid extraction method has the problems of large sample requirement, long time, poor extraction effect and the like. Therefore, how to develop a nucleic acid extraction method and an extraction kit with high extraction efficiency is a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a lysate and a nucleic acid extraction kit, the whole extraction process is only 10 minutes, and the supernatant can be directly used for subsequent target gene detection, so that the extraction time is greatly shortened, and the experimental steps are simplified. The method realizes the aims of no loss and no pollution in nucleic acid extraction.
In order to achieve the above object, one object of the present invention is to provide a nucleic acid extraction method comprising: taking a sample to be detected, adding cell lysate, uniformly mixing, standing at room temperature to separate solid from liquid, and obtaining supernatant, wherein the supernatant is extracted nucleic acid;
the volume ratio of the sample to be detected to the cell lysate is 4: 4-6;
the formula of the lysis solution is as follows: 60 to 70mM Tris-HCl with pH 8 to 9, 15 to 18mM ammonium sulfate, 5 to 8mM MgCl2.6H2O, 5-8 mu M EDTA, 1-2 mu M SDS and 40-60 mu g/ml proteinase K.
Further, the formula of the lysis solution is as follows: 67mM Tris-HCl pH 8.5, 16.6mM ammonium sulfate, MgCl2.6H2O6.7 mM, 6.7. mu.M EDTA, 1.7. mu.M SDS and 50. mu.g/ml proteinase K.
Further, the volume ratio of the sample to be tested to the cell lysate is 4: 5.
further, the added lysis solution is mixed evenly by reversing the upside down.
Further, the room-temperature standing time is 8-12 min.
The invention also provides a nucleic acid extraction kit, which comprises lysate, wherein the lysate comprises the following formula: 60 to 70mM Tris-HCl with pH 8 to 9, 15 to 18mM ammonium sulfate, 5 to 8mM MgCl2.6H2O, 5-8 mu M EDTA, 1-2 mu M SDS and 40-60 mu g/ml proteinase K.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
according to the nucleic acid extraction method provided by the invention, after a sample to be detected is cracked by the cracking liquid provided by the invention, the supernatant can be directly used for subsequent target gene detection, so that the extraction time is greatly shortened, and the experimental steps are simplified. The method realizes the aims of no loss and no pollution in nucleic acid extraction. The reagent can avoid the recovery step of nucleic acid, avoid the loss or pollution of nucleic acid, and can realize the release and detection of nucleic acid by using the reagent.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a drawing showing the detection of a gene of interest and the agarose gel electrophoresis of nucleic acid in Experimental example 1 of the present invention; wherein the numbers of lanes 1 to 5 represent comparative example 1 to comparative example 5, respectively, the number of lane 6 represents example 1, and the numbers of lanes 7 to 14 represent comparative example 6 to comparative example 13, respectively;
FIG. 2 is a drawing showing the detection of a gene of interest and the agarose gel electrophoresis of nucleic acid in Experimental example 1 of the present invention; wherein numbers 1 to 12 represent example 1 to example 12, respectively, and NC represents a negative control group;
FIG. 3 is a graph showing the nucleic acid concentration and agarose gel electrophoresis in example 1.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
according to an exemplary embodiment of the present invention, there is provided a nucleic acid extraction method including: taking a sample to be detected, adding cell lysate, uniformly mixing, standing at room temperature to separate solid from liquid, and obtaining supernatant, wherein the supernatant is extracted nucleic acid;
the volume ratio of the sample to be detected to the cell lysate is 4: 4-6;
the formula of the lysis solution is as follows: 60 to 70mM Tris-HCl with pH 8 to 9, 15 to 18mM ammonium sulfate, 5 to 8mM MgCl2.6H2O, 5-8 mu M EDTA, 1-2 mu M SDS and 40-60 mu g/ml proteinase K.
Preferably, the formulation of the lysis solution is: 67mM Tris-HCl pH 8.5, 16.6mM ammonium sulfate, MgCl2.6H2O6.7 mM, 6.7. mu.M EDTA, 1.7. mu.M SDS and 50. mu.g/ml proteinase K.
The lysis solution can enable a sample to be detected to release more nucleic acid, does not influence subsequent detection, and reduces the nucleic acid loss and pollution in the extraction process; the whole extraction process is only 10 minutes, and the supernatant can be directly used for subsequent target gene detection, so that the extraction time is greatly shortened, and the experimental steps are simplified. The method realizes the aims of no loss and no pollution in nucleic acid extraction. The reagent can avoid the recovery step of nucleic acid, avoid the loss or pollution of nucleic acid, and can realize the release and detection of nucleic acid by using the reagent.
The innovation of the invention is that: through the coordination and coordination among the components and the control on the use amount of the components, the cell can be fully cracked at normal temperature, and the nucleic acid is released; but also can maintain the stability of nucleic acid and does not influence the subsequent target gene detection.
In the formulation of the lysate:
adding 60-70 mM Tris-HCl with the pH value of 8-9 as a buffer solution to ensure that the pH value is stable during cell lysis;
adding protease K with the concentration of 40-60 mu g/ml to dissociate nucleic acid binding protein;
adding 15-18 mM ammonium sulfate to reduce the content of protein in the extracting solution and effectively remove protein impurities;
adding 5-8 mu M of EDTA which is a metal chelating agent;
adding 1-2 mu M of surfactant SDS, wherein the SDS has polar and nonpolar groups, so that nucleic acid is released, and the subsequent PCR reaction cannot be inhibited by the addition of 1-2 mu M;
adding 5-8 mM MgCl2.6H2O, which can increase the solubility of nucleic acids in the lysate;
adding proteinase K in the prior art usually requires inactivation at high temperature, otherwise proteinase K can affect the subsequent PCR reaction (such as digestion of Taq enzyme in a PCR system), and the conventional lysate in the prior art is difficult to be fully cracked;
according to the invention, protease K does not need to be inactivated, the lysate is used at normal temperature, the components are cooperated, and the research of the invention shows that 1-2 mu M of surfactant SDS and 5-8 mM of MgCl are mixed together2.6H2And the synergistic cooperation of O can reduce the influence of proteinase K on the subsequent PCR reaction, and the cleavage is sufficient.
Because RNA is extremely easy to hydrolyze in an alkaline environment, the invention combines the optimum pH for cracking and the pH for storing nucleic acid, can fully crack and keep the stability of the nucleic acid, and the concentration and the purity of the RNA are still stable when the RNA is stored at 4 ℃ for three days under the condition of the solution with the pH of 8-9.
The volume ratio of the sample to be detected to the cell lysate is 4: the reasons for 4-6 are: if the volume ratio is too large, the nucleic acid extraction and cell lysis are not facilitated; if the volume ratio is too small, the RNA preservation and the subsequent target gene detection are not favorable. Most preferably, the volume ratio of the sample to be tested to the cell lysate is 4: 5.
according to another exemplary embodiment of the present invention, a nucleic acid extraction kit is provided, which comprises the lysate.
The invention provides a nucleic acid extraction kit which can extract all genomes including DNA and RNA; in the present example, saliva genomic DNA was extracted.
However, in other embodiments of the nucleic acid extraction kit, isopropanol, an adsorption column, a washing solution and an eluent are also included. The disadvantage of this embodiment is the long time required for extraction. Specifically, the washing solution comprises a solution A and a solution B, wherein the solution A is: 120-130 mmol/l NaCl, and absolute ethyl alcohol with volume concentration of 80-90%; the solution B is as follows: 25-35 mmol/l Tris, and 80-90% volume concentration absolute ethyl alcohol. The formula of the eluent is as follows: 9.5 to 10.5mM Tris-HCl; 0.8 to 1.2mM EDTA. The concentration of the isopropanol is 200-400 ul/ml. The kit is used for extracting a genome, and the method comprises the following steps: adding a sample to be detected into a lysis solution, uniformly mixing, and then adding isopropanol to obtain a mixed solution; adding the mixed solution into an adsorption column, centrifuging and pouring out supernatant to obtain a first adsorption column; adding a washing solution into the first adsorption column, centrifuging, and pouring out a supernatant to obtain a second adsorption column; centrifuging the second adsorption column, pouring out supernatant, and standing at room temperature to obtain a third adsorption column; and transferring the third adsorption column into a new centrifugal tube, dropwise adding an elution buffer solution, and standing at room temperature or centrifuging to obtain the genome.
The detection principle of the nucleic acid extraction kit provided by the invention is as follows: the function of the lysis solution is to cause the cell to be lysed, and the genomic DNA in the cell is released; the invention improves the components and the content of the cracking liquid, the combination does not need to be heated, the complete cell cracking and nucleic acid release can be ensured at normal temperature, and the combination of the nucleic acid and the adsorption column is facilitated. The lysate can be used for extracting nucleic acid in cells, bacteria, serum, plasma, saliva, urine or pleural effusion, preferably serum or plasma, and more preferably extracting nucleic acid in cells, bacteria, serum, plasma, saliva, urine or pleural effusion and saliva of a sample to be detected in PCR detection.
The method for preparing the nucleic acid extraction kit of the present application will be described in detail below with reference to examples, comparative examples, and experimental data.
Example 1
1. The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: 67mM Tris-HCl pH 8.5, 16.6mM ammonium sulfate, MgCl2.6H2O6.7 mM, 6.7. mu.M EDTA, 1.7. mu.M SDS and 50. mu.g/ml proteinase K.
2. The extraction method comprises the following steps: (1) cracking: 400ul of cell sample is taken, 500ul of cell lysate is added, the mixture is evenly mixed by turning upside down, and the mixture is placed for 10min at room temperature. (2) Detecting the purity and integrity of the extracted nucleic acid concentration, and detecting the target gene.
Example 2
1. The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl67mM, ammonium sulfate 15mM, MgCl2.6H2O6.7 mM, EDTA 6.7. mu. M, SDS 1.7.7. mu.M, and proteinase K50. mu.g/ml.
2. The extraction method comprises the following steps: (1) cracking: 400ul of cell lysate is added into 400ul of cell sample, the mixture is turned upside down and mixed evenly, and the mixture is placed for 10min at room temperature. (2) Detecting the purity and integrity of the extracted nucleic acid concentration, and detecting the target gene.
Example 3
1. The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl67mM, ammonium sulfate 18mM, MgCl2.6H2O6.7 mM, EDTA 6.7. mu. M, SDS 1.7.7. mu.M, and proteinase K50. mu.g/ml. The extraction method was the same as in example 1.
2. The extraction method comprises the following steps: (1) cracking: 400ul of cell sample is taken, 600ul of cell lysate is added, the mixture is evenly mixed by turning upside down, and the mixture is placed for 10min at room temperature. (2) Detecting the purity and integrity of the extracted nucleic acid concentration, and detecting the target gene.
Example 4
The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl67mM, ammonium sulfate 16.6mM, MgCl2.6H2O5 mM, EDTA 6.7. mu. M, SDS 1.7.7. mu.M, and proteinase K50. mu.g/ml. The extraction method was the same as in example 1.
Example 5
The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl67mM, ammonium sulfate 16.6mM, MgCl2.6H2O8 mM, EDTA 6.7. mu. M, SDS 1.7.7. mu.M and proteinase K50. mu.g/ml. The extraction method was the same as in example 1.
Example 6
The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl67mM, ammonium sulfate 16.6mM, MgCl2.6H2O6.7 mM, EDTA 6.7. mu. M, SDS 1.7.7. mu.M and proteinase K50. mu.g/ml. The extraction method was the same as in example 1.
Example 7
The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: tris-HCl67mM, ammonium sulfate 16.6mM, MgCl2.6H2O6.7 mM, EDTA 5. mu. M, SDS 1.7.7. mu.M and proteinase K50. mu.g/ml. The extraction method was the same as in example 1.
Example 8
The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl67mM, ammonium sulfate 16.6mM, MgCl2.6H2O6.7 mM, EDTA 8. mu. M, SDS 1.7.7. mu.M, and proteinase K50. mu.g/ml. The extraction method was the same as in example 1.
Example 9
The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl67mM, ammonium sulfate 16.6mM, MgCl2.6H2O6.7 mM, EDTA 8. mu. M, SDS1. mu.M and proteinase K50. mu.g/ml. The extraction method was the same as in example 1.
Example 10
The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl67mM, ammonium sulfate 16.6mM, MgCl2.6H2O6.7 mM, EDTA 8. mu. M, SDS 2. mu.M and proteinase K50. mu.g/ml. The extraction method was the same as in example 1.
Example 11
The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl 60mM, ammonium sulfate 16.6mM, MgCl2.6H2O6.7 mM, EDTA 8. mu. M, SDS 1.7.7. mu.M, and proteinase K40. mu.g/ml. The extraction method was the same as in example 1.
Example 12
The lysis solution provided by the embodiment of the invention comprises the following components in parts by weight: Tris-HCl 70mM, ammonium sulfate 16.6mM, MgCl2.6H2O6.7 mM, EDTA 8. mu. M, SDS 1.7.7. mu.M, and proteinase K60. mu.g/ml. The extraction method was the same as in example 1.
Comparative examples 1 to 13
The formulations of the lysates in comparative examples 1-13 are different from those of example 1, and the other components are the same as those of example 1, as shown in Table 1.
TABLE 1 lysate formulations
Comparative example 14
The lysate in this comparative example does not contain SDS, the other examples are the same as example 1, and specifically the formulation of the lysate is: 67mM Tris-HCl pH 8.5, 16.6mM ammonium sulfate, MgCl2.6H2O6.7 mM, 6.7. mu.M EDTA and 50. mu.g/ml proteinase K. The extraction method was the same as in example 1.
Comparative example 15
MgCl was not contained in the cracking solution in this comparative example2.6H2And O, the rest is the same as the embodiment 1, and the formula of the lysis solution is specifically as follows: 67mM, pH 8.5 Tris-HCl, 6.7. mu.M EDTA, 1.7. mu.M SDS and 50. mu.g/ml proteinase K. The extraction method was the same as in example 1.
Comparative example 16
The cracking solution in the comparative example contains 0.5 mu MSDS, the rest is the same as example 1, and the formula of the cracking solution is specifically as follows: 67mM Tris-HCl pH 8.5, 16.6mM ammonium sulfate, MgCl2.6H2O6.7 mM, 6.7. mu.M EDTA, 0.5. mu.M SDS and 50. mu.g/ml proteinase K. The extraction method was the same as in example 1.
Comparative example 17
The cracking solution in the comparative example contains 2.5 mu MSDS, the rest is the same as example 1, and the formula of the cracking solution is specifically as follows: 67mM Tris-HCl pH 8.5, 16.6mM ammonium sulfate, MgCl2.6H2O6.7 mM, 6.7. mu.M EDTA, 2.5. mu.M SDS and 50. mu.g/ml proteinase K. The extraction method was the same as in example 1.
Comparative example 18
In this comparative example, the cracking liquid contained 4mM MgCl2.6H2And O, the rest is the same as the embodiment 1, and the formula of the lysis solution is specifically as follows: 67mM Tris-HCl, MgCl at pH 8.52.6H2O4 mM, 6.7. mu.M EDTA, 1.7. mu.M SDS and 50. mu.g/ml proteinase K. The extraction method was the same as in example 1.
Comparative example 19
In this comparative example, the cracking liquid contained 9M MgCl2.6H2And O, the rest is the same as the embodiment 1, and the formula of the lysis solution is specifically as follows: 67mM Tris-HCl, MgCl at pH 8.52.6H2O9 mM, 6.7. mu.M EDTA, 1.7. mu.M SDS and 50. mu.g/ml proteinase K. The extraction method was the same as in example 1.
Comparative example 20
The formulation of the cracking solution in this comparative example is the same as that of example 1. The extraction method comprises the following steps: (1) cracking: 400ul of cell sample is taken, 300ul of cell lysate is added, the mixture is evenly mixed by turning upside down, and the mixture is placed for 10min at room temperature. (2) Detecting the purity and integrity of the extracted nucleic acid concentration, and detecting the target gene. The extraction method was the same as in example 1.
Comparative example 21
The formulation of the cracking solution in this comparative example is the same as that of example 1. The extraction method comprises the following steps: (1) cracking: 400ul of cell sample is taken, 700ul of cell lysate is added, the mixture is evenly mixed by turning upside down, and the mixture is placed for 10min at room temperature. (2) Detecting the purity and integrity of the extracted nucleic acid concentration, and detecting the target gene. The extraction method was the same as in example 1.
Experimental example 1
The same number of human cells were subjected to nucleic acid extraction using the kits of each example and comparative example, and the nucleic acid concentration, purity, and expression level of the target gene were measured, and the results are shown in FIG. 1, FIG. 2, and Table 2.
TABLE 2-concentration and purity of nucleic acids extracted from different groups of lysates
From the data results in table 2, it can be seen that:
the nucleic acids of examples 1 to 12 were higher in concentration and purity as compared with those of comparative examples 1 to 13;
comparative example 14 does not contain SDS, and the concentration and purity of nucleic acid are not high;
comparison ofExample 15 contains no MgCl2.6H2O, the concentration and purity of nucleic acid are not high;
in comparative example 16, the concentration of SDS was 0.5. mu.M, which is less than the range of 1 to 2. mu.M of the present invention, and the disadvantage of incomplete cell lysis existed;
in comparative example 17, the concentration of SDS was 2.5. mu.M, which is larger than the range of 1 to 2. mu.M in the present invention, there was a disadvantage that nucleic acid was unstable;
comparative example 18, MgCl2.6H2The concentration of O is 4mM and is less than the range of 5-8 mM, and the defects of unstable nucleic acid and low nucleic acid purity exist;
in comparative example 19, MgCl2.6H2The concentration of O is 9mM, and is more than the range of 5-8 mM, so that the defects of nucleic acid instability and low nucleic acid purity exist;
in comparative example 20, the formulation of the lysate is the same as in example 1, but the volume ratio of the sample to be tested to the cell lysate in the extraction method is 4: 3, volume ratio of the invention is more than 4: 4-6, the defects of unstable nucleic acid and low nucleic acid purity exist;
in comparative example 21, the formulation of the lysate is the same as in example 1, but the volume ratio of the sample to be tested to the cell lysate in the extraction method is 4: 7, less than the volume ratio of the invention 4: 4-6, which is unfavorable for RNA preservation and subsequent target gene detection.
Comparative examples 14 to 19 As compared with examples of the present invention, it can be seen that surfactants SDS and MgCl2.6H2The addition concentration of the O and the O is too high or too low to generate good synergistic effect, and the components are cooperatively matched, so that a sample to be detected releases more nucleic acid, the subsequent detection is not influenced, and the nucleic acid loss and pollution in the extraction process are reduced; the whole extraction process is only 10 minutes, and the supernatant can be directly used for subsequent target gene detection, so that the extraction time is greatly shortened, and the experimental steps are simplified. The method realizes the aims of no loss and no pollution in nucleic acid extraction. The reagent can avoid the recovery step of nucleic acid, avoid the loss or pollution of nucleic acid, and can realize the release and detection of nucleic acid by using the reagent.
As can be seen from FIG. 1, the band of the target gene of example 1 is brighter than those of comparative examples 1 to 13;
as is clear from FIG. 2, in examples 1 to 12, since the nucleic acid concentration, purity, integrity and target gene were detected by extracting the same amount of nucleic acid as the sample, the most suitable ammonium sulfate content was 16.6mM and MgCl, since the nucleic acid concentration and the most suitable target gene were the highest and the most suitable target gene was the brightest in example 12.6H2The optimal content of O is 6.7mM, the optimal content of EDTA is 6.7 mu M, SDS is 1.7 mu M, and the optimal content of protease is K50 mu g/ml.
FIG. 3 is a graph showing the concentration and purity of nucleic acids in lanes 1 to 6 in FIG. 3, in agarose gel electrophoresis in example 1.
TABLE 3
| Lane lane | Nucleic acid concentration (ng/ul) | Purity of nucleic acid (260/280) |
| 1 | 323 | 1.91 |
| 2 | 320 | 1.90 |
| 3 | 347 | 1.89 |
| 4 | 338 | 1.81 |
| 5 | 354 | 1.62 |
| 6 | 352 | 1.61 |
As can be seen from FIG. 3, the whole extraction process is only 10 minutes, and the supernatant can be directly used for the subsequent target gene detection, thereby greatly shortening the extraction time and simplifying the experimental steps. The method realizes the aims of no loss and no pollution in nucleic acid extraction.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (7)
1. A method for extracting nucleic acid, comprising: taking a sample to be detected, adding cell lysate, uniformly mixing, standing at room temperature to separate solid from liquid, and obtaining supernatant, wherein the supernatant is extracted nucleic acid;
the volume ratio of the sample to be detected to the cell lysate is 4: 4-6;
the formula of the lysis solution is as follows: 60 to 70mM Tris-HCl with pH 8 to 9, 15 to 18mM ammonium sulfate, 5 to 8mM MgCl2.6H2O, 5-8 mu M EDTA, 1-2 mu M SDS and 40-60 mu g/ml proteinase K.
2. The method for extracting nucleic acid according to claim 1, wherein the formulation of the lysis solution is: 67mM Tris-HCl pH 8.5, 16.6mM ammonium sulfate, MgCl2.6H2O6.7 mM, 6.7. mu.M EDTA, 1.7. mu.M SDS and 50. mu.g/ml proteinase K.
3. The method for extracting nucleic acid according to claim 1, wherein the volume ratio of the sample to be tested to the cell lysate is 4: 5.
4. the method for extracting nucleic acid according to claim 1, wherein the adding of the lysis buffer and the mixing are performed in a manner of upside down mixing.
5. The method for extracting nucleic acid according to claim 1, wherein the standing time at room temperature is 8 to 12 min.
6. A nucleic acid extraction kit is characterized by comprising a lysate, wherein the lysate has the following formula: 60 to 70mM Tris-HCl with pH 8 to 9, 15 to 18mM ammonium sulfate, 5 to 8mM MgCl2.6H2O, 5-8 mu M EDTA, 1-2 mu M SDS and 40-60 mu g/ml proteinase K.
7. The nucleic acid extraction kit of claim 6, wherein the formulation of the lysis solution is: 67mM Tris-HCl pH 8.5, 16.6mM ammonium sulfate, MgCl2.6H2O6.7 mM, 6.7. mu.M EDTA, 1.7. mu.M SDS and 50. mu.g/ml proteinase K.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010997444.2A CN112063616A (en) | 2020-09-21 | 2020-09-21 | Nucleic acid extraction method and extraction kit |
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| CN112626174A (en) * | 2020-12-16 | 2021-04-09 | 广州源井生物科技有限公司 | Cell lysis solution and application thereof |
| CN116949036A (en) * | 2023-09-11 | 2023-10-27 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from ascites and extraction method |
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| CN112626174A (en) * | 2020-12-16 | 2021-04-09 | 广州源井生物科技有限公司 | Cell lysis solution and application thereof |
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| CN116949036A (en) * | 2023-09-11 | 2023-10-27 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from ascites and extraction method |
| CN116949036B (en) * | 2023-09-11 | 2023-12-15 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from ascites and extraction method |
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