CN112899266A - Cracking binding solution for nucleic acid extraction, kit and application thereof - Google Patents
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Abstract
The invention relates to a lysis binding solution for extracting bacterial nucleic acid, which consists of aqueous solutions of guanidine hydrochloride, EDTA, triton X-100, Tris-HCl, NP-40 and CTAB. The lysis binding solution can organically combine the steps of lysis first and then section after nucleic acid extraction by the traditional magnetic bead method, and can ensure that the extracted nucleic acid has good purity and high concentration. When the nucleic acid extraction kit prepared by the lysis binding solution provided by the invention is used for extracting nucleic acid, only 3 steps are needed in the whole nucleic acid extraction process, the specific nucleic acid extraction time is short, and the defect of multiple steps in the existing magnetic bead method nucleic acid extraction method is overcome; the steps of cracking and combining are completed in one step, so that the operation steps are reduced, and the method is more suitable for full-automatic nucleic acid extraction application. The sample processing amount and the sample processing speed are increased, and the nucleic acid in the biological sample is quickly extracted in high flux.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a lysis binding solution for nucleic acid extraction, and a nucleic acid extraction kit prepared by using the lysis binding solution.
Background
Nucleic acid extraction is a very critical step in the field of nucleic acid molecule detection. Since Miescher discovered and isolated DNA for the first time in 1869 years, and Meselson M et al isolated DNA for the first time by density gradient centrifugation in 1957, various nucleic acid extraction methods were reported, many researchers have diligently searched for methods for extracting nucleic acid, various materials and reagents for nucleic acid were improved, various reagents such as sodium dodecylsulfate, phenol, urea, and guanidine salt were applied to nucleic acid extraction experiments, and various commercial kits for nucleic acid extraction were also produced. The traditional extraction method mainly comprises the following steps: phenol extraction, alkaline lysis, CTAB extraction and EtBr-CsCl gradient centrifugation. These conventional extraction methods can separate DNA and RNA from different tissue samples, but these techniques include the operation steps of precipitation, centrifugation, etc., which require a large amount of biological samples, and the extraction steps are complicated, time-consuming, labor-consuming, and low in yield, which are difficult to realize automation, and in addition, most of the conventional methods also require toxic chemical reagents, which are potentially harmful to the health of operators, so that the conventional techniques for separating nucleic acids from liquid phase systems are gradually replaced by new methods based on solid phase carriers, along with the development of molecular biology and polymer materials, and the common solid phase materials at present include silica gel, glass particles, diatomaceous earth, anion exchange carriers, etc. However, these solid phase methods usually require several steps such as rapid centrifugation and vacuum filtration to achieve separation, and are inconvenient for high-throughput and automated operation due to large sample demand and high sample consumption, thereby severely limiting the application in the field of clinical gene diagnosis.
With the development of molecular biology technology, in order to meet the experimental requirements of high-throughput sample processing and high-quality nucleic acid acquisition, the nucleic acid extraction and separation technology tends to be developed more simply, conveniently, quickly, with high quality, high purity and high throughput, and the quality of nucleic acid directly determines the success or failure of subsequent experiments. In recent years, molecular diagnostic techniques have been rapidly developed, and with the maturity of techniques such as gene sequencing, the cost is further reduced, and the application in clinic is more and more common. The types of samples for molecular diagnosis and detection are dozens of samples, and the detection items suitable for the processed products also cover various molecular biology technical platforms such as fluorescent quantitative PCR, gene sequencing, gene chips, biological mass spectrometry and the like. However, regardless of the type of test item, the accurate test result is undoubtedly dependent on the specimen of high quality and the specimen pretreatment process. Therefore, the automation trend of clinical molecular diagnosis will gradually become dominant in the domestic clinical laboratory, and the automation is firstly realized on the nucleic acid extraction which is popular in the current manual operation and has the greatest influence on the result.
By adopting the magnetic bead extraction method, the extraction rate and the extraction purity of nucleic acid are mainly influenced by extracting solutions, such as lysis solution, binding solution, washing solution, eluent and the like, and if the amount and the purity of the extracted sample are not too high, the nucleic acid cannot be effectively detected, even the detection result is influenced, and even the subsequent experimental requirements cannot be completely met. The traditional magnetic bead method nucleic acid extraction method is also improved in an enhanced magnetic bead method nucleic acid extraction method of application No. 201110124322.3, and the current magnetic bead method nucleic acid extraction method has two defects: firstly, the extraction efficiency of RNA is low, secondly, the nucleic acid extraction steps are multiple, the cracking and combining are divided into two steps, and magnetic beads can be added into a solution to combine nucleic acid after the cracking is finished, so that the full-automatic application is not facilitated. The invention combines the nucleic acid cracking of a sample with nucleic acid magnetic beads in one step, free RNA is adsorbed on the surfaces of the magnetic beads under the action of high-concentration salt and isopropanol in a cracking solution, finally, the magnetic beads in the centrifugal tube are tightly attached to the wall of the centrifugal tube under the action of magnetic force, and solution in the centrifugal tube is sucked away, thereby achieving the purposes of cracking and combining. The invention is characterized in that the nucleic acid extraction only needs 4 steps, namely cracking and combining, cleaning 1, cleaning 2 and eluting, and the first 3 steps are all to enhance the action of magnetic beads in a solution for adsorbing nucleic acid by adding isopropanol, thereby enhancing the efficiency of nucleic acid extraction by a magnetic bead method, wherein the volume ratio of a biological sample, the isopropanol and a lysis solution is 1:1:2 when the biological sample, the isopropanol and the lysis solution are combined for cracking. And provides a method for efficiently extracting RNA when being applied to RNA extraction. So that the cracking and the combination are completed in one step, and the reaction reagent of each step can be filled into the reaction cabin corresponding to the step in advance when the method is applied to the automatic nucleic acid extraction, so that the method is more suitable for the full-automatic application. But the isopropanol has stimulation effect on mucous membranes of eyes and respiratory tracts, can damage retina and optic nerve and brings nausea and uncomfortable feeling to people when being inhaled in a volatile manner. It is more toxic, anesthetic and irritant to the upper respiratory mucosa than ethanol. Headache, drowsiness and eye, nose and throat irritation symptoms occur when exposed to high concentration steam. Ingestion or inhalation of large amounts of steam can cause flushing, headaches, mental depression, nausea, coma, and the like.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a simple and effective lysis-binding solution for bacterial nucleic acid extraction, and a bacterial nucleic acid extraction kit containing the lysis-binding solution.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a lysis binding solution for nucleic acid extraction, which consists of: guanidine hydrochloride, EDTA, triton X-100, Tris-HCl, NP-40, CTAB, water.
Further, the lysis binding solution consists of the following substances: 3-6M guanidine hydrochloride, 5-20mM EDTA, 1-20% TritonX-100, 0.1-1M Tris-HCl, 0.5-2% NP-40, 0.1-0.5% CTAB, and the balance of water.
Further, the lysis binding solution consists of the following substances: 4M guanidine hydrochloride, 5mM EDTA, 10% TritonX-100, 0.7M Tris-HCl, 1% NP-40, 0.1% CTAB, and the balance water.
Further, the lysis binding solution also comprises 3% -8% of magnetic beads.
Further, the pH value of the cracking binding solution is 6.0-6.5.
2. The use of any of the above lysis-binding solutions for nucleic acid extraction in the preparation of a reagent or kit for nucleic acid extraction.
3. A nucleic acid extraction kit comprising the lysis-binding solution for nucleic acid extraction as described in any of the above, said nucleic acid extraction kit comprising a proteinase K solution, a lysis-binding solution, a washing buffer and an elution buffer.
Further, the concentration of the proteinase K solution is 10-30 mg/ml.
Further, the washing buffer comprises a washing buffer 1 and a washing buffer 2, wherein the washing buffer 1 consists of the following components: 0.5-2M guanidine hydrochloride, 10mM Tris-HCl, 0.1-1M NaCl, pH 6.0-6.5, 30-60% ethanol solution; the washing buffer 2 is 70-85% ethanol water solution.
Further, the elution buffer consisted of the following components: 1-5mM EDTA, 5-20mM Tris-HCl, pH 7.0-10.
4. The extraction method for extracting nucleic acid by using the nucleic acid extraction kit comprises the following specific steps:
1) adding a lysis binding solution into a biological sample, adding proteinase K, carrying out lysis at 70-95 ℃ for 5-15min, releasing nucleic acid from the biological sample, binding the nucleic acid with magnetic beads in the lysis binding solution, aggregating the magnetic beads under the action of an external magnetic field to form a magnetic bead-nucleic acid compound, and collecting the magnetic bead-nucleic acid compound;
2) adding a washing buffer solution into the magnetic bead-nucleic acid compound, washing to remove impurities on the magnetic bead-nucleic acid compound, and collecting the washed magnetic bead-nucleic acid compound under the action of an external magnetic field;
3) and adding an elution buffer solution into the washed magnetic bead-nucleic acid compound, and eluting at 65-85 ℃ for 3-8min to elute the nucleic acid bound on the magnetic bead, thereby obtaining the extracted nucleic acid.
Further, the concentration of the proteinase K solution is 10-30 mg/ml.
Further, the washing buffer comprises a washing buffer 1 and a washing buffer 2, wherein the washing buffer 1 consists of the following components: 0.5-2M guanidine hydrochloride, 10mM Tris-HCl, 0.1-1M NaCl, pH 6.0-6.5, 30-60% ethanol solution; the washing buffer 2 is 70-85% ethanol water solution.
Further, the elution buffer consisted of the following components: 1-5mM EDTA, 5-20mM Tris-HCl, pH 7.0-10.
Further, the biological sample comprises tissue, cells, plasma, serum, body fluid, swab, virus culture fluid or feces.
The invention has the beneficial effects that: the lysis binding solution provided by the invention can organically combine the steps of lysis first and then step later in the traditional magnetic bead method nucleic acid extraction, does not need a separate combination step, can carry out lysis and adsorption simultaneously, and reduces the operation steps. And can ensure the purity of the extracted nucleic acid to be good and the concentration to be high. And the components used in the lysis binding solution abandon the binding adsorption of organic solvents such as isopropanol used in the traditional magnetic bead method nucleic acid extraction, and ensure that no organic solvent with the inhibiting effect on enzyme exists in the extracted nucleic acid sample. Usually, the sample needs to be heated for cracking during cracking, so that the volatility of the organic solvent is accelerated, the health hazard to operators is large, and the sample cannot be stored for a long time at normal temperature by adding the organic reagent. The invention does not contain organic solvent, avoids the opportunity of sucking isopropanol for nucleic acid extraction users, and increases good experimental experience. The cracking experiment of the cracking liquid is researched, the proportion of each reagent is adjusted, and the formula of the cracking liquid for efficiently extracting the nucleic acid is obtained through a large amount of experimental improvement of an inventor.
When the bacterial nucleic acid extraction kit prepared by the lysis binding solution provided by the invention is used for extracting nucleic acid, only 3 steps are needed in the whole nucleic acid extraction process, the specific nucleic acid extraction time is short, and the defect of multiple steps in the existing magnetic bead method nucleic acid extraction method is overcome; the steps of cracking and combining are completed in one step, so that the operation steps are reduced, and the method is more suitable for full-automatic nucleic acid extraction application; the sample processing amount and the sample processing rate are increased, the nucleic acid in the biological sample is rapidly extracted in high flux, and the method can particularly exert technical advantages and efficiency in clinical application and reduce tedious repeated operation steps of clinical workers. The bacterial nucleic acid extraction kit has low cost, simple nucleic acid extraction method and high purity of extracted nucleic acid, and can be directly used for molecular biological experimental research such as PCR and clinical detection.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a graph showing the results of fluorescent quantitative PCR of 10IU/ml HBV-extracted nucleic acid;
FIG. 2 is a graph showing the results of fluorescent quantitative PCR of 100IU/ml HBV-extracted nucleic acid;
FIG. 3 shows the results of fluorescent quantitative PCR of 2100 IU/mL FluA-extracted nucleic acids in example.
FIG. 4 shows the results of fluorescent quantitative PCR of the nucleic acid extracted in 210 IU/mL FluA of example.
Detailed Description
The technical solution of the present invention will be further explained with reference to the drawings and the embodiments. The described embodiments are only some embodiments of the invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 hepatitis B Virus nucleic acid extraction
1) Diluting a Hepatitis B Virus (HBV) nucleic acid quantitative standard substance to 100IU/mL and 10IU/mL by using negative serum, putting 200uL of the serum into a centrifugal tube, adding 500uL of lysis binding buffer solution into the centrifugal tube, adding 20uL of 10mg/mL proteinase K solution, heating and uniformly mixing for 5 minutes at 90 ℃, putting the centrifugal tube on a magnetic frame, carrying out magnetic separation for 20s, and discarding the supernatant;
2) continuously adding 500ul of washing buffer solution 1 into the centrifuge tube, uniformly mixing for 1 minute, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 20s, and sucking the supernatant;
3) continuously adding 500ul of washing buffer solution 2 into the centrifuge tube, uniformly mixing for 1 minute, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 20s, and sucking the supernatant;
4) and finally, adding 100ul of elution buffer solution into the centrifugal tube, heating and uniformly mixing for 5 minutes at 80 ℃, placing the centrifugal tube on a magnetic frame, performing magnetic separation for 20 seconds, transferring the supernatant into another clean centrifugal tube, and storing at the temperature of-20 ℃ to obtain the finally extracted and purified HBV virus nucleic acid solution.
The composition of the lysis binding buffer described in the examples was: guanidine hydrochloride 4M, EDTA 5mM, triton X-10010% (v/v), Tris-HCl 0.7M, NP-401% (w/v), CTAB 0.1% (w/v), magnetic beads 5% (w/v), pH 6.0. All relevant concentrations in the specification are the same, and the solvent is water.
The composition of wash buffer 1 was: 0.5M guanidinium hydrochloride, 10mM Tris-HCl, 0.1M NaCl, pH 6.0, 40% (v/v) ethanol solution.
The washing buffer 2 comprises the following components: 75% (v/v) ethanol in water.
The composition of the elution buffer was: 1mM EDTA,5mM Tris-HCl, pH 8.
The magnetic beads are superparamagnetic silicon oxide nano microspheres, the diameter of the magnetic beads is 100-1000 nm, and the preferred diameter of the magnetic beads is 500 nm.
The invention can also be matched with an automatic instrument to rapidly extract the nucleic acid of the HBV virus serum in high flux, and the automatic nucleic acid extractor is used, and the program is shown in the following table 1:
table 1:
the QIAamp DSP Virus Kit was used as a control reagent, the amount of sample extracted was 200ul, and the elution volume was 100 ul. The extraction method is operated according to the description thereof.
After extraction, 25uL of the HBV nucleic acid DNA extracting solution is taken as a template, 25uL of HBV reaction solution and 50uL of total reaction volume are subjected to amplification detection by using an ABI7500 fluorescence quantitative PCR instrument. The results of detecting HBV by using the nucleic acid extracting reagent of the present invention and the comparative reagent are shown in Table 2, FIG. 1 is a graph showing the results of PCR of nucleic acid extracted from 10IU/ml HBV, and FIG. 2 is a graph showing the results of PCR of nucleic acid extracted from 100IU/ml HBV.
TABLE 2 results of HBV detection using the nucleic acid extraction reagent of the present invention and the comparative reagent
As shown in the results in Table 2 and FIGS. 1 and 2, the results of the detection of hepatitis B virus nucleic acid extracted by the reagent of the present invention are superior to the results of the detection of hepatitis B virus nucleic acid extracted by the control reagent.
Example 2 influenza A Virus
1) Diluting a commercially available influenza A virus quality control product (FluA virus) to 100IU/mL and 10IU/mL by using a negative sample, taking 200uL of the sample, placing the sample in a centrifugal tube, adding 500uL of lysis binding buffer solution into the centrifugal tube, adding 20uL of 10mg/mL protease K solution, heating and uniformly mixing for 5 minutes at 90 ℃, placing the centrifugal tube on a magnetic frame, performing magnetic separation for 20s, and discarding the supernatant;
2) continuously adding 500ul of washing buffer solution 1 into the centrifuge tube, uniformly mixing for 1 minute, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 20s, and sucking the supernatant;
3) continuously adding 500ul of washing buffer solution 2 into the centrifuge tube, uniformly mixing for 1 minute, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 20s, and sucking the supernatant;
4) and finally, adding 100ul of elution buffer solution into the centrifugal tube, heating and uniformly mixing for 5 minutes at 80 ℃, placing the centrifugal tube on a magnetic frame, performing magnetic separation for 20 seconds, transferring the supernatant into another clean centrifugal tube, and storing at the temperature of-20 ℃ to obtain the final FluA virus nucleic acid solution for extraction and purification.
The composition of the lysis binding buffer described in the examples was: guanidine hydrochloride 3M, EDTA 5mM, triton X-10020%, Tris-HCl 0.2M, NP-402%, CTAB 0.5%, magnetic beads 3%, pH 6.0.
The washing buffer comprises a washing buffer 1 and a washing buffer 2, wherein the washing buffer 1 consists of the following components: 0.8M guanidine hydrochloride, 10mM Tris-HCl, 0.6M NaCl, pH 6.0, 50% ethanol solution; washing buffer 2 was 75% aqueous ethanol.
The composition of the elution buffer was: 1mM EDTA,5mM Tris-HCl, pH 8.
The magnetic beads are superparamagnetic silicon oxide nano microspheres, the diameter of the magnetic beads is 100-1000 nm, and the preferred diameter of the magnetic beads is 500 nm.
The invention can also be matched with an automatic instrument to rapidly extract FluA virus nucleic acid with high flux, and the automatic nucleic acid extractor is used, and the program is shown in the following table 3:
TABLE 3
The QIAamp DSP Virus Kit was used as the control reagent, the amount of sample extracted was 200ul, the elution volume was 100ul, and the extraction was performed according to the instructions.
After extraction, a fluorescent quantitative PCR detection kit of commercially available FLUA is adopted, 2uL of the nucleic acid extracting solution is taken as a template, 18uL of reaction solution and 20uL of total reaction volume are obtained, and amplification detection is carried out by using a fluorescent quantitative PCR instrument. Results of fluorescent quantitative PCR As shown in Table 4, FIGS. 3 and 4, FIG. 3 shows the results of PCR with 100IU/mL FluA-extracted nucleic acid, and FIG. 4 shows the results of PCR with 10IU/mL FluA-extracted nucleic acid. The detection result of the reagent for extracting the nucleic acid of the influenza A virus is fully shown to be superior to the detection result of the contrast reagent for extracting the nucleic acid of the influenza A virus.
Table 4: the nucleic acid extraction reagent and the contrast reagent of the invention detect the FluA result
EXAMPLE 3 clinical samples
The method comprises the steps of collecting 6 nasopharyngeal swab clinical samples of novel coronavirus infected persons and 10 nasopharyngeal swab samples collected at random in clinic, taking 200ul of samples to carry out nucleic acid extraction, and respectively adopting the kit and the extraction method to carry out nucleic acid extraction, wherein the lysis binding buffer, the washing buffer and the elution buffer used in the embodiment 3 are the same as those used in the embodiment 1, namely the kit is the same as that in the embodiment 1. The sample size of the QIAamp viral RNA Mini Kit used as a control reagent was 200ul, and the elution volume was 100 ul. After extraction, the novel coronavirus nucleic acid detection kit (fluorescence PCR method) of Shanghai Berger is adopted for detection, 5ul of nucleic acid samples are taken, the total reaction volume is 25ul, the detection results are shown in Table 3, the results of detecting the double-target ORF1ab and the N gene of the novel coronavirus show that the negative and positive rates of the detection results of the extraction kit and the extraction method of the invention and the comparison reagent are 100 percent after extraction.
TABLE 5 results of detection of novel coronavirus using the nucleic acid extraction reagent of the present invention and the comparative reagent
-indicates no detection
Example 4
1. With the method of the present invention, washing and elution of nucleic acids are carried out after direct lysis and binding.
1) Diluting the quantitative standard substance of Hepatitis B Virus (HBV) nucleic acid to 100IU/mL with negative serum, placing 200u of the serum into a centrifugal tube (3 parallel samples are set), adding 500uL of lysis buffer solution into the centrifugal tube, adding 20uL of 20mg/mL proteinase K solution, heating and uniformly mixing for 5 minutes at 90 ℃; placing the centrifuge tube on a magnetic frame, magnetically separating for 20s, and discarding the supernatant;
2) continuously adding 600ul of washing buffer solution 1 into the centrifuge tube, uniformly mixing for 1 minute, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 20s, and sucking the supernatant;
3) continuously adding 500ul of washing buffer solution 2 into the centrifuge tube, uniformly mixing for 1 minute, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 20s, and sucking the supernatant;
4) finally, 100ul of elution buffer solution is added into the centrifugal tube, the centrifugal tube is placed on a magnetic frame after being heated and uniformly mixed for 5 minutes at 80 ℃, magnetic separation is carried out for 20 seconds, and the supernatant is transferred into another clean centrifugal tube, so that the HBV virus nucleic acid solution which is finally extracted and purified can be obtained, and the HBV virus nucleic acid solution can be stored at the temperature of minus 20 ℃.
Wherein, the lysis binding buffer comprises the following components: guanidine hydrochloride 4M, EDTA 5mM, triton X-10010%, Tris-HCl 0.7M, NP-401%, CTAB 0.1%, magnetic beads 5%, pH 6.0. Wherein the percentage (%) is mass percent concentration, all relevant concentrations in the specification are the same, and the solvent is water.
The composition of wash buffer 1 was: 0.5M guanidine hydrochloride, 10mM Tris-HCl, 0.1M NaCl, pH 6.0, 40% ethanol solution.
The washing buffer 2 comprises the following components: 75% ethanol aqueous solution.
The composition of the elution buffer was: 1mM EDTA,5mM Tris-HCl, pH 8.
The diameter of the magnetic beads is 500 nm.
2. The traditional method is adopted, firstly, the lysis is carried out, then 200ul-500ul isopropanol is added as binding solution for binding, and then the washing and the elution of nucleic acid are carried out. Lysis buffer: 2M sodium iodide, 3M guanidine hydrochloride, 10mM EDTA, Tween-205%, 5% nano magnetic beads and 2% SDS.
3. The procedure was as mentioned in the enhanced magnetic bead method nucleic acid extraction method of application No. 201110124322.3, 5M guanidine hydrochloride, guanidine isothiocyanate, sodium iodide, potassium iodide: the lysis solution comprises the following components: 1-2% of triton X-100, 1-2% of nonylphenol polyoxyethylene ether (NP 40), 1-2% of Tween 20, 10-20 mM Tris-HCl, pH7-8, and 1-2mM EDTA. And the volume ratio of the biological sample, the isopropanol and the lysate is 1:1: 2.
After extraction, 25uL of HBV nucleic acid DNA extracting solution is taken as a template, 25uL of HBV reaction solution and 50uL of total reaction volume are taken by adopting a DAAN gene hepatitis B virus nucleic acid determination kit (PCR-fluorescent probe method), and amplification detection is carried out by utilizing an ABI7500 fluorescent quantitative PCR instrument. The purity and concentration of the extract were measured by UV spectrophotometer and the results are shown in Table 6 below.
TABLE 6
As shown in Table 6, the purity of the extract was satisfactory between 1.7 and 2.1, but the concentration and Ct value of the extract were inferior to those of the present invention, as a result of the method of the present invention and the addition of the binding solution.
Finally, the basic principles, principal features and advantages of the invention have been shown and described. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A lysis binding solution for nucleic acid extraction, which is characterized by comprising the following substances: guanidine hydrochloride, EDTA, triton X-100, Tris-HCl, NP-40, CTAB, water.
2. The lysis binding solution for nucleic acid extraction according to claim 1, wherein the lysis binding solution comprises: 3-6M guanidine hydrochloride, 5-20mM EDTA, 1-20% TritonX-100, 0.1-1M Tris-HCl, 0.5-2% NP-40, 0.1-0.5% CTAB, and the balance of water.
3. The lysis binding solution for nucleic acid extraction according to claim 1, wherein the lysis binding solution comprises: 4M guanidine hydrochloride, 5mM EDTA, 10% TritonX-100, 0.7M Tris-HCl, 1% NP-40, 0.1% CTAB, and the balance water.
4. The nucleic acid extraction lysis binding solution according to any one of claims 1 to 3, wherein the lysis binding solution further comprises 3% to 8% magnetic beads.
5. Use of the cleavage conjugate solution for nucleic acid extraction according to any one of claims 1 to 4 for preparing a reagent or a kit for nucleic acid extraction.
6. A nucleic acid extraction kit comprising the cleavage binding solution for nucleic acid extraction according to any one of claims 1 to 4, wherein the nucleic acid extraction kit comprises a proteinase k solution, the cleavage binding solution, a washing buffer and an elution buffer.
7. The nucleic acid extraction kit according to claim 6, wherein the wash buffer comprises a wash buffer 1 and a wash buffer 2, and the wash buffer 1 is composed of: 0.5-2M guanidine hydrochloride, 10mM Tris-HCl, 0.1-1M NaCl, pH 6.0-6.5, 30-60% ethanol solution; the washing buffer 2 is 70-85% ethanol water solution.
8. The nucleic acid extraction kit according to claim 6 or 7, wherein the elution buffer consists of: 1-5mM EDTA, 5-20mM Tris-HCl, pH 7.0-10.
9. The method for extracting nucleic acid by using the nucleic acid extraction kit according to any one of claims 6 to 8, characterized in that the extraction method comprises the following specific steps:
1) adding a lysis binding solution and a proteinase k solution into a biological sample, carrying out lysis at 70-95 ℃ for 5-15min, releasing nucleic acid from the biological sample, binding the nucleic acid with magnetic beads in the lysis binding solution, aggregating the magnetic beads under the action of an external magnetic field to form a magnetic bead-nucleic acid compound, and collecting the magnetic bead-nucleic acid compound;
2) adding a washing buffer solution into the magnetic bead-nucleic acid compound, washing to remove impurities on the magnetic bead-nucleic acid compound, and collecting the washed magnetic bead-nucleic acid compound under the action of an external magnetic field;
3) and adding an elution buffer solution into the washed magnetic bead-nucleic acid compound, and eluting at 65-85 ℃ for 3-8min to elute the nucleic acid bound on the magnetic bead, thereby obtaining the extracted nucleic acid.
10. The extraction method according to claim 9, wherein the biological sample comprises tissue, cells, plasma, serum, body fluid, swab, virus culture fluid or feces.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113652419A (en) * | 2021-08-17 | 2021-11-16 | 宁波上格石医用材料有限公司 | Cracking binding solution for nucleic acid extraction and extraction method |
| CN113755489A (en) * | 2021-10-13 | 2021-12-07 | 江苏溢纳生物科技有限公司 | Lysis binding solution for nucleic acid extraction and extraction method thereof |
| CN114438076A (en) * | 2022-03-07 | 2022-05-06 | 江苏迅睿生物技术有限公司 | A magnetic bead method virus nucleic acid extraction kit and using method |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150275269A1 (en) * | 2012-10-26 | 2015-10-01 | The Emerther Company | Method for purifying nucleic acid and kit |
| CN106754890A (en) * | 2017-01-24 | 2017-05-31 | 南方医科大学 | The extracts kit and extracting method of a kind of viral RNA |
| CN110951725A (en) * | 2019-12-30 | 2020-04-03 | 申友基因组研究院(南京)有限公司 | One-step nucleic acid extraction process based on paramagnetic particle method |
| CN111607590A (en) * | 2020-06-08 | 2020-09-01 | 申翌生物科技(杭州)有限公司 | Nucleic acid extraction method and kit applicable to whole blood, serum or plasma by virtue of magnetic bead method |
| CN111662901A (en) * | 2020-06-09 | 2020-09-15 | 佛山市博朋生物科技有限公司 | Method for extracting virus nucleic acid from animal low nucleic acid content sample |
-
2021
- 2021-02-04 CN CN202110153524.4A patent/CN112899266A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150275269A1 (en) * | 2012-10-26 | 2015-10-01 | The Emerther Company | Method for purifying nucleic acid and kit |
| CN106754890A (en) * | 2017-01-24 | 2017-05-31 | 南方医科大学 | The extracts kit and extracting method of a kind of viral RNA |
| CN110951725A (en) * | 2019-12-30 | 2020-04-03 | 申友基因组研究院(南京)有限公司 | One-step nucleic acid extraction process based on paramagnetic particle method |
| CN111607590A (en) * | 2020-06-08 | 2020-09-01 | 申翌生物科技(杭州)有限公司 | Nucleic acid extraction method and kit applicable to whole blood, serum or plasma by virtue of magnetic bead method |
| CN111662901A (en) * | 2020-06-09 | 2020-09-15 | 佛山市博朋生物科技有限公司 | Method for extracting virus nucleic acid from animal low nucleic acid content sample |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113652419A (en) * | 2021-08-17 | 2021-11-16 | 宁波上格石医用材料有限公司 | Cracking binding solution for nucleic acid extraction and extraction method |
| CN113755489A (en) * | 2021-10-13 | 2021-12-07 | 江苏溢纳生物科技有限公司 | Lysis binding solution for nucleic acid extraction and extraction method thereof |
| CN114517196A (en) * | 2022-02-25 | 2022-05-20 | 杭州诺辉健康科技有限公司 | Extraction method and application of plasma free miRNA |
| CN114438076A (en) * | 2022-03-07 | 2022-05-06 | 江苏迅睿生物技术有限公司 | A magnetic bead method virus nucleic acid extraction kit and using method |
| CN114457069A (en) * | 2022-03-07 | 2022-05-10 | 江苏迅睿生物技术有限公司 | A kind of magnetic bead method pathogen nucleic acid extraction kit and using method |
| CN119193578A (en) * | 2024-11-27 | 2024-12-27 | 山东省农业科学院畜牧兽医研究所 | A tissue lysis solution and a method for extracting DNA from animal tissue |
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