CN112941067A - Lysis binding solution for whole blood nucleic acid extraction and kit and application thereof - Google Patents
Lysis binding solution for whole blood nucleic acid extraction and kit and application thereof Download PDFInfo
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention relates to a lysis binding solution for extracting whole blood nucleic acid, which consists of aqueous solutions of the following substances: guanidine hydrochloride, sodium iodide, EDTA, triton X-100, Tween20, Tris-HCl, SDS, NP-40. The cracking combination liquid provided by the invention can be used for cracking and adsorbing at the same time, so that the operation steps are reduced, and the cracking combination liquid does not contain organic solvents, and further can be combined with an instrument to perform automatic extraction, and can treat samples in batches. The invention obtains the formula of the lysis solution for efficiently extracting nucleic acid by researching the lysis performance experiment of the lysis solution, adjusting the composition and the proportion of each reagent and carrying out a large amount of experimental improvement of an inventor. Can ensure that the purity of the nucleic acid of the extracted whole blood is good, the concentration is high, the nucleic acid strip is clearer and brighter than the nucleic acid strip extracted by a contrast reagent, and the extraction efficiency is higher.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a lysis binding solution for whole blood nucleic acid extraction, and a nucleic acid extraction kit prepared by using the lysis binding solution.
Background
Nucleic acid is genetic material of all organisms and is widely present in all animals, plants and microorganisms, and comprises deoxyribonucleic acid (RNA) and ribonucleic acid (DNA). With the wide application of molecular biology techniques in biomedicine and related fields, the extraction and purification techniques of nucleic acids have been further developed. The extraction of nucleic acid mainly refers to the separation of nucleic acid from biological macromolecular substances such as protein, polysaccharide, fat and the like. Nucleic acid extraction and purification are key links of nucleic acid detection, and the efficiency and purity of nucleic acid extraction play an important role in downstream nucleic acid detection.
Since the first successful discovery and isolation of DNA by Miescher in 1869 and the first isolation of DNA by density gradient centrifugation by MeselsonM et al in 1957, various nucleic acid extraction methods were reported, such as: such as phenol chloroform method, centrifugal column extraction method, glass powder adsorption method, alkali cracking method and EtBr-CsCl gradient centrifugation method. Many researchers have diligently searched for methods for extracting nucleic acids, and have improved various materials and reagents for nucleic acids, and various reagents such as sodium dodecyl sulfate, phenol, urea, guanidine salt and the like are applied to nucleic acid extraction experiments, and various commercial kits for nucleic acid extraction are also produced. 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. The magnetic bead method can specifically identify and efficiently combine nucleic acid molecules by using nano magnetic beads, can separate nucleic acid from samples such as blood, tissues, pathogenic microorganisms and the like under the action of an external magnetic field and guanidine salt, and can be used for diagnosing clinical diseases. The magnetic bead method is convenient for automation, simple and convenient to operate, short in extraction time, safe and nontoxic.
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, the present invention provides a lysis binding solution for whole blood nucleic acid extraction based on magnetic beads, a kit containing the lysis binding solution, and a method for whole blood nucleic acid extraction, so as to achieve rapid, high-throughput, and automated nucleic acid extraction.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a lysis binding solution for whole blood nucleic acid extraction, which consists of the following substances: guanidine hydrochloride, sodium iodide, EDTA, surfactant, Tris-HCl, SDS, NP-40, water.
Further, the lysis binding solution consists of the following substances: 2-5M guanidine hydrochloride, 0.5-2M sodium iodide, 5-20mM EDTA, 0.1-1M Tris-HCl, 20-30% of surfactant, NP-401-2%, 0.1-0.5% of SDS and the balance of water, wherein the surfactant is triton X-100 or/and Tween-20.
Further, the lysis binding solution also comprises 3% -8% of magnetic beads.
Further, the lysis binding solution consists of the following substances: 3M guanidine hydrochloride, 0.5M sodium iodide, 15mM EDTA, 10% triton X-100, 10% Tween20, 0.2M Tris-HCl, 5% magnetic beads, 0.1% SDS, 1% NP-40, and the balance water.
2. Use of the lysis conjugate according to any of the above in the preparation of a reagent or kit for nucleic acid extraction.
3. The whole blood nucleic acid extraction kit containing the lysis-binding solution for whole blood nucleic acid extraction according to any of the above items, comprising the lysis-binding solution, a proteinase K solution, a washing buffer solution, and an elution buffer solution.
Further, the washing buffer comprises a washing buffer 1 and a washing buffer 2, wherein the washing buffer 1 consists of the following components: 1-3M guanidine hydrochloride, 0.1-1M NaAc, 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 7.5-8.
The concentration of the proteinase K in the proteinase K solution is 10-30 mg/ml.
4. The extraction method for extracting the whole blood nucleic acid comprises the following specific steps:
1) adding a lysis binding solution and a proteinase K solution into a biological sample, carrying out lysis for 5-15min at 20-85 ℃, 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 55-85 ℃ for 3-8min to elute the nucleic acid bound on the magnetic bead, thereby obtaining the extracted nucleic acid.
Further, the washing buffer includes washing buffer 1 and washing buffer 2, which are washed once with washing buffer 1 and washing buffer 2, respectively.
Further, wash buffer 1 consisted of the following components: 1-3M guanidine hydrochloride, 0.1-1M NaAc, 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 7.5-8.
The concentration of the proteinase K in the proteinase K solution is 10-30 mg/ml.
Further, the biological sample includes tissue, cells, whole blood, plasma, serum, body fluid, swab wash.
The invention has the beneficial effects that: the cracking combination liquid provided by the invention can be used for cracking and adsorbing at the same time, so that the operation steps are reduced, and the cracking combination liquid does not contain organic solvents, and further can be combined with an instrument to perform automatic extraction, and can treat samples in batches. Compared with a silica gel membrane adsorption column method, the magnetic bead method for extracting nucleic acid has low extraction efficiency, and mostly adopts two steps of cracking and combining, namely, the magnetic bead combined nucleic acid is added after the cracking is finished, so that the method is not beneficial to automatic nucleic acid extraction application, and the magnetic bead method for extracting nucleic acid has the main advantages of automation, and has no advantages compared with other methods if the automation is not carried out or the automation cost is high. The invention obtains the formula of the lysis solution for efficiently extracting nucleic acid by researching the lysis performance experiment of the lysis solution, adjusting the composition and the proportion of each reagent and carrying out a large amount of experimental improvement of an inventor. Can ensure that the purity of the nucleic acid of the extracted whole blood is good, the concentration is high, the nucleic acid strip is clearer and brighter than the nucleic acid strip extracted by a contrast reagent, and the extraction efficiency is higher.
And the components used in the lysis binding solution abandon the isopropanol used as an organic solvent in the traditional magnetic bead method for nucleic acid extraction, ensure that no organic solvent with an inhibiting effect on enzyme exists in the extracted nucleic acid sample, avoid the opportunity of sucking the isopropanol for the nucleic acid extraction user, and increase the good experimental experience.
When the whole blood 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 whole blood nucleic acid extraction kit has low cost, the nucleic acid extraction method is simple, the purity of the extracted nucleic acid is high, and the kit can be directly used for molecular biological experimental research such as PCR and the like 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 shows the electrophoresis results of the reagent of the present invention for extracting nucleic acid from whole blood and the control reagent for extracting nucleic acid from whole blood;
FIG. 2 shows the results of fluorescent quantitative PCR of the nucleic acids extracted in example 1.
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
1) Putting 200uL of EDTA anticoagulation blood into a centrifugal tube, adding 600uL of lysis buffer solution and 20uL of 10-30mg/ml proteinase K solution into the centrifugal tube, heating and uniformly mixing for 3 minutes at 65 ℃, putting the centrifugal tube on a magnetic frame, performing magnetic separation for 20s, and removing supernatant;
wherein, the components added in the lysis buffer solution are respectively: guanidine hydrochloride 3M, sodium iodide 0.5M, EDTA 15mM, triton X-10010% (v/v), Tween 2010% (v/v), Tris-HCl 0.2M, nano magnetic beads 5% (w/v), SDS 0.1% (w/v), NP-401% (w/v), the balance of water, and the pH value of the lysis buffer is adjusted to 7.0.
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 absorbing and discarding the supernatant;
wherein, the components added in the washing buffer solution 1 are respectively: 2M guanidine hydrochloride, NaAc 0.3M, 40% (v/v) ethanol solution; the rest components are water, and the pH value of the washing buffer solution is adjusted to 6.2;
3) continuously adding 600ul 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 absorbing and discarding the supernatant; the washing buffer solution 2 is 75% (v/v) ethanol water solution;
4) and finally, adding 100ul of elution buffer solution into the centrifugal tube, heating and uniformly mixing for 5 minutes at 65 ℃, placing the centrifugal tube on a magnetic frame, performing magnetic separation for 20 seconds, transferring the supernatant into another clean non-enzyme centrifugal tube, and storing at the temperature of-20 ℃ to obtain the finally extracted and purified whole blood genome DNA solution.
The elution buffer contained EDTA 2mM, Tris-HCl 8mM, adjusted to pH 8.
The sample size of the contrast reagent was 200ul extracted from QIAamp DNA Blood Mini Kit, and the elution volume was 100 ul.
After the extraction was completed, 5ul of each of the whole blood genomic DNA solutions was subjected to 1% agarose gel electrophoresis, and the results are shown in FIG. 1. Compared with the electrophoresis result of the whole blood nucleic acid extracted by the contrast reagent, the electrophoresis result of the whole blood nucleic acid extracted by the reagent has the advantages of clearer and brighter band and higher extraction efficiency.
The QIAamp DNA Blood Mini Kit utilizes the selective adsorption property of silica gel membrane, nucleic acid in a sample can be specifically bound to the silica gel membrane of QIAamp, and PCR inhibitors such as: divalent cations and proteins are completely removed and finally the pure nucleic acid bound to the spin column can be collected by elution with water or buffer in a kit.
Example 2
The results of the fluorescent quantitative PCR using 2ul of the inventive reagent and the control reagent extracted in example 1 as targets for β -globin are shown in Table 1 and FIG. 2.
TABLE 1 Whole blood genome fluorescent PCR results of inventive and control reagents
Example 3
EDTA anticoagulated whole blood samples (principle of collector volunteers) were collected from the local hospital in the corridor for 10 cases. Putting 200uL of whole blood sample into a centrifugal tube, adding 600uL of lysis buffer solution and 20uL of 20mg/ml proteinase K solution into the centrifugal tube, heating and uniformly mixing for 5 minutes at 60 ℃, then putting the centrifugal tube on a magnetic frame, carrying out magnetic separation for 20s, and discarding the supernatant;
2) continuously adding 700ul 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; the washing buffer 1 was: 2M guanidine hydrochloride, NaAc 0.3M, 40% ethanol solution; the rest components are water, and the pH value of the washing buffer solution is adjusted to 6.2;
3) continuously adding 700ul 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 absorbing and discarding the supernatant; the washing buffer 2 is 75% (v/v) ethanol water solution;
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 human genome nucleic acid solution.
Wherein the lysis buffer, the washing buffer and the elution buffer used in example 3 of the present invention are the same as those used in example 1, i.e., the kit used is the same as that used in example 1.
The invention can also use the kit to cooperate with an automatic instrument to rapidly extract the nucleic acid of the whole blood sample with high flux, and the automatic nucleic acid extractor is used, and the procedures are as follows:
TABLE 2
After extraction, 2uL of the nucleic acid is taken as a template, 18uL of reaction liquid and 20uL of total reaction volume, a commercially available CYP2C19 drug metabolizing enzyme gene polymorphism detection Kit is adopted, amplification detection is carried out by using an ABI7500 fluorescence quantitative PCR instrument, nucleic acid samples of the same sample extracted by using a QIAamp DNA Blood Mini Kit are subjected to first-generation sequencing comparison consistency, and detection results are shown in the following table 3. As a result, the consistency rate of the genotype of 30 samples of three sites of CYP2C19 detected by nucleic acid extracted by the kit disclosed by the invention and the sequencing result is 100%.
TABLE 3 detection of CYP2C19 genotype by nucleic acid extracted from kit of the invention
Example 4 comparative data with conventional addition of binding solution
1. With the method of the present invention, washing and elution of nucleic acids are carried out after direct lysis and binding.
Putting 200uL of EDTA anticoagulation blood into a centrifugal tube (3 parallel samples are arranged), adding 600uL of lysis buffer solution and 20uL of 30mg/ml proteinase K solution into the centrifugal tube, and heating and uniformly mixing for 3 minutes at 65 ℃;
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; adding 600ul 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 20 seconds, and absorbing and removing the supernatant;
and finally, adding 100ul of elution buffer solution into the centrifuge tube, heating and uniformly mixing for 5 minutes at 80 ℃, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 20 seconds, transferring the supernatant into another clean centrifuge tube to obtain a finally extracted and purified nucleic acid solution, and storing at the temperature of-20 ℃.
The lysis buffer comprises guanidine hydrochloride 3M, sodium iodide 0.5M, EDTA 15mM, triton X-10010%, Tween 2010%, Tris-HCl 0.2M, nano magnetic beads 5%, SDS 0.1%, NP-401%, and the balance of water, and the pH value of the lysis buffer is adjusted to 7.0.
The washing buffer 2 was 70% ethanol aqueous solution.
The elution buffer comprises EDTA 1mM, Tris-HCl 10mM and pH value 8;
the nano magnetic beads are superparamagnetic silicon oxide nano magnetic microbeads with the diameter of 500 nm.
2. The traditional method is adopted, firstly, cracking is carried out, then isopropanol is added as binding liquid for binding, and then, washing and 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 Tween20 tween-20, 10-20 mM Tris-HCl, 1-2mM EDTA, and pH7-8, wherein the volume ratio of the biological sample, isopropanol and lysate is 1:1: 2.
After extraction, the purity and concentration of the extract were measured by an ultraviolet spectrophotometer, and the results are shown in Table 4 below.
Table 4:
as shown in Table 4, the purity of the extraction between 1.7 and 2.1 was satisfactory as a result of the method of the present invention and the addition of the binding solution, but the concentration of the extraction was far lower than the result of the present invention.
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 whole blood nucleic acid extraction, which is characterized by comprising the following substances: guanidine hydrochloride, sodium iodide, EDTA, surfactant, Tris-HCl, SDS, NP-40, water.
2. The lysis binding solution for whole blood nucleic acid extraction according to claim 1, wherein the lysis binding solution is composed of: 2-5M guanidine hydrochloride, 0.5-2M sodium iodide, 5-20mM EDTA, 0.1-1M Tris-HCl, 20-30% of surfactant, NP-401-2%, 0.1-0.5% of SDS and the balance of water, wherein the surfactant is TritonX-100 or/and Tween-20.
3. The lysis binding solution for nucleic acid extraction from whole blood according to claim 1 or 2, wherein the lysis binding solution further comprises 3% to 8% magnetic beads.
4. The lysis solution for whole blood nucleic acid extraction according to claim 3, wherein the lysis solution comprises: 3M guanidine hydrochloride, 0.5M sodium iodide, 15mM EDTA, 10% TritonX-100, 10% Tween20, 0.2M Tris-HCl, 5% magnetic beads, 0.1% SDS, 1% NP-40, and the balance water.
5. Use of the lysis-binding solution for whole blood nucleic acid isolation according to any one of claims 1 to 4 for preparing a reagent or a kit for nucleic acid isolation.
6. The kit for whole blood nucleic acid extraction comprising the lysis-binding solution for whole blood nucleic acid extraction according to any one of claims 1 to 4, wherein the kit for whole blood nucleic acid extraction comprises the lysis-binding solution, a proteinase K solution, a washing buffer, and an elution buffer.
7. The whole blood nucleic acid extraction kit according to claim 6, wherein the washing buffer comprises a washing buffer 1 and a washing buffer 2, and the washing buffer 1 is composed of the following components: 1-3M guanidine hydrochloride, 0.1-1M NaAc, pH 6.0-6.5, 30-60% ethanol solution; the washing buffer 2 is 70-85% ethanol water solution.
8. The whole blood nucleic acid extraction kit according to claim 6 or 7, wherein the elution buffer is composed of: 1-5mM EDTA,5-20mM Tris-HCl, pH 7.5-8.
9. The method for extracting nucleic acid from whole blood using the kit for extracting nucleic acid from whole blood according to any one of claims 6 to 9, comprising the steps of:
1) adding a lysis binding solution into a biological sample, lysing for 5-15min at 20-85 ℃, 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 55-85 ℃ for 3-8min to elute the nucleic acid bound on the magnetic bead, thereby obtaining the extracted nucleic acid.
10. The extraction method of claim 3, wherein the biological sample comprises tissue, cells, whole blood, plasma, serum, body fluid, swab wash.
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| 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 |
| CN114672481A (en) * | 2022-04-21 | 2022-06-28 | 叶晓君 | Novel high-efficiency nucleic acid extraction method of plant fiber adsorption matrix |
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