CN116355894A - Magnetic bead method nucleic acid extraction kit and extraction method suitable for oral swab samples - Google Patents
Magnetic bead method nucleic acid extraction kit and extraction method suitable for oral swab samples Download PDFInfo
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- CN116355894A CN116355894A CN202310326689.6A CN202310326689A CN116355894A CN 116355894 A CN116355894 A CN 116355894A CN 202310326689 A CN202310326689 A CN 202310326689A CN 116355894 A CN116355894 A CN 116355894A
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- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 18
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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
- 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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Abstract
The invention discloses a magnetic bead method nucleic acid extraction kit suitable for an oral swab sample, which comprises the following components: the kit can extract genome DNA from an oral swab stored in a dry or stored liquid, and can realize high-flux nucleic acid extraction by matching with an automatic nucleic acid extraction device.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a magnetic bead method nucleic acid extraction kit and an extraction method suitable for an oral swab sample.
Background
Gene testing is a test method for evaluating whether a subject is a disease, constitution or personal trait related to genetic inheritance from the aspect of chromosome structure, DNA sequence, DNA mutation site or gene expression level, and is also a method for accurate medical analysis. Gene detection can be used for diagnosing diseases and predicting the risk of the diseases. The main application directions of gene detection today include: identification/paternity identification/traceability of ancestral sources, single gene/chromosome genetic disease diagnosis, screening of genes, clinical preventive medicine, clinical personalized medicine, congenital constitution/trait potential analysis and the like. The basis of these studies is not isolated from the extraction of genomic DNA.
The sample used for obtaining DNA in gene testing is usually blood, other body fluids, tissue cells, or the like. Among them, collection of oral epithelial mucosal cells by oral swab is the most convenient way. The oral cavity is convenient to wipe, the method is simple and easy to understand, the sample storage is also convenient, and a painless and noninvasive DNA acquisition mode suitable for people of all ages is provided for disease control and prevention.
Different genetic testing projects have different requirements on the preservation mode of oral swab samples, and the method generally comprises two steps of drying and preserving oral swab samples with preserving fluid. At present, a commercial oral swab nucleic acid extraction kit adopts a centrifugal column method to extract and extract dry-stored oral swab sample nucleic acid, and cannot extract the swab sample nucleic acid stored in a preservation solution. Moreover, for the detection of a large number of gene detection samples, the extraction by the centrifugal column method is very time-consuming and labor-consuming, and the excessive time is required to cause the loss of nucleic acid collected by the oral swab, thereby affecting the accuracy of the result. Therefore, an extraction method for extracting and preserving nucleic acid of an oral swab sample by drying and preserving the nucleic acid of the oral swab sample by extracting and preserving solution is urgently needed, and the method can be matched with automatic nucleic acid extraction equipment to realize high-flux extraction and rapidly obtain nucleic acid with high concentration and good quality.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problems of the prior art, and provides a magnetic bead method nucleic acid extraction kit and an extraction method suitable for an oral swab sample, which can extract genome DNA from an oral swab stored in a dry or preservation liquid and can realize high-flux nucleic acid extraction by matching with automatic nucleic acid extraction equipment.
In order to solve the technical problems, the invention discloses a magnetic bead method nucleic acid extraction kit suitable for an oral swab sample, which comprises the following components: digest a, digest B, deproteinized solution, wash, eluent, proteinase K, bead suspension, wherein:
the digestive juice A consists of the following components in concentration: 1-5% of sarcosyl, 0.5-2.0% of polyethylene glycol octyl phenyl ether, 50-300mM of sodium chloride, 20-100mM of tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM of disodium edetate, wherein the solvent is pure water. The digestive juice A acts to digest cells on the dry preserved swab sample and release nucleic acids within the cells.
The digestive juice B consists of the following components in concentration: 1-5M guanidine thiocyanate, 0.5-2.0% of polyethylene glycol octyl phenyl ether, 1-3% of polysorbate-20, 0.02-0.5% of acetylcysteine, 50-300mM of sodium chloride, 20-100mM of tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM of disodium ethylenediamine tetraacetate, wherein the solvent is pure water. The digestive juice B has the function of supplementing the digestive lysis effect and promoting the combination of DNA and magnetic beads.
The deproteinized solution consists of the following components in concentration: 1-5M guanidine hydrochloride, 0.5-2.0% of polyethylene glycol octyl phenyl ether, 0.5-1.5% of polysorbate-20, 30-70% of absolute ethyl alcohol, 50-300mM sodium chloride, 20-100mM tris (hydroxymethyl) aminomethane hydrochloride, 0.1-1mM disodium ethylenediamine tetraacetate, and pure water as solvent. The deproteinizing liquid serves to wash off proteins adhering to the magnetic beads.
Wherein the washing liquid comprises the following components in concentration: ethanol with the volume fraction of 70-80%, 20-50mM of tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM of disodium ethylenediamine tetraacetate, and the solvent is pure water. The washing liquid serves to wash away salt ions remaining on the magnetic beads.
The magnetic bead suspension is formed by mixing magnetic beads and a preservative, the magnetic beads are used for adsorbing DNA, and the concentration of the magnetic bead solution is 50-100mg/mL.
The eluent is deionized water.
Preferably, the digestive juice A consists of the following components in concentration: 2-3% of sarcosyl, 0.5-1.0% of polyethylene glycol octyl phenyl ether, 100-200mM sodium chloride, 20-50mM tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM disodium edetate, wherein the solvent is pure water.
The digestive juice B comprises the following components in concentration: 2-4M guanidine thiocyanate, 1.5-2.0% of polyethylene glycol octyl phenyl ether, 2-2.5% of polysorbate-20, 0.1-0.3% of acetylcysteine, 100-200mM sodium chloride, 20-50mM tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM disodium ethylenediamine tetraacetate, wherein the solvent is pure water.
The deproteinized solution comprises the following components in concentration: 1-3M guanidine hydrochloride, 0.5-1.0% of polyethylene glycol octyl phenyl ether, 0.8-1.0% of polysorbate-20, 45-60% of ethanol, 50-150mM of sodium chloride, 20-50mM of tris (hydroxymethyl) aminomethane hydrochloride and 0.5-1mM of disodium ethylenediamine tetraacetate, wherein the solvent is pure water.
The invention further provides application of the kit in extracting and drying preservation or extracting genome DNA from an oral swab preserved by preservation solution.
Specifically, the invention provides a magnetic bead method nucleic acid extraction method for an oral swab sample stored by drying or storing liquid by using the kit, which is characterized by comprising the following steps of:
(1) For a dry preserved swab sample, transferring the swab sample into a centrifuge tube, adding 300 mu L of digestive juice A, 20 mu L of proteinase K, incubating for 30min at 56 ℃, adding 300 mu L of digestive juice B, incubating for 10min at 56 ℃, and transferring the digestive products into a clean 2mL centrifuge tube; for the swab sample stored by the preservation solution, transferring the swab sample into a 2mL centrifuge tube, adding 300 mu L of preservation solution for preserving the swab sample, 200 mu L of digestive juice B and 20 mu L of proteinase K, incubating at 56 ℃ for 30min, and transferring the digestive products into a clean 2mL centrifuge tube;
(2) For a dry and preserved swab sample, adding 200 mu L of isopropanol and 10 mu L of magnetic bead suspension into the digestion product of the previous step, carrying out vortex mixing, placing a centrifuge tube on a rotary mixing instrument for 10min, taking down the centrifuge tube, carrying out short centrifugation, placing the centrifuge tube on a magnetic rack for magnetic attraction for 1min, and discarding liquid; adding 300 mu L of isopropanol and 10 mu L of magnetic bead suspension into the digestion product of the previous step for the swab sample stored in the preservation solution, carrying out vortex mixing, placing a centrifuge tube on a rotary mixing instrument for 10min, carrying out short centrifugation, placing the centrifuge tube on a magnetic rack for magnetic attraction for 1min, and discarding the liquid;
(3) Adding 500 mu L deproteinized liquid into the magnetic beads in the previous step, swirling for 30 seconds, centrifuging briefly, then placing the magnetic beads on a magnetic rack for magnetic attraction for 1min, and discarding the liquid;
(4) Repeating the step (3) for one time;
(5) Adding 500 mu L of washing liquid into the magnetic beads in the previous step, swirling for 30 seconds, placing the magnetic beads on a magnetic rack for magnetic attraction for 1min after short centrifugation, discarding the liquid, repeating the rinsing process operation once, opening a centrifugal tube cover and placing the centrifugal tube cover at room temperature for 5min;
(6) Repeating step (5) for one time;
(7) Opening a centrifuge tube on the magnetic rack in the previous step, and standing for 5min at room temperature;
(8) Adding 100 μl of eluent, vortexing for 30 seconds, incubating at 56 deg.C for 10min, centrifuging briefly, magnetically absorbing on a magnetic rack for 1min, and collecting supernatant containing extracted nucleic acid
The beneficial effects are that: the invention provides a magnetic bead method nucleic acid extraction kit and an extraction method suitable for an oral swab sample, which can extract genome DNA from an oral swab preserved by drying or preserving liquid, and the magnetic bead method extraction reagent can be matched with automatic nucleic acid extraction equipment to realize high-flux nucleic acid extraction.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is the result of electrophoresis of the nucleic acid extracted in example 1;
FIG. 2 is the result of qPCR detection of the nucleic acid extracted in example 1;
FIG. 3 shows the result of electrophoresis of nucleic acids extracted in example 2
Detailed Description
The invention discloses a magnetic bead method nucleic acid extraction kit and an extraction method suitable for an oral swab sample, and particularly provides the following embodiments.
Swab samples include two types: a dry preserved swab sample, a swab preservation solution preserved swab sample. The dry preservation of the swab sample has the following advantages: 1) Convenient to store and transport: the swab sample stored in a drying way does not need preservation liquid, so that the swab sample is more convenient to transport and store, and does not need refrigeration; 2) Long-term preservation: the preserved swab sample is dried, and cells and DNA are in a dormant state in a dried state, so that the swab sample has longer stability; 3) The cost is reduced: the dry preserved swab sample does not need preservation liquid, so that the transportation and storage cost can be reduced; 4) Avoiding the influence of preservation solution on nucleic acid: some preservation solutions may have an effect on the quality of the nucleic acid, which can be avoided by drying the preserved swab sample.
The swab preservation solution has the following advantages that: 1) The procedure for extracting nucleic acids is relatively simple: the swab preservation solution is used for preserving the swab sample without using the digestive juice A, and is used for lysing cells on the swab and releasing nucleic acid in the cells; 2) The requirements on the sample are relatively low: the dry preserved swab sample has no protection of the swab preservation solution, so the requirements on the collection and preservation of the sample are high, the sample needs to be prevented from being influenced by pollution and humidity, and the swab preservation solution is not needed to consider the sample.
According to the invention, the two swab samples with different preservation modes are respectively subjected to optimization test, and the optimized reagent formula is integrated into a kit, so that the genome DNA can be extracted from the dried preserved swab samples and the genome DNA can be extracted from the swab samples preserved by the swab preservation solution.
In the following examples, the composition of digestive juice a is: 4% of sodium dodecyl sarcosinate, 2% of polyethylene glycol octyl phenyl ether, 150mM sodium chloride, 50mM tris (hydroxymethyl) aminomethane hydrochloride and 1mM disodium ethylenediamine tetraacetate by mass-volume ratio, and the solvent is pure water.
The composition of the digestive juice B is as follows: 5M guanidine thiocyanate, 0.8% of polyethylene glycol octyl phenyl ether, 1% of polysorbate-20, 0.05% of acetylcysteine, 150mM sodium chloride, 50mM tris (hydroxymethyl) aminomethane hydrochloride and 1mM disodium ethylenediamine tetraacetate, wherein the solvent is pure water.
The deproteinized liquid comprises the following components: 1M guanidine hydrochloride, 1% of polyethylene glycol octyl phenyl ether, 0.5% of polysorbate-20, 55% of absolute ethyl alcohol, 150mM of sodium chloride, 50mM of tris (hydroxymethyl) aminomethane hydrochloride, 1mM of disodium edetate, and pure water as a solvent.
The composition of the washing liquid is as follows: 80% ethanol, 20mM tris (hydroxymethyl) aminomethane hydrochloride and 0.1mM disodium edetate, and pure water as solvent.
The composition of the eluent is as follows: deionized water.
The magnetic bead suspension consists of silicon hydroxyl magnetic beads with the mass-volume ratio of 50mg/mL, cresol with the mass-volume ratio of 0.1%, benzoic acid with the mass-volume ratio of 0.05%, and pure water as a solvent.
EXAMPLE 1 Manual extraction of nucleic acids from oral swab samples
8 2mL centrifuge tubes, labeled No. 1, no. 2, no. 3, no. 4, no. 5, no. 6, no. 7, no. 8, were taken. Placing 4 dry-stored oral swabs into a No. 1-4 centrifuge tube respectively, adding 300 mu L of digestive juice A and 20 mu L of proteinase K respectively, incubating for 30min at 56 ℃, adding 300 mu L of digestive juice B, incubating for 10min at 56 ℃, transferring digestive products into a clean 2mL centrifuge tube, and marking according to the previous number; taking 4 preservation solutions (from Tiangen corporation, product number: RK 112) and placing the preserved oral swab samples into 5-8 centrifuge tubes respectively, respectively absorbing 300uL of the preservation solutions, adding into the centrifuge tubes, adding 200uL of digestion solution B and 20 uL of proteinase K, incubating at 56 ℃ for 30min, transferring the digestion products into clean 2mL centrifuge tubes, and marking according to the previous numbers; adding 200 mu L of isopropanol and 10 mu L of magnetic bead suspension into the digestion product of the previous step for a centrifuge tube No. 1-4, carrying out vortex mixing, placing the centrifuge tube on a rotary mixing instrument for 10min, taking down the centrifuge tube, carrying out short centrifugation, placing the centrifuge tube on a magnetic rack for magnetic attraction for 1min, and discarding liquid; adding 300 mu L of isopropanol and 10 mu L of magnetic bead suspension into the digestion product of the previous step for a No. 5-8 centrifuge tube, carrying out vortex mixing, placing the centrifuge tube on a rotary mixing instrument for 10min, carrying out short centrifugation, placing the centrifuge tube on a magnetic rack for magnetic attraction for 1min, and discarding liquid; adding 500 mu L deproteinized liquid into a No. 1-8 centrifuge tube, swirling for 30 seconds, placing on a magnetic rack for magnetic attraction for 1min after short centrifugation, discarding the liquid, and repeating the operation for one time; adding 500 mu L of washing liquid into a No. 1-8 centrifuge tube, swirling for 30 seconds, placing on a magnetic rack for magnetic attraction for 1min after short centrifugation, discarding the liquid, repeating the rinsing process operation once, opening a centrifuge tube cover, placing at room temperature for 5min, and repeating the operation once; opening a No. 1-8 centrifuge tube on a magnetic rack, and standing at room temperature for 5min; adding 100 μl of eluent, vortexing for 30 seconds, incubating at 56 ℃ for 10min, centrifuging briefly, magnetically attracting on a magnetic rack for 1min, and sucking supernatant to obtain the extract containing the extracted nucleic acid, and labeling.
Taking 2 mu L of No. 1-8 DNA samples, detecting the concentration and purity of the DNA samples after the DNA samples are blanked by using ultrapure water by a micro-spectrophotometer, wherein the results are shown in a table 1, 1-4 are extraction groups for dry preservation of oral swab samples, and 5-8 are extraction groups for preservation of oral swab samples by preservation solution; respectively taking 5 mu L of DNA samples for electrophoresis, and detecting the size and the integrity of the nucleic acid fragments, wherein the result is shown in the figure 1; respectively taking two groups of obtained DNA samples, performing qPCR detection, and measuring the difference of Ct values, wherein the result is shown in figure 2.
In view of the concentration and purity of the DNA in table 1, the present method can extract DNA of higher concentration and higher purity from both the dry preserved swab sample and the preserved fluid preserved swab sample by manual extraction. The qPCR result amplification curve of FIG. 2 is normal, which shows that the DNA extracted by the embodiment has high concentration and good quality, and can be normally used for downstream experiments.
TABLE 1 DNA concentration and purity of oral swab samples in example 1
| Numbering device | Concentration (ng/. Mu.L) | A260/280 | A260/230 |
| 1 | 54.9 | 1.83 | 1.41 |
| 2 | 54.7 | 1.80 | 1.37 |
| 3 | 52.5 | 1.82 | 1.43 |
| 4 | 53.0 | 1.81 | 1.42 |
| 5 | 132.7 | 1.93 | 1.71 |
| 6 | 133.1 | 1.93 | 1.67 |
| 7 | 141.8 | 1.92 | 1.74 |
| 8 | 126.8 | 1.91 | 1.76 |
EXAMPLE 2 full automatic nucleic acid extraction Instrument for extracting nucleic acid from oral swab sample
8 2mL centrifuge tubes, labeled No. 1, no. 2, no. 3, no. 4, no. 5, no. 6, no. 7, no. 8, were taken. Placing 4 dry-stored oral swabs into a No. 1-4 centrifuge tube respectively, adding 300 mu L of digestive juice A and 20 mu L of proteinase K respectively, incubating for 30min at 56 ℃, adding 300 mu L of digestive juice B, incubating for 10min at 56 ℃, transferring digestive products into a clean 2mL centrifuge tube, adding 200 mu L of magnetic bead suspension respectively, marking according to the previous number, obtaining a pretreated sample, and storing for later use; similarly, taking 4 preservation solutions (from Tiangen corporation, product number: RK 112) and placing the preserved oral swab samples into 5-8 centrifuge tubes respectively, respectively sucking 300 mu L of corresponding preservation solution, adding into the centrifuge tubes, adding 200 mu L of digestion solution B and 20 mu L of proteinase K, incubating at 56 ℃ for 30min, transferring the digestion products into clean 2mL centrifuge tubes, respectively adding 300 mu L of magnetic bead suspension, marking according to the previous number, and obtaining pretreated samples for preservation. Adding the following corresponding reagents into a 96 deep-hole plate according to the table 2, placing the deep-hole plate into an instrument according to the operation of a purification instrument Purifier HT of Jifan full-automatic nucleic acid extraction and purification instrument, installing a magnetic rod sleeve, and running a preset extraction program, wherein the extraction program is shown in the table 3; the DNA samples in plate 6 were collected after the end of the procedure.
TABLE 2 deep well plate reagent sample volume
TABLE 3 purification apparatus Purifier HT extraction procedure for Jifan fully automatic nucleic acid extraction
Taking 2 mu L of No. 1-8 DNA samples respectively, detecting the concentration and purity of the DNA samples after the DNA samples are blanked by using ultrapure water by a micro-spectrophotometer, and obtaining the results shown in a table 4, wherein 1-4 is an extraction group for dry preservation of oral swab samples, and 5-8 is an extraction group for preservation of oral swab samples by preservation solution; respectively taking 5 mu L of DNA sample for electrophoresis, and detecting the size and the integrity of the nucleic acid fragment, wherein the result is shown in figure 3.
In view of the DNA concentration and purity of table 4, the method of this example can extract DNA of higher concentration and better purity from both the dry preserved swab sample and the preserved fluid preserved swab sample by means of extraction with a fully automatic nucleic acid extractor. The electrophoresis band is complete and clear in combination with the attached figure 3, and the sample application hole has no protein residue, which shows that the DNA extracted by the embodiment has high concentration and good quality.
TABLE 4 DNA concentration and purity of oral swab samples in example 1
| Numbering device | Concentration (ng/. Mu.L) | A260/280 | A260/230 |
| 1 | 54.9 | 1.84 | 1.69 |
| 2 | 59.7 | 1.83 | 1.63 |
| 3 | 58.5 | 1.83 | 1.60 |
| 4 | 60.1 | 1.85 | 1.64 |
| 5 | 152.7 | 1.94 | 1.82 |
| 6 | 154.1 | 1.95 | 1.84 |
| 7 | 156.8 | 1.94 | 1.82 |
| 8 | 155.8 | 1.96 | 1.85 |
The magnetic bead method nucleic acid extraction kit and the extraction method suitable for the oral swab sample can extract genome DNA from the oral swab preserved by drying or preserved in preservation solution, and can realize high-flux nucleic acid extraction by matching with automatic nucleic acid extraction equipment, so that the principle is popular and easy to understand, and the operation is simple. The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (9)
1. A magnetic bead method nucleic acid extraction kit suitable for an oral swab sample, which is characterized by comprising the following components: digest a, digest B, deproteinized solution, wash, eluent, proteinase K, bead suspension, wherein:
the digestive juice A consists of the following components in concentration: 1-5% of sarcosyl, 0.5-2.0% of polyethylene glycol octyl phenyl ether, 50-300mM sodium chloride, 20-100mM tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM disodium edetate, wherein the solvent is pure water;
the digestive juice B consists of the following components in concentration: 1-5M guanidine thiocyanate, 0.5-2.0% of polyethylene glycol octyl phenyl ether, 1-3% of polysorbate-20, 0.02-0.5% of acetylcysteine, 50-300mM of sodium chloride, 20-100mM of tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM of disodium ethylenediamine tetraacetate, wherein the solvent is pure water;
the deproteinized solution consists of the following components in concentration: 1-5M guanidine hydrochloride, 0.5-2.0% of polyethylene glycol octyl phenyl ether, 0.5-1.5% of polysorbate-20, 30-70% of absolute ethyl alcohol, 50-300mM sodium chloride, 20-100mM tris (hydroxymethyl) aminomethane hydrochloride, 0.1-1mM disodium ethylenediamine tetraacetate, and pure water as solvent.
2. The kit of claim 1, wherein the wash solution comprises the following concentrations of components: ethanol with the volume fraction of 70-80%, 20-50mM of tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM of disodium ethylenediamine tetraacetate, and the solvent is pure water.
3. The kit according to claim 1, wherein the magnetic bead suspension is formed by mixing magnetic beads and a preservative, the magnetic beads are used for adsorbing DNA, and the concentration of the magnetic bead solution is 50-100mg/mL.
4. The kit of claim 1, wherein the eluent is deionized water.
5. The kit according to claim 1, wherein the digestive juice a is composed of the following concentration components: 2-3% of sarcosyl, 0.5-1.0% of polyethylene glycol octyl phenyl ether, 100-200mM sodium chloride, 20-50mM tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM disodium edetate, wherein the solvent is pure water.
6. The kit according to claim 1, wherein the digestive juice B comprises the following components in concentration: 2-4M guanidine thiocyanate, 1.5-2.0% of polyethylene glycol octyl phenyl ether, 2-2.5% of polysorbate-20, 0.1-0.3% of acetylcysteine, 100-200mM sodium chloride, 20-50mM tris (hydroxymethyl) aminomethane hydrochloride and 0.1-1mM disodium ethylenediamine tetraacetate, wherein the solvent is pure water.
7. The kit of claim 1, wherein the deproteinized solution comprises the following concentrations of components: 1-3M guanidine hydrochloride, 0.5-1.0% of polyethylene glycol octyl phenyl ether, 0.8-1.0% of polysorbate-20, 45-60% of ethanol, 50-150mM of sodium chloride, 20-50mM of tris (hydroxymethyl) aminomethane hydrochloride and 0.5-1mM of disodium ethylenediamine tetraacetate, wherein the solvent is pure water.
8. Use of a kit according to any one of claims 1 to 7 for extracting genomic DNA from an oral swab stored dry or in a stock solution.
9. A method for extracting nucleic acid from a dry-stored or stored oral swab sample by a kit according to any one of claims 1 to 8, comprising the steps of:
(1) For a dry preserved swab sample, transferring the swab sample into a centrifuge tube, adding 300 mu L of digestive juice A, 20 mu L of proteinase K, incubating for 30min at 56 ℃, adding 300 mu L of digestive juice B, incubating for 10min at 56 ℃, and transferring the digestive products into a clean 2mL centrifuge tube; for the swab sample stored by the preservation solution, transferring the swab sample into a 2mL centrifuge tube, adding 300 mu L of preservation solution for preserving the swab sample, 200 mu L of digestive juice B and 20 mu L of proteinase K, incubating at 56 ℃ for 30min, and transferring the digestive products into a clean 2mL centrifuge tube;
(2) For a dry and preserved swab sample, adding 200 mu L of isopropanol and 10 mu L of magnetic bead suspension into the digestion product of the previous step, carrying out vortex mixing, placing a centrifuge tube on a rotary mixing instrument for 10min, taking down the centrifuge tube, carrying out short centrifugation, placing the centrifuge tube on a magnetic rack for magnetic attraction for 1min, and discarding liquid; adding 300 mu L of isopropanol and 10 mu L of magnetic bead suspension into the digestion product of the previous step for the swab sample stored in the preservation solution, carrying out vortex mixing, placing a centrifuge tube on a rotary mixing instrument for 10min, carrying out short centrifugation, placing the centrifuge tube on a magnetic rack for magnetic attraction for 1min, and discarding the liquid;
(3) Adding 500 mu L deproteinized liquid into the magnetic beads in the previous step, swirling for 30 seconds, centrifuging briefly, then placing the magnetic beads on a magnetic rack for magnetic attraction for 1min, and discarding the liquid;
(4) Repeating the step (3) for one time;
(5) Adding 500 mu L of washing liquid into the magnetic beads in the previous step, swirling for 30 seconds, placing the magnetic beads on a magnetic rack for magnetic attraction for 1min after short centrifugation, discarding the liquid, repeating the rinsing process operation once, opening a centrifugal tube cover and placing the centrifugal tube cover at room temperature for 5min;
(6) Repeating step (5) for one time;
(7) Opening a centrifuge tube on the magnetic rack in the previous step, and standing for 5min at room temperature;
(8) Adding 100 μl of eluent, vortexing for 30 seconds, incubating at 56 ℃ for 10min, centrifuging briefly, magnetically attracting on a magnetic rack for 1min, and sucking supernatant to obtain the extract containing the extracted nucleic acid.
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