Inhibitor remover suitable for whole blood DNA extraction and nucleic acid extraction kit

Abstract

The invention relates to the technical field of nucleic acid extraction, in particular to an inhibitor remover suitable for whole blood DNA extraction and a nucleic acid extraction kit. The inhibitor remover comprises the following components in percentage by mass: 20%-40% of a thiol compound, 30%-50% of lower alcohol, 1%-2% of a nonionic surfactant and organic weak acid with the concentration of 10mM-30mM. The nucleic acid extraction kit comprises a lysis binding solution, a first washing solution, a second washing solution, an eluent and the inhibitor remover. When the kit disclosed by the invention is used for extracting whole blood DNA, inhibitors in a whole blood sample can be effectively removed, and the sensitivity of related PCR (Polymerase Chain Reaction) detection is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid extraction, and in particular to an inhibitor remover suitable for extracting DNA from whole blood and a nucleic acid extraction kit. Background Art

[0002] Nucleic acid extraction is one of the key steps in molecular diagnostic experiments. In clinical diagnosis, the detection results of nucleic acids are helpful for the diagnosis and classification of diseases; in the scientific research field, nucleic acid extraction is an important prerequisite for technologies such as gene cloning, PCR reaction, and whole-genome sequencing. There are two main purposes of nucleic acid extraction. One is purification, that is, to eliminate interfering substances in the sample; the other is concentration, that is, to increase the nucleic acid concentration in the sample. The concentration and purity of nucleic acids will directly affect the results of downstream experiments. Therefore, nucleic acid extraction is very important.

[0003] Blood is a tissue that circulates in the heart and blood vessel cavities and plays an important role in maintaining the homeostasis of the internal environment of the body. Therefore, some blood indicators are commonly used clinically as the basis for disease examination. With the continuous improvement of the molecular diagnostic system, blood is gradually applied to disease detection and genetic disease screening. For blood samples, its stable nucleic acids are particularly important.

[0004] Blood contains various inhibitors, such as fibrinogen, heme, etc. These inhibitors have similar physical and chemical properties to DNA. When using conventional DNA extraction methods (such as phenol-chloroform extraction method, guanidine hydrochloride lysis column method, sodium chloride precipitation method, etc.) to extract whole blood samples, these inhibitors will be enriched together with DNA, resulting in low purity of the DNA sample and poor subsequent detection results. Therefore, it is crucial to invent a reagent that can effectively remove inhibitors from whole blood samples. Summary of the Invention

[0005] In order to solve a series of problems caused by inhibitor residues during the extraction of whole blood DNA in the prior art, the purpose of the present invention is to provide an inhibitor remover suitable for extracting DNA from whole blood and a nucleic acid extraction kit. The inhibitor remover of the present invention includes a thiol compound with a mass fraction of 20% - 40%, a lower alcohol with a mass fraction of 30% - 50%, a non-ionic surfactant with a mass fraction of 1% - 2%, and an organic weak acid with a concentration of 10 mM - 30 mM. The nucleic acid extraction kit of the present invention includes a lysis-binding solution, a first washing solution, a second washing solution, an elution solution, and the above-mentioned inhibitor remover. Using the kit of the present invention can effectively remove inhibitors from whole blood samples during the extraction of whole blood DNA and improve the sensitivity of related PCR detections.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] The first object of the present invention is to provide an inhibitor remover applicable to the extraction of whole blood DNA, wherein the inhibitor remover comprises a thiol compound with a mass fraction of 20% - 40%, a lower alcohol with a mass fraction of 30% - 50%, a nonionic surfactant with a mass fraction of 1% - 2%, and an organic weak acid with a concentration of 10 mM - 30 mM.

[0008] In one embodiment of the present invention, the thiol compound is selected from one of 2-mercapto-3-butanol or 3-mercapto-2-butanol;

[0009] The lower alcohol is selected from one of ethanol, isopropanol or 1,2-propanediol;

[0010] The nonionic surfactant is selected from one of Tween-20, Triton X-100 or NP-40;

[0011] The organic weak acid is selected from one of boric acid, citric acid or acetic acid.

[0012] The second object of the present invention is to provide an application of an inhibitor remover applicable to the extraction of whole blood DNA in extracting DNA from whole blood samples.

[0013] The third object of the present invention is to provide an application of an inhibitor remover applicable to the extraction of whole blood DNA in preparing a nucleic acid extraction kit.

[0014] The fourth object of the present invention is to provide a nucleic acid extraction kit, wherein the nucleic acid extraction kit comprises a lysis-binding solution, a first washing solution, a second washing solution, an elution solution and the above-mentioned inhibitor remover.

[0015] In one embodiment of the present invention, the lysis-binding solution comprises a nonionic surfactant with a mass fraction of 5% - 10%, a chaotropic salt with a mass fraction of 60% - 70%, a Tris-HCl buffer solution with a concentration of 0.1 M, disodium ethylenediaminetetraacetate with a concentration of 10 - 20 mM, and 0.5 - 1 mg of silica hydroxyl magnetic beads;

[0016] The nonionic surfactant is selected from one of Tween-20, Triton X-100 or NP-40;

[0017] The chaotropic salt is selected from one of guanidine hydrochloride, guanidine thiocyanate or sodium perchlorate;

[0018] The pH value of the Tris-HCl buffer solution is 7.0 - 8.0.

[0019] In one embodiment of the present invention, the first washing solution comprises a lower alcohol with a mass fraction of 20% - 40% and a chaotropic salt with a mass fraction of 20% - 30%;

[0020] The lower alcohol is selected from one of ethanol, isopropanol or 1,2-propanediol;

[0021] The chaotropic salt is selected from one of guanidine hydrochloride, guanidine thiocyanate or sodium perchlorate.

[0022] In one embodiment of the present invention, the second washing solution is ethanol with a mass fraction of 70% to 80%.

[0023] In one embodiment of the present invention, the eluent is 10 mM Tris-HCl buffer (pH = 8.0).

[0024] The fifth object of the present invention is to provide an application of a nucleic acid extraction kit in extracting DNA from whole blood samples.

[0025] The steps for extracting whole blood DNA by the nucleic acid extraction kit containing an inhibitor remover of the present invention are as follows:

[0026] (S1) Add 200 - 400 μL of whole blood sample, 400 - 500 μL of lysis-binding solution, and 20 - 40 μL of inhibitor remover to a 2 mL centrifuge tube. After vortexing and mixing, place the centrifuge tube on a metal bath at 70 °C and incubate at 1500 rpm for 10 - 20 minutes.

[0027] (S2) Briefly centrifuge to collect the solution on the tube cap and tube wall to the bottom of the tube, then place the centrifuge tube on a magnetic rack for 1 - 2 minutes. After the magnetic beads are completely adsorbed, discard the solution (do not aspirate the magnetic beads).

[0028] (S3) Add 600 - 800 μL of washing solution 1 to the centrifuge tube. After vortexing and mixing, briefly centrifuge to collect the solution on the tube cap and tube wall to the bottom of the tube, then place the centrifuge tube on a magnetic rack for 1 - 2 minutes. After the magnetic beads are completely adsorbed, discard the solution (do not aspirate the magnetic beads).

[0029] (S4) Add 600 - 800 μL of washing solution 2 to the centrifuge tube. After vortexing and mixing, briefly centrifuge to collect the solution on the tube cap and tube wall to the bottom of the tube, then place the centrifuge tube on a magnetic rack for 1 - 2 minutes. After the magnetic beads are completely adsorbed, discard the solution (do not aspirate the magnetic beads).

[0030] (S5) Briefly centrifuge to collect the solution on the tube cap and tube wall to the bottom of the tube, place the centrifuge tube on a magnetic rack for 1 - 2 minutes. After the magnetic beads are completely adsorbed, discard the solution (do not aspirate the magnetic beads). Open the tube cap of the centrifuge tube and air-dry at room temperature for 3 - 5 minutes.

[0031] (S6) Add 50 - 200 μL of eluent (preheated at 65°C) to the centrifuge tube, vortex and mix well, then incubate at 65°C for 2 - 3 minutes. Briefly centrifuge to collect the solution on the tube cap and tube wall to the bottom of the tube. Then place the centrifuge tube on the magnetic rack for 3 - 5 minutes. After the magnetic beads are completely adsorbed, transfer the nucleic acid solution to a new 1.5 mL centrifuge tube (do not aspirate the magnetic beads).

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) It can more comprehensively remove inhibitors in whole blood samples: Thiol compounds can reduce the disulfide bonds of fibrinogen in whole blood samples; organic weak acids can effectively chelate metal ions and sugars in whole blood samples; non-ionic surfactants and lower alcohols can make thiol compounds fully miscible with water, all of which can effectively reduce the residue of inhibitors in whole blood samples.

[0034] (2) It can significantly reduce the odor of the inhibitor remover: Thiol compounds are commonly used meat flavors in the food industry, showing meaty and onion flavors at appropriate concentrations and being not easily volatile, which is significantly different from commonly used compounds such as mercaptoethanol and mercaptoacetic acid on the market with strong odors and easy volatility, and can improve the acceptance of users during use. Description of the Drawings

[0035] Figure 1 It is a fluorescence PCR result diagram after treating a whole blood sample containing excessive inhibitors according to the requirements of Example 5 without using an inhibitor remover.

[0036] Figure 2 It is a fluorescence PCR result diagram after treating a whole blood sample containing excessive inhibitors according to the requirements of Example 5 using β-mercaptoethanol as an inhibitor remover.

[0037] Figure 3 It is a fluorescence PCR result diagram after treating a whole blood sample containing excessive inhibitors according to the requirements of Example 5 using the inhibitor remover described in Example 1. Detailed Embodiments

[0038] The present invention provides an inhibitor remover suitable for extracting DNA from whole blood. The inhibitor remover includes a thiol compound with a mass fraction of 20% - 40%, a lower alcohol with a mass fraction of 30% - 50%, a non-ionic surfactant with a mass fraction of 1% - 2%, and an organic weak acid with a concentration of 10 mM - 30 mM.

[0039] In one embodiment of the present invention, the thiol compound is selected from one of 2-mercapto-3-butanol or 3-mercapto-2-butanol;

[0040] The lower alcohol is selected from one of ethanol, isopropanol or 1,2-propanediol;

[0041] The non-ionic surfactant is selected from one of Tween-20, Triton X-100 or NP-40;

[0042] The organic weak acid is selected from one of boric acid, citric acid or acetic acid.

[0043] The present invention provides an application of an inhibitor remover suitable for extracting whole blood DNA in extracting whole blood sample DNA.

[0044] The present invention provides an application of an inhibitor remover suitable for extracting whole blood DNA in preparing a nucleic acid extraction kit.

[0045] The present invention provides a nucleic acid extraction kit, which includes a lysis-binding solution, a first washing solution, a second washing solution, an elution solution and the above-mentioned inhibitor remover.

[0046] In one embodiment of the present invention, the lysis-binding solution includes a non-ionic surfactant with a mass fraction of 5% - 10%, a chaotropic salt of 60% - 70%, a Tris-HCl buffer solution with a concentration of 0.1M, disodium ethylenediaminetetraacetate of 10 - 20 mM, and 0.5 - 1 mg of silica hydroxyl magnetic beads;

[0047] The non-ionic surfactant is selected from one of Tween-20, Triton X-100 or NP-40;

[0048] The chaotropic salt is selected from one of guanidine hydrochloride, guanidine thiocyanate or sodium perchlorate;

[0049] The pH value of the Tris-HCl buffer solution is 7.0 - 8.0.

[0050] In one embodiment of the present invention, the first washing solution includes a lower alcohol with a mass fraction of 20% - 40% and a chaotropic salt of 20% - 30%;

[0051] The lower alcohol is selected from one of ethanol, isopropanol or 1,2-propanediol;

[0052] The chaotropic salt is selected from one of guanidine hydrochloride, guanidine thiocyanate or sodium perchlorate.

[0053] In one embodiment of the present invention, the second washing solution is ethanol with a mass fraction of 70% - 80%.

[0054] In one embodiment of the present invention, the elution solution is 10 mM Tris-HCl buffer solution (pH = 8.0).

[0055] The present invention provides an application of a nucleic acid extraction kit in extracting whole blood sample DNA.

[0056] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0057] In the following embodiments, unless otherwise specified, the reagents used are commercially available reagents, and the detection means and methods are conventional detection means and methods in the art.

[0058] Example 1

[0059] This example provides an inhibitor remover suitable for extracting DNA from whole blood.

[0060] The inhibitor remover contains 2-mercapto-3-butanol with a mass fraction of 40%, ethanol with a mass fraction of 30%, Triton X-100 with a mass fraction of 1%, and boric acid with a concentration of 10 mM.

[0061] Example 2

[0062] This example provides an inhibitor remover suitable for extracting DNA from whole blood.

[0063] The inhibitor remover contains 2-mercapto-3-butanol with a mass fraction of 30%, ethanol with a mass fraction of 50%, Triton X-100 with a mass fraction of 2%, and boric acid with a concentration of 30 mM.

[0064] Example 3

[0065] This example provides a kit suitable for extracting DNA from whole blood.

[0066] The kit includes the following substances:

[0067] Inhibitor remover: 2-mercapto-3-butanol at 40%, ethanol at 30%, boric acid at 10 mM, Triton X-100 at 1%;

[0068] Lysis and binding solution: NP-40 at 10%, guanidine thiocyanate at 60%, 0.1 M Tris-HCl buffer (pH = 7.0), disodium ethylenediaminetetraacetate at 20 mM, 0.5 mg of silica hydroxyl magnetic beads;

[0069] Washing solution 1: isopropanol at 40%, guanidine hydrochloride at 20%;

[0070] Washing solution 2: ethanol at 70%;

[0071] Elution solution: 10 mM Tris-HCl buffer (pH = 8.0).

[0072] Example 4

[0073] This example provides a kit suitable for extracting DNA from whole blood.

[0074] The kit includes the following substances:

[0075] Inhibitor remover: 30% 2-mercapto-3-butanol, 50% ethanol, 30 mM boric acid, 2% Triton X-100;

[0076] Lysis binding solution: 5% NP-40, 70% guanidine thiocyanate, 0.1 M Tris-HCl buffer (pH = 8.0), 10 mM disodium ethylenediaminetetraacetate, 1 mg silica hydroxyl magnetic beads;

[0077] Washing solution 1: 20% isopropanol, 30% guanidine hydrochloride;

[0078] Washing solution 2: 80% ethanol;

[0079] Elution solution: 10 mM Tris-HCl buffer (pH = 8.0).

[0080] Example 5

[0081] Effect of inhibitor remover in whole blood DNA extraction reagent

[0082] Add 12 mg fibrinogen (from human plasma) and 0.6 mg bilirubin as excessive inhibitors to 3 mL whole blood samples, and add EBV pseudovirus with a final concentration of 1×10 3 Copies / mL as the model virus, and perform whole blood DNA extraction using the following three methods respectively:

[0083] A. Without using inhibitor remover:

[0084] Add 200 μL whole blood sample, 500 μL lysis binding solution, and 20 μL ultrapure water to a 2 mL centrifuge tube. After vortex mixing, place the centrifuge tube on a metal bath at 70 °C and incubate at 1500 rpm for 20 minutes.

[0085] B. Using common mercaptoethanol on the market as inhibitor remover

[0086] Add 200 μL whole blood sample, 500 μL lysis binding solution, and 20 μL β-mercaptoethanol to a 2 mL centrifuge tube. After vortex mixing, place the centrifuge tube on a metal bath at 70 °C and incubate at 1500 rpm for 20 minutes.

[0087] C. Using the inhibitor remover combination of the present invention

[0088] Add 200 μL whole blood sample, 500 μL lysis binding solution, and 20 μL inhibitor remover (Example 1) to a 2 mL centrifuge tube. After vortex mixing, place the centrifuge tube on a metal bath at 70 °C and incubate at 1500 rpm for 20 minutes.

[0089] After the above different de-suppression treatments, the same extraction operation was performed on each group:

[0090] (1) Briefly centrifuge to collect the solution on the tube cap and the tube wall to the bottom of the tube, then place the centrifuge tube on the magnetic stand for 1 - 2 minutes. After the magnetic beads are completely adsorbed, aspirate and discard the solution (do not aspirate the magnetic beads).

[0091] (2) Add 600 μL of Wash Buffer 1 to the centrifuge tube, vortex to mix well, then briefly centrifuge to collect the solution on the tube cap and the tube wall to the bottom of the tube. Then place the centrifuge tube on the magnetic stand for 1 minute. After the magnetic beads are completely adsorbed, aspirate and discard the solution (do not aspirate the magnetic beads).

[0092] (3) Add 600 μL of Wash Buffer 2 to the centrifuge tube, vortex to mix well, then briefly centrifuge to collect the solution on the tube cap and the tube wall to the bottom of the tube. Then place the centrifuge tube on the magnetic stand for 1 minute. After the magnetic beads are completely adsorbed, aspirate and discard the solution (do not aspirate the magnetic beads).

[0093] (4) Briefly centrifuge to collect the solution on the tube cap and the tube wall to the bottom of the tube, place the centrifuge tube on the magnetic stand for 1 minute. After the magnetic beads are completely adsorbed, aspirate and discard the solution (do not aspirate the magnetic beads). Open the tube cap of the centrifuge tube and air-dry at room temperature for 3 minutes.

[0094] (5) Add 100 μL of Elution Buffer (preheated at 65 °C) to the centrifuge tube, vortex to mix well, incubate at 65 °C for 3 minutes, briefly centrifuge to collect the solution on the tube cap and the tube wall to the bottom of the tube. Then place the centrifuge tube on the magnetic stand for 3 minutes. After the magnetic beads are completely adsorbed, transfer the nucleic acid solution to a new 1.5 mL centrifuge tube (do not aspirate the magnetic beads).

[0095] Among them, the formula of each component of the whole blood nucleic acid extraction kit involved in the above process:

[0096] Lysis Binding Buffer: 10% NP - 40, 60% guanidine thiocyanate, 0.1 M Tris - HCl buffer (pH = 7.0), 20 mM disodium ethylenediaminetetraacetate, 0.5 mg silica hydroxyl magnetic beads;

[0097] Wash Buffer 1: 40% isopropanol, 20% guanidine hydrochloride;

[0098] Wash Buffer 2: 70% ethanol;

[0099] Elution Buffer: 10 mM Tris - HCl buffer (pH = 8.0).

[0100] After the extraction was completed, the extraction products of each group were subjected to PCR amplification on ABI7500 using an Epstein - Barr virus nucleic acid detection kit (fluorescent PCR method). The composition of the amplification system and the amplification program are as follows:

[0101] Amplification system (per sample):

[0102] 9 μL of PCR buffer, 3 μL of Taq enzyme, and 8 μL of EB virus primer probe

[0103] Amplification program:

[0104] Step 1: React at 50 °C for 2 min, incubate at 94 °C for 5 min, and cycle 1 time;

[0105] Step 2: 94 °C for 10 s → 60 °C for 45 s, cycle 5 times;

[0106] Step 3: 94 °C for 10 s → 60 °C for 45 s, cycle 40 times, and collect FAM channel signals at 60 °C.

[0107] The detection results are as Figure 1 , Figure 2 and Figure 3 shown. It can be seen from the results that when the inhibitor remover of Example 1 is used to process the whole blood sample, the Ct value of its fluorescence PCR amplification is significantly better than that when the commonly used mercaptoethanol on the market is used as the inhibitor remover or no inhibitor remover is used to process the whole blood sample.

[0108] Example 6

[0109] This example provides the application of the kit described in Example 4 and a similar product (Blood DNA Kit D3392, Omega) in the extraction of whole blood DNA.

[0110] The extraction is carried out according to the following steps:

[0111] (1) Add 200 μL of whole blood sample, 500 μL of lysis-binding solution, and 20 μL of inhibitor remover to a 2 mL centrifuge tube. After vortexing and mixing, place the centrifuge tube on a metal bath at 70 °C and incubate at 1500 rpm for 20 minutes.

[0112] (2) Centrifuge briefly to collect the solution on the tube cap and the tube wall to the bottom of the tube, then place the centrifuge tube on a magnetic rack for 1 - 2 minutes. After the magnetic beads are completely adsorbed, discard the solution (do not aspirate the magnetic beads).

[0113] (3) Add 600 μL of washing solution 1 to the centrifuge tube. After vortexing and mixing, centrifuge briefly to collect the solution on the tube cap and the tube wall to the bottom of the tube, then place the centrifuge tube on a magnetic rack for 1 minute. After the magnetic beads are completely adsorbed, discard the solution (do not aspirate the magnetic beads).

[0114] (4) Add 600 μL of washing solution 2 to the centrifuge tube. After vortexing and mixing, centrifuge briefly to collect the solution on the tube cap and the tube wall to the bottom of the tube, then place the centrifuge tube on a magnetic rack for 1 minute. After the magnetic beads are completely adsorbed, discard the solution (do not aspirate the magnetic beads).

[0115] (5) Briefly centrifuge to collect the solution on the tube cap and the tube wall to the bottom of the tube. Place the centrifuge tube on the magnetic stand for 1 minute. After the magnetic beads are completely adsorbed, aspirate and discard the solution (do not aspirate the magnetic beads). Open the tube cap of the centrifuge tube and air-dry at room temperature for 3 minutes.

[0116] (6) Add 100 μL of elution buffer (preheated at 65 °C) to the centrifuge tube. Vortex to mix well and incubate at 65 °C for 3 minutes. Briefly centrifuge to collect the solution on the tube cap and the tube wall to the bottom of the tube. Then place the centrifuge tube on the magnetic stand for 3 minutes. After the magnetic beads are completely adsorbed, transfer the nucleic acid solution to a new 1.5 mL centrifuge tube (do not aspirate the magnetic beads).

[0117] While the above experiment is being completed, use the control kit Blood DNA Kit D3392 to extract 200 μL of the same whole blood sample, and the elution volume is 100 μL.

[0118] After extraction using the kit described in Example 4 and the control kit (Blood DNA Kit D3392, Omega), use a NanoDrop MD one ultra-micro ultraviolet spectrophotometer to detect the DNA concentration and purity. The results are shown in Table 1.

[0119] Table 1 Comparison of extraction results of the kit described in Example 4 and the control reagent

[0120]

[0121]

[0122] As can be seen from Table 1, the extraction concentration and purity of the kit of the present invention are both higher than those of the control kit (Blood DNA Kit D3392, Omega).

[0123] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. Those skilled in the art can obviously make various modifications to these embodiments easily and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the interpretation of the present invention should be within the protection scope of the present invention.

Claims

1. An inhibitor remover applicable to whole blood DNA extraction, characterized in that, The inhibitor remover comprises a thiol compound with a mass fraction of 20% to 40%, a lower alcohol with a mass fraction of 30% to 50%, a nonionic surfactant with a mass fraction of 1% to 2%, and an organic weak acid with a concentration of 10 mM to 30 mM.

2. The inhibitor removing agent for whole blood DNA extraction according to claim 1, characterized in that, The thiol compound is selected from one of 2-mercapto-3-butanol or 3-mercapto-2-butanol; The lower alcohol is selected from one of ethanol, isopropanol or 1,2-propanediol; The nonionic surfactant is selected from one of Tween-20, Triton X-100 or NP-40; The organic weak acid is selected from one of boric acid, citric acid or acetic acid.

3. Application of an inhibitor remover for extracting whole blood DNA as described in any one of claims 1 to 2 in extracting whole blood sample DNA.

4. Application of an inhibitor remover for extracting whole blood DNA as described in any one of claims 1 to 2 in preparing a nucleic acid extraction kit.

5. A nucleic acid extraction kit, characterized in that, The nucleic acid extraction kit comprises a lysis binding solution, a first washing solution, a second washing solution, an elution solution and an inhibitor remover as described in any one of claims 1 to 2.

6. The nucleic acid extraction kit according to claim 5, wherein, The lysis binding solution comprises a nonionic surfactant with a mass fraction of 5% to 10%, a chaotropic salt with a mass fraction of 60% to 70%, a Tris-HCl buffer solution with a concentration of 0.1 M, disodium ethylenediaminetetraacetate with a concentration of 10 to 20 mM, and silica hydroxyl magnetic beads with a mass of 0.5 to 1 mg; The nonionic surfactant is selected from one of Tween-20, Triton X-100 or NP-40; The chaotropic salt is selected from one of guanidine hydrochloride, guanidine thiocyanate or sodium perchlorate; The pH value of the Tris-HCl buffer solution is 7.0 to 8.

0.

7. A nucleic acid extraction kit according to claim 5, wherein, The first washing solution comprises a lower alcohol with a mass fraction of 20% to 40% and a chaotropic salt with a mass fraction of 20% to 30%; The lower alcohol is selected from one of ethanol, isopropanol or 1,2-propanediol; The chaotropic salt is selected from one of guanidine hydrochloride, guanidine thiocyanate or sodium perchlorate.

8. A nucleic acid extraction kit according to claim 5, characterized in that, The second washing solution is ethanol with a mass fraction of 70% to 80%.

9. A nucleic acid extraction kit according to claim 5, characterized in that, The elution solution is a 10 mM Tris-HCl buffer solution.

10. Application of a nucleic acid extraction kit as described in claim 5 in extracting whole blood sample DNA.

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