A nucleic acid extraction lysis / binding solution, a nucleic acid extraction solution system and a kit

Abstract

The present invention discloses a nucleic acid extraction lysis / binding solution, a nucleic acid extraction solution system and a kit. The nucleic acid extraction lysis / binding solution includes: 2-5M guanidine isothiocyanate, 0.1-4% SDS, 5-40% N-methylacetamide, 10-50% tetraethylene glycol; further includes: 5-20% Triton X-100, 10-100mM Tris-HCl, 0-50mM EDTA·2Na, 0.1-5mg / mL silicon hydroxy magnetic beads, and the pH range of the Tris-HCl is 6-9. The nucleic acid extraction solution system includes: nucleic acid extraction lysis / binding solution, proteinase K, washing solution 1, washing solution 2, and eluent. The lysis / binding solution provided by the present invention realizes better mutual solubility of guanidine isothiocyanate and SDS, so that the lysis of plasma samples is more sufficient, and the extraction efficiency of plasma cfDNA is improved. In addition, the combination of tetraethylene glycol and N-methylacetamide in the present invention effectively realizes the replacement of ethanol or isopropanol as a binding system, solves the flammable and explosive problems in the prior art, greatly reduces transportation costs, and simplifies the production process.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid extraction, and in particular to a nucleic acid extraction lysis / binding solution, a nucleic acid extraction solution system and a kit. Background Art

[0002] Molecular diagnosis uses nucleic acid as the detection object and is mainly used in the diagnosis of various clinical departments, such as tumors, infections, genetics, etc. The basis of all work is nucleic acid extraction. Under specific conditions, the surface of the solid phase carrier magnetic beads is coated with silicon, amino or carboxyl groups, or solid phase silica gel membrane carriers. Relying on electrostatic, hydrophobic and hydrogen bonding effects, the specific binding of nucleic acids and magnetic beads can be achieved. Non-specific impurities, salts, etc. can be removed through several washes, and then the nucleic acids are eluted from the solid phase carrier to achieve purification.

[0003] A certain amount of protein, macromolecules and salts usually remain on magnetic beads and silica gel membrane solid phase carriers, and the washing step helps to remove these impurities. There are usually two ways of washing, which vary depending on the sample. For the extraction of plasma cfDNA (free DNA), it is usually divided into two steps of washing. The first step is to remove proteins with medium concentrations of chaotropic salts and alcohols, and then wash with ethanol to remove salts. Through relevant product manuals and public chemical safety data sheets (MSDS), Table 1 summarizes the operation and components of the common cfDNA extraction kits currently on the market.

[0004] Table 1

[0005]

[0006] Conventional nucleic acid extraction kits based on solid phase carrier method usually use high concentrations of guanidine salts and proteinase K to lyse the sample, and then use monohydric alcohols such as ethanol and isopropanol as the binding solution, and the subsequent washing solution basically contains ethanol. For the lysate, the anionic detergent sodium dodecyl sulfate (SDS) can effectively denature the protein, destroy the electrostatic interaction between DNA and protein, and separate the two. As a highly chaotropic salt, the presence of guanidine isothiocyanate helps the denaturation and lysis of proteins, and is also crucial for the binding of nucleic acids to the silicon matrix. In addition, in order to enhance the adsorption strength of the two, ethanol or isopropanol is usually added during the binding stage. Since SDS and guanidine isothiocyanate are difficult to dissolve in each other, and directly adding ethanol during the binding stage will interfere with the lysis effect of SDS, guanidine isothiocyanate and protein, these processes are usually carried out step by step, such as firstly through the initial lysis of SDS and proteinase K, then adding a guanidine salt solution for further lysis, and then adding an alcohol-containing binding solution for binding to complete in sequence.

[0007] SDS is difficult to dissolve in high concentrations of guanidine salts, and SDS is a strong protein denaturant that can destroy its hydrophobic effect, open hydrogen bonds and combine with it to form a protein-SDS complex structure. Guanidine salts can dissolve proteins, causing damage to cell structures and the secondary structure of nucleoproteins, thereby removing impurities such as proteins as much as possible to obtain high-purity DNA. However, under normal conditions, due to the low solubility of SDS in high-salt solutions, it is difficult for SDS and guanidine salts to work synergistically to achieve the optimal lysis state. The current extraction reagents for plasma cfDNA are difficult to achieve the fusion of SDS and guanidine salt lysis in one step to achieve the best synergistic effect, and the operation steps are numerous and complicated. On the other hand, the lysis / binding solution and washing solution usually contain a large amount of ethanol, which is flammable, explosive, and volatile, which is very unfavorable for production and transportation, and adds additional costs. Summary of the invention

[0008] The object of the present invention is to provide a nucleic acid extraction lysis / binding solution and a kit and application. One of the technical problems to be solved by the present invention is to prepare a solution system of SDS and guanidine salt miscible to form an efficient nucleic acid extraction lysis / binding solution. The second technical problem to be solved by the present invention is to propose a reagent system for plasma cfDNA extraction, in which each component does not contain any flammable liquids such as ethanol, thereby reducing the complexity of production and transportation and reducing costs.

[0009] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows:

[0010] A nucleic acid extraction lysis / binding solution, the lysis / binding solution comprises: 2-5M guanidine isothiocyanate, 0.1-4% SDS, 5-40% N-methylacetamide, 10-50% tetraethylene glycol. Guanidine isothiocyanate is also called guanidine thiocyanate; the solution system of the lysis / binding solution is an aqueous solution; the percentage involved is a mass volume ratio for solid substances and a volume volume ratio for liquid substances. Here, SDS and N-methylacetamide are solids, and tetraethylene glycol is a liquid.

[0011] As a preferred embodiment, the lysis / binding solution comprises: 3-5M guanidine thiocyanate, 1-2% SDS, 15-20% N-methylacetamide, and 20-30% tetraethylene glycol.

[0012] As a preferred embodiment, the lysis / binding solution further comprises: 5-20% Triton X-100, 10-100 mM Tris-HCl, 0-50 mM EDTA·2Na, 0.1-5 mg / mL silicon hydroxy magnetic beads, and the pH range of the Tris-HCl is 6-9.

[0013] As a preferred embodiment, the lysis / binding solution further comprises: 7.5-10% Triton X-100, 25-50mM Tris-HCl, 10-15mM EDTA·2Na, 0.5-1mg / mL silicon hydroxy magnetic beads, and the pH range of the Tris-HCl is 7.5-8.5.

[0014] The present invention also provides a nucleic acid extraction solution system, which comprises: the nucleic acid extraction lysis / binding solution, proteinase K, washing solution 1, washing solution 2, and eluent;

[0015] The concentration range of the proteinase K is 15-25 mg / mL;

[0016] The composition and concentration range of the washing solution 1 are: 1-3M guanidine thiocyanate, 100-500mM NaCl, 0-2% Triton X-100, 15-40% tetraethylene glycol;

[0017] The composition and concentration range of the washing solution 2 are: 50-200 mM NaCl, 5-20 mM Tris-HCl, 50-80% tetraethylene glycol, and the pH range of the Tris-HCl is 6-7;

[0018] The composition and concentration range of the eluent are: 5-20 mM Tris-HCl, and the pH range of the Tris-HCl is 6-8.

[0019] The present invention also provides a method for extracting plasma cfDNA using the nucleic acid extraction solution system, comprising the following steps:

[0020] 1) Lysis / binding: Proteinase K and the nucleic acid extraction lysis / binding solution of the present invention are added to the plasma sample, and the sample is incubated for lysis at 0-60° C.; the nucleic acid extraction lysis / binding solution contains silicon hydroxy magnetic beads.

[0021] 2) Washing step 1: collect the magnetic beads, remove the lysis solution, add washing solution 1, and perform shaking washing;

[0022] 3) Washing step 2: collect the magnetic beads, remove the washing solution 1, add the washing solution 2, and perform shaking washing;

[0023] 4) Washing step 2: collect the magnetic beads, remove the washing solution 2, add the eluent, and let stand;

[0024] 5) Elution step: Collect the magnetic beads, remove all the solution, add the eluent, shake and elute, and then collect the eluent, which is the extracted cfDNA.

[0025] As a preferred embodiment, in step 1), the volume of proteinase K added is 0.04-0.06 times that of the plasma sample, and the volume of nucleic acid extraction lysis / binding solution added is 1-1.5 times that of the plasma sample.

[0026] As a preferred embodiment, in the washing steps 2-4), the volume of washing solution 1, washing solution 2 and eluent added is 0.1-0.5 times of that of the plasma sample, respectively.

[0027] As a preferred embodiment, the volume of the eluent added in the elution step 5) is 0.01-0.05 times that of the plasma sample.

[0028] As a preferred embodiment, the method for extracting plasma cfDNA with the nucleic acid extraction solution system comprises the following specific steps:

[0029] 1) Lysis / binding: Add 1-5 ml of treated plasma sample to the tube, add 0.05 times the volume of plasma sample proteinase K, add 1.25 times the volume of plasma sample nucleic acid extraction lysis / binding solution (the nucleic acid extraction lysis / binding solution contains silicon hydroxy magnetic beads), and then incubate vigorously at a temperature of 0-60°C for 10-30 minutes;

[0030] 2) Washing step 1: Place the tube on the magnetic rack and wait until the magnetic beads are completely aggregated on the side wall, then remove all the solution; remove the magnetic rack, add 0.5-1 mL of washing solution 1 to the tube, and shake vigorously at room temperature for 1 minute;

[0031] 3) Washing step 2: Place the tube on the magnetic rack until the magnetic beads are completely aggregated on the side wall, and then remove all the solution; remove the magnetic rack, add 0.5-1 mL of washing solution 2 to the tube, and shake vigorously at room temperature for 1 minute;

[0032] 4) Washing step 3: Place the tube on the magnetic rack and wait until the magnetic beads are completely aggregated on the side wall, then remove all the solution; keep the tube on the magnetic rack, add 0.5-1 mL of eluent to the tube, let it stand for 10 seconds, and remove as much liquid as possible;

[0033] 5) Elution: Remove the magnetic rack, add 25-100 μL of elution solution to the tube, and shake vigorously at 60°C for 3-5 minutes; place the tube on the magnetic rack and let it stand until the magnetic beads are completely aggregated to the side wall, then transfer the solution to a new centrifuge tube to obtain the purified cfDNA.

[0034] The present invention also provides a nucleic acid extraction kit, which comprises the nucleic acid extraction lysis / binding solution.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The nucleic acid extraction lysis / binding solution provided by the present invention realizes the miscibility of guanidine isothiocyanate and SDS. The lysis solution lyses the plasma sample more fully, can complete the lysis and binding in one step, simplifies the operation process, improves the extraction efficiency of plasma cfDNA to a greater extent, shortens the total operation time, and easily realizes automated operation.

[0037] 2. The present invention utilizes a combination of tetraethylene glycol and N-methylacetamide to effectively replace ethanol or isopropanol as a binding system, and simultaneously utilizes tetraethylene glycol to replace ethanol as an important component in the washing system, thereby solving the flammable and explosive problems of the prior art, greatly reducing transportation costs, and simplifying the production process. DETAILED DESCRIPTION

[0038] The technical scheme of the present invention is described below by specific examples. The raw materials and reagents used in the present invention are all commercially available.

[0039] Example 1 Dissolution of Nucleic Acid Extraction Lysis / Binding Solution at Various Temperatures and Concentrations

[0040] Lysis / binding solutions were prepared according to the following concentration groups. After being fully dissolved, the dissolution of the solutions was observed at room temperature (about 25°C) and -20°C. The specific data are shown in Table 2.

[0041] Table 2

[0042]

[0043] It can be seen from the data results in Table 2 that in the absence of the combination of N-methylacetamide and tetraethylene glycol, or in the presence of only a single component, the mixed solution containing guanidine isothiocyanate and SDS is in a white suspension state and cannot be completely dissolved. When N-methylacetamide and tetraethylene glycol exist in a certain ratio, guanidine isothiocyanate and SDS can be well dissolved in each other, so that the solution system can be well used for subsequent actions on the sample.

[0044] Example 2 Comparison of the extraction yield of plasma samples using the method of the present invention and similar cfDNA extraction kits

[0045] The nucleic acid extraction solution system of the present invention is prepared according to the scheme in Table 3.

[0046] Table 3

[0047]

[0048] According to the above plasma cfDNA extraction method, 8 processed plasma samples were extracted and Qiagen Circulating Nucleic Acid kit was used as a control reagent for extraction under the same conditions, with 2 mL of each sample. The control reagent was extracted according to the standard protocol provided in the instructions. The extraction method of the present invention is as follows:

[0049] 1) Add 2 ml of treated plasma sample, 100 μL of proteinase K, and 2.5 mL of lysis / binding solution to the tube, and then incubate vigorously at 37°C for 20 minutes.

[0050] 2) Place the tube on the magnetic rack until the magnetic beads are completely aggregated on the side wall, then remove all the solution. Remove the magnetic rack, add 700 μL of washing solution 1 to the tube, and shake vigorously at room temperature for 1 minute.

[0051] 3) Place the tube on the magnetic rack until the beads are completely aggregated on the side wall, then remove all the solution. Remove the magnetic rack, add 700 μL of washing solution 2 to the tube, and shake vigorously at room temperature for 1 minute.

[0052] 4) Place the tube on the magnetic rack and wait until the magnetic beads are completely gathered on the side wall, then remove all the solution. Keep the tube on the magnetic rack, add 700 μL of elution solution to the tube, let it stand for 10 seconds, and remove the liquid completely.

[0053] 5) Remove the magnetic rack, add 50 μL of elution buffer to the tube, and shake vigorously at 60°C for 5 minutes. Leave the tube on the magnetic rack until the magnetic beads are completely aggregated on the side wall, and transfer the solution to a new centrifuge tube for later use.

[0054] After the extraction was completed by the method of the present invention and the control reagent, the concentration was measured using Qubit 4.0 to compare the recovery rate. The data results are shown in Table 4.

[0055] Table 4

[0056] Sample No. The method of the present invention Control reagent / ng 1 43.7 38.8 2 24.4 22.6 3 33.1 27.8 4 25.0 20.8 5 46.4 39.9 6 57.0 49.5 7 59.9 50.9 8 27.6 29.2

[0057] It can be seen from the data in Table 4 that compared with the control reagent, the recovery rate of the extraction method of the present invention is significantly improved.

[0058] Regarding the concentration range of each component in the nucleic acid extraction lysis / binding solution, after further experimental testing, the preferred range of each component is 3-5M guanidine thiocyanate, 1-2% SDS, 15-20% N-methylacetamide, and 20-30% tetraethylene glycol. When these components are mixed within the corresponding concentration range, the nucleic acid extraction recovery rate obtained is good.

[0059] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims

1. A nucleic acid extraction lysis / binding solution, characterized in that: The components of the lysis / binding solution are: 3-5M guanidine thiocyanate, 1-2% SDS, 15-20% N-methylacetamide, 20-30% tetraethylene glycol; 7.5-10% Triton X-100, 25-50 mM Tris-HCl, 10-15 mM EDTA·2Na, 0.5-1 mg / mL silicon hydroxy magnetic beads, wherein the pH range of the Tris-HCl is 7.5-8.

5.

2. A nucleic acid extraction solution system, characterized in that: The solution system comprises: the nucleic acid extraction lysis / binding solution of claim 1, proteinase K, washing solution 1, washing solution 2, and elution solution; The concentration range of the proteinase K is 15-25 mg / mL; The composition and concentration range of the washing solution 1 are: 1-3M guanidine thiocyanate, 100-500mM NaCl, 0-2% TritonX-100, 15-40% tetraethylene glycol; The composition and concentration range of the washing solution 2 are: 50-200 mM NaCl, 5-20 mM Tris-HCl, 50-80% tetraethylene glycol, and the pH range of the Tris-HCl is 6-7; The composition and concentration range of the eluent are: 5-20 mM Tris-HCl, and the pH range of the Tris-HCl is 6-8.

3. The method for extracting plasma cfDNA using the nucleic acid extraction solution system according to claim 2, comprising the following steps: 1) Lysis / binding: Proteinase K and nucleic acid extraction lysis / binding solution were added to the plasma sample and incubated for lysis at 0-60°C; 2) Washing step 1: collect the magnetic beads, remove the lysis solution, add washing solution 1, and perform shaking washing; 3) Washing step 2: collect the magnetic beads, remove the washing solution 1, add the washing solution 2, and perform shaking washing; 4) Washing step 3: Collect the magnetic beads, remove the washing solution 2, add the eluent, and let stand; 5) Elution step: Collect the magnetic beads, remove all the solution, add the eluent, shake and elute, and then collect the eluent, which is the extracted cfDNA.

4. The method for extracting plasma cfDNA using a nucleic acid extraction solution system according to claim 3, characterized in that: In the step 1), the volume of proteinase K added is 0.04-0.06 times that of the plasma sample, and the volume of nucleic acid extraction lysis / binding solution added is 1-1.5 times that of the plasma sample.

5. The method for extracting plasma cfDNA using a nucleic acid extraction solution system according to claim 3, characterized in that: In the washing steps 2-4), the volume of washing solution 1, washing solution 2 and eluent added is 0.1-0.5 times of the plasma sample, respectively.

6. The method for extracting plasma cfDNA using a nucleic acid extraction solution system according to claim 3, characterized in that: The volume of the eluent added in the elution step 5) is 0.01-0.05 times that of the plasma sample.

7. A nucleic acid extraction kit, characterized in that: The kit comprises the nucleic acid extraction lysis / binding solution according to claim 1.

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